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Gene families evolve by the processes of speciation (creating orthologs), gene duplication (paralogs), and horizontal gene transfer (xenologs), in addition to sequence divergence and gene loss. Orthologs in particular play an essential role in comparative genomics and phylogenomic analyses. With the continued sequencing of organisms across the tree of life, the data are available to reconstruct the unique evolutionary histories of tens of thousands of gene families. Accurate reconstruction of these histories, however, is a challenging computational problem, and the focus of the Quest for Orthologs Consortium. We review the recent advances and outstanding challenges in this field, as revealed at a symposium and meeting held at the University of Southern California in 2017. Key advances have been made both at the level of orthology algorithm development and with respect to coordination across the community of algorithm developers and orthology end-users. Applications spanned a broad range, including gene function prediction, phylostratigraphy, genome evolution, and phylogenomics. The meetings highlighted the increasing use of meta-analyses integrating results from multiple different algorithms, and discussed ongoing challenges in orthology inference as well as the next steps toward improvement and integration of orthology resources.
The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.
The ability to vocalize is ubiquitous in vertebrates, but neural networks underlying vocal control remain poorly understood. Here, we performed simultaneous neuronal recordings in the frontal cortex and dorsal striatum (caudate nucleus, CN) during the production of echolocation pulses and communication calls in bats. This approach allowed us to assess the general aspects underlying vocal production in mammals and the unique evolutionary adaptations of bat echolocation. Our data indicate that before vocalization, a distinctive change in high-gamma and beta oscillations (50–80 Hz and 12–30 Hz, respectively) takes place in the bat frontal cortex and dorsal striatum. Such precise fine-tuning of neural oscillations could allow animals to selectively activate motor programs required for the production of either echolocation or communication vocalizations. Moreover, the functional coupling between frontal and striatal areas, occurring in the theta oscillatory band (4–8 Hz), differs markedly at the millisecond level, depending on whether the animals are in a navigational mode (that is, emitting echolocation pulses) or in a social communication mode (emitting communication calls). Overall, this study indicates that fronto-striatal oscillations could provide a neural correlate for vocal control in bats.
In recent environmental research, relational values (RVs) have emerged as a new group of values to explain environmental behavior. Although this new concept is attracting attention, empirical studies on the subject are still rare. On this basis, we have conducted three studies to analyze an existing measurement tool for RVs and compared the construct with the concept of connection to nature. In study 1, we confirmed convergent and discriminant validity of the RV scale by comparing it with the Two Factor Model of Environmental Values (2-MEV) model using a sample of n = 350 university students. Additionally, study 1 verified reliability using test–retest reliability on three different groups of students (n1 = 53; n2 = 37; n3 = 48). In study 2, principal component analyses were performed to examine the structure of RVs and to compare it to the concept of connection to nature by reusing the sample 350 university students from study 1. The results show that RVs and connection to nature are not fundamentally distinct constructs, but overlap. However, if the structure of the RV measurement is forced to a single factor, no perfect fit is found, making a multidimensional solution more likely. A third study was conducted to review the results from study 2 using confirmatory factor analysis on a new sample of 878 university and high school students. Study 3 confirmed RVs as a multidimensional construct with three factors: care, community, and connection. It also proved the overlap of the connection to nature and RV concepts to some extent.
The balance between peripheral T-cell reactivity and self-tolerance is achieved during T-cell development in the thymus. During thymic development T-cell sensitivity to self-antigens drives their selection and is dynamically regulated via multiple mechanisms. The microRNA miR-181 has been implicated as a post-transcriptional modulator of T-cell sensitivity due to its suppression of several negative regulators of T-cell receptor (TCR) signalling. By tuning developing thymocytes to be exquisitely sensitive to signals transduced through their TCR, miR-181 has previously been shown to be essential for the agonist selection of invariant natural killer T (iNKT) cells. In this thesis, we extend the knowledge on the developmental control elicited by miR-181 in the thymus to cover mucosal-associated invariant T (MAIT), regulatory T (Treg) and conventional T cells. Using a germline knock-out of mature miR-181a/b-1, we could show that all agonist-selected T cell populations are critically dependant on miR-181a/b-1, noting an absence of MAIT and a reduction of thymic-derived Tregs in miR-181a/b-1-deficient mice. Furthermore, we provided evidence that miR-181 is also required for the negative selection of conventional T cells, with miR-181a/b-1-deficient mice presenting with a near absence of apoptotic markers. Therefore, by heightening the TCR sensitivity to self-antigens, miR-181a/b-1 aids in the detection and subsequent elimination of autoreactive thymocytes. In addition, we characterised the murine primary miR-181a/b-1 transcript, which surprisingly has a transcription start site (TSS) more than 70kB upstream of the mature miRNA sequences. This shall hopefully lead to future research aimed at deciphering the upstream regulatory networks that promote dynamic miR-181a/b-1 expression in developing thymocytes. In summary, we present here a single miRNA subset with broad implications in T-cell development. In disagreement with central dogma that individual miRNAs generally provide weak to moderate modulation over cellular pathways, we showcase the miR-181 family subset, miR-181a/b-1, as an efficient regulator of TCR signalling pathways. Due to the sensitive nature of TCR signalling during thymocyte selection, miR-181a/b-1 elicits gross effects, which are essential for agonist selection, central tolerance and generating a functional self-tolerant peripheral T cell repertoire. We therefore conclude that miR-181a/b-1 is fundamental in T-cell development as a whole.
Zeit ist einer jener Begriffe, für die man die Augustinische Charakterisierung gelten lassen wollte, es sei klar, was sie bedeuten, solange nicht danach gefragt werde (Augustinus Confessiones Lib. XI, 17). Die Frage aber nach dem, was "Zeit" eigentlich ist, erscheint umso berechtigter, als es insbesondere die Naturwissenschaften sind, die für sich in Anspruch nehmen, hier Antworten geben zu können. Die zu erwartenden Antworten wären danach wesentlich empirischer Natur – also direkt oder indirekt experimentell gestützt und mithin Ergebnis dieser Forschung. ...
Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are by individual molecules such as cytokines carried by blood, by neural transmission, or, in more severe pathologies, by the entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs), but the parameters that determine their uptake are unknown. Using varied animal models that stimulate neuronal activity by peripheral inflammation, optogenetics, and selective proteasome inhibition of dopaminergic (DA) neurons, we show that the transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object placement. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.
The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.
Humans and other primates are highly visual animals. Our daily visual activities such as recognizing familiar faces, interacting with objects, or reading, are supported by an extensive system of interacting brain areas. The interactions between the many individual nerve cells both within and between brain areas need to be coordinated. One possible solution to achieve flexible coordination between cells in the network is rhythmic activity, or oscillations. The focus of the thesis will be activity in the largest visual area, V1, in non-human primates. In V1, high-frequency activity, so-called gamma-band activity (“gamma”, ca. 30-90 Hz) can be frequently observed and has been suggested to play a role in coordinating activity in the visual system. In Chapter 1, the coordination problem, the primate visual system and gamma-band oscillations are introduced in detail. The following chapters explore the dependence of gamma on contextual influences. Does V1 use contextual information to optimize co-ordination? In the first part, the short-term consequences of repeated encounters with visual stimuli on V1 responses are explored (Chapters 2 and 3). Inspired by results from colored, naturalistic images in the first part, the second part tests the dependence of gamma on spatial and chromatic stimulus aspects (Chapters 4 and 5).
Stimulus repetition is a simple yet powerful way to tap into our brains’ ability to learn and adapt to our environment. Repeated presentation of a visual stimulus tends to decrease responses to this stimulus. Is this accompanied by changes in the coordination of brain activity? In Chapter 2, the stimulus-specificity of repetition effects on gamma was tested using naturalistic stimuli. V1 is most typically studied using black-and-white, artificial stimuli that are very familiar to the animals. Here, colored natural images were repeatedly presented that were initially novel to the animals, to provide a wider and more naturalistic range of stimulation. Both multi-unit spiking activity (MUA) and gamma showed stimulus-specific repetition effects. MUA responses de-creased most strongly for initial repetitions and less for later repetitions. In contrast, gamma could increase or decrease for initial repetitions, but tended to increase for later repetitions. This points to the operation of multiple plasticity mechanisms. One process may rapidly decrease MUA and gamma and be related to initial novelty or adaptation. The other increases gamma, is active for more repetitions, and could constitute a form of refinement of coordination over time. Moreover, based on the spacing of stimulus repetitions, stimulus memory in V1 persisted for tens of seconds.
In the following Chapter 3, the stimulus location specificity and persistence of the repetition effects for longer timescales were tested. To this end, the observation that the increase in gamma with repetition was strongest for the first tens of repetitions was used to test for location specificity and memory. Using simple artificial stimuli that were repeated many times at two alternating locations, both location specificity and memory on the order of minutes was observed. Due to the structure of the primate visual system, location specificity suggests that the repetition effects involve early to mid-level visual areas such as V1. Memory for previous stimulus presentations on the order of minutes has not been previously reported for V1 gamma. Taken together, these experiments demonstrate short-term plasticity of gamma that is stimulus- and location specific and persists on the timescale of minutes.
In Chapter 2, the average gamma-band response to the large, naturalistic stimuli was highly stimulus dependent. Relative increases in gamma-band activity scaled between tens and thousands of percent change depending on the stimulus. Particularly the color of the stimuli appeared to play a strong role, although the stimulus set was too limited and uncontrolled to draw strong conclusions. In Chapters 4 and 5, underlying mechanisms for the stimulus specificity of gamma were explored using more well-controlled, artificial stimuli that varied in color and spatial structure.
Much of vision relies on the analysis of spatial structure. Each nerve cell in V1 only responds to visual stimuli in a particular, small part of the visual field, its so-called “receptive field” (RF). Compared to isolated RF stimulation, nearby cells that are stimulated by a similar structure from different parts of visual space can show response decreases, commonly known as “surround suppression”, and may show coordinated activity in the gamma band. In Chapter 3, responses to large, uniformly colored disks are contrasted with responses to black or white (achromatic) disks. A first experiment showed that gamma-band responses were stronger for colored than achromatic stimuli, whereas MUA responses could decrease below baseline for colored stimuli. To test whether these phenomena were related to surround suppression, stimulus size was manipulated in a second experiment. When stimuli were of sufficient size to induce surround suppression, clear gamma-band responses emerged. Surround suppression and gamma were stronger for chromatic stimuli. However, the change of stimulus size could have changed not only surround suppression but also stimulus saliency. Therefore, in a third experiment, the overall size of the stimulus was kept constant, and the spatial structure of the stimulus was manipulated. In comparison to uniform, predictable stimulus structure, mismatches between the center of the stimulus and the surrounding visual space led to strong increases in MUA responses and strong de-creases in gamma-band activity. These effects were restricted to the recording sites with RFs at the mismatch location. These experiments underpin the strong role of both spatial structure and color for gamma in V1.
In Chapter 4, responses to different color hues are studied in more detail. Gamma response strength depended on hue, being strongest for red compared to blue and green stimuli when measured with a gray background. To better understand the underlying mechanisms of the differential responses, the spatio-temporal context in the form of the background color was manipulated. Background color had a strong influence on gamma strength. Using differently colored backgrounds, different parts of the color signaling pathways could be adapted. Response differences to different color hues could be explained well with a model that incorporates differences in adaptation between pathways involving long- compared to medium-wavelength cone signals.
Taken together, these experiments indicate a strong role of both spatial context (stimulus size and structure) and temporal context and drive (repetition, adaptation) for the generation of gamma-band activity in V1. Functional implications of these dependencies are considered in the final Chapter 6, and a role for gamma-band syn-chronization in a coding regime for visual inputs that generate strong drive and high predictability is suggested.
Sorting nexins are a conserved protein family involved in vesicle transport, membrane trafficking and protein sorting. The sorting nexin ATG24/SNX4 has been demonstrated to be involved in different autophagy pathways and in endosomal trafficking. However, its impact on cellular quality control and on aging and development is still elusive. Here we report studies analyzing the function of PaATG24 in the aging model Podospora anserina. Ablation of PaATG24 leads to a reduced growth rate, infertility, and to a pronounced lifespan reduction. These characteristics are accompanied by alterations of the morphology and size distribution of vacuoles and severe impairments in non-selective and selective autophagy of peroxisomes (pexophagy) and mitochondria (mitophagy). While general autophagy and pexophagy are almost completely blocked, a PaATG24-independent form of mitophagy is induced during aging. In the ΔPaAtg24 mutant a strong accumulation of peroxisomes occurs while mitochondrial abundance is only slightly increased. These mitochondria are partially affected in function. Most strikingly, although some PaATG24-independent mitophagy exists, it appears that this is not sufficient to remove dysfunctional mitochondria efficiently enough to prevent premature aging. Overall our data emphasize the key role of mitochondria in aging and of mitophagy in quality control to keep a population of “healthy” mitochondria during aging.
The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of β‐barrel‐type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of β‐barrel‐type proteins. At present, only the processing of Toc75‐III, the β‐barrel‐type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75‐V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75‐V/OEP80 is processed as well, we reinvestigated the processing in a protoplast‐based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N‐terminal domain. Thus, Toc75‐V contains a cleavable N‐terminal signal and exposes its polypeptide transport‐associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.
Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3’ UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3’ UTR.
The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.
Brain metastases are the most common intracranial tumor in adults and are associated with poor patient prognosis and median survival of only a few months. Treatment options for brain metastasis patients remain limited and largely depend on surgical resection, radio- and/or chemotherapy. The development and pre-clinical testing of novel therapeutic strategies require reliable experimental models and diagnostic tools that closely mimic technologies that are used in the clinic and reflect histopathological and biochemical changes that distinguish tumor progression from therapeutic response. In this study, we sought to test the applicability of magnetic resonance (MR) spectroscopy in combination with MR imaging to closely monitor therapeutic efficacy in a breast-to-brain metastasis model. Given the importance of radiotherapy as the standard of care for the majority of brain metastases patients, we chose to monitor the post-irradiation response by magnetic resonance spectroscopy (MRS) in combination with MR imaging (MRI) using a 7 Tesla small animal scanner. Radiation was applied as whole brain radiotherapy (WBRT) using the image-guided Small Animal Radiation Research Platform (SARRP). Here we describe alterations in different metabolites, including creatine and N-acetylaspartate, that are characteristic for brain metastases progression and lactate, which indicates hypoxia, while choline levels remained stable. Radiotherapy resulted in normalization of metabolite levels indicating tumor stasis or regression in response to treatment. Our data indicate that the use of MR spectroscopy in addition to MRI represents a valuable tool to closely monitor not only volumetrical but also metabolic changes during tumor progression and to evaluate therapeutic efficacy of intervention strategies. Adapting the analytical technology in brain metastasis models to those used in clinical settings will increase the translational significance of experimental evaluation and thus contribute to the advancement of pre-clinical assessment of novel therapeutic strategies to improve treatment options for brain metastases patients.
Die vorliegende kumulative, publikationsbasierte Disserationsschrift zum Thema „Diversität und Zoogeographie metazoischer Fischparasiten aus dem Südpolarmeer“ gibt einen zusammenfassenden Überblick über die von mir verfasseten ausgewählten drei (ISI-)Publiaktionen. Diese sind im Anhang (Kapitel 6) in chronologischer Reihenfolge aufgeführt. Die Verweise zu den Publikationen sind im Text mit den römischen Ziffern I-III (s.u.) gekennzeichnet. Die für die Promotion relevanten Publikationen wurden wie folgt publiziert:
I Münster J, Kochmann J, Klimpel S, Klapper R, Kuhn T (2016) Parasite fauna of Antarctic Macrourus whitsoni (Gadiformes: Macrouridae) in comparison with closely related macrourids. Parasites & Vectors 9:403
II Münster J, Kochmann J, Grigat J, Klimpel S, Kuhn T (2017) Parasite fauna of the Antarctic dragonfish Parachaenichthys charcoti (Perciformes: Bathydraconidae) and closely related Bathydraconidae from the Antarctic Peninsula, Southern Ocean. Parasites & Vectors 10:235
III Kuhn T, Zizka VMA, Münster J, Klapper R, Mattiucci S, Kochmann J, Klimpel S (2018) Lighten up the dark: metazoan parasites as indicators for the ecology of Antarctic crocodile icefish (Channichthyidae) from the north-west Antarctic Peninsula. PeerJ 6, e4638
Diese drei Publikationen sind im Ergebnisteil (Kapitel 2) separat zusammengefasst und folgend im gemeinsamen Kontext diskutiert (Kapitel 3).
To survive and thrive in nature, animals need to adapt their behavior to their environment. Behavioral adaptation is primarily due to changes within the brain and involves changes in the brain proteome (the collection of proteins in the brain). However, thus far very few studies have examined the proteomic changes during behavioral adaptation. Hence, with this work I set out to determine the proteomic changes induced in the brain of zebrafish larvae undergoing behavioral adaptation. Specifically, I examined the changes induced by adaptation to the natural challenge of strong water currents. To this end I took advantage of an assay developed by my collaborators Luis Castillo and Soojin Ryu. In this assay 5 days old zebrafish larvae were exposed to strong water currents. Subsequently they exhibited a reduction in cortisol response and initial locomotion, and increased rheotaxis, as defined by increased swimming directly against the water current when re-exposed to the water current. I employed this assay to investigate the changes to the larval zebrafish brain proteome during behavioral adaptation. Furthermore, I developed a method for extracting larval brains and prepare them for mass-spectrometric analysis. This work not only allowed the comparison of the brain proteome of naïve and behaviorally-adapted larvae, but also resulted in the most comprehensive proteome of the zebrafish brain observed to date and the first proteome of the larval zebrafish brain. In total 4309 proteins were identified in the brain. When the proteome of naïve and behaviorally adapted larvae were compared 41 proteins were found to be more abundant and 16 to be less abundant in the pre-exposed larvae. Of these 57 proteins, 28 have previously been found to have functions in the brain, 17 with functions identified in other tissues, and 12 proteins that have yet to be described. From examining the most relevant function of each protein I propose a speculative model in which the larval brain undergoes behavioral adaptation and becomes less susceptible to stress (reduction in mecp2 and hsp90 protein), form new neuronal connections (regulation of arid1b, fmn2b, ptpra, mycbp2, and pcyt2), modulate existing connections (regulation of asic1b, calsenilin, ptpra, aplp2, dag1, olfm1b, mycbp2, smad3a, and acvr2a abundance), undergo spatial learning in form of navigating the water vortex (increases in calsenilin, ptpra, and pcyt2), show an elevation in protein turnover (increases in lamp2, Ublcp1, larp4b, and ublcp1), have increased and regulated energy production (increases or reduction in rpia, ldhbb, and mitochondrial proteins; nfs1, eci1, MRPS2B, MRPL4, and mrps2), and a decrease in neurogenesis (reduction in smad3a, and ric8a).
To further investigate proteomic changes during behavioral adaptation, I investigated the translational response by metabolically labeling the larval forebrain with ANL and visualizing the labeled proteins using the fluorescent non-canonical amino acid tagging (FUNCAT). I detected a general increase in translation within the forebrain as a result of the water vortex adaptation, which correlated well with the range of changes observed in the brain proteome. Specifically, a region within the forebrain correlated with a region in the adult zebrafish that is homologous to the mammalian limbic region.
Taken together these results show that during behavioral adaptation, protein synthesis is significantly increased in the larval forebrain, and that throughout the brain regulation of the proteome includes proteins that could support the following functions: changes or modifications in neuronal connectivity, the stress response, spatial learning, changes in energy metabolism and changes in neurogenesis.
Lastly, I set out to provide a new tool for zebrafish researchers. Together with Güney Akbalik I introduced metabolic labeling of newly synthesized RNA using 5-ethynyluridine (EU) and subsequent visualization with a copper catalyzed clickreaction to the zebrafish larvae. With 5 hours of EU incubation I was able to visualize newly synthesized RNA and identify pentylenetetrazole-induced transcriptional increases. With this I showed that EU labeling could be implemented to examining transcriptional changes within the brain of zebrafish larvae.
Metabolic engineering can serve to convert microorganisms to
microbial cell factories with the goal of producing various chemicals. Commonly used strategies to modify metabolic pathways include deletions and overexpression of genes, as well as the introduction of heterologous genes or genes which have been optimized for the host organism or for a reaction of interest. Aside from these classic metabolic engineering strategies, researchers have also implemented pathway compartmentalization strategies, which mimic nature’s strategies of colocalizing enzymes for pathway optimization.
In this thesis, classic metabolic engineering strategies were combined with pathway compartmentalization strategies. For pathway compartmentalization, mitochondria and peroxisomes were harnessed, and additionally a new strategy to create artificial subcellular organelles was evaluated. In the latter approach, the so-called Zera peptide was fused to the enzymes of interest. Zera consists of the first 113 amino acids of the plant storage protein γ-Zein (Zea mays). Natively, plant storage proteins accumulate in endoplasmic reticulum (ER)-derived vesicles in plant seeds and serve as an amino acid source for the germinating plant. In this thesis, it was shown that Zera also induces the formation of artificial, ER-derived vesicles in Saccharomyces cerevisiae. Furthermore, it was shown that Zera fusion enzymes remained active, albeit with sometimes reduced activity.
In line with the goal of compartmentalizing pathways in these artificial, Zera-induced vesicles, a new tool was developed to determine the pH in the ER of S. cerevisiae and in the ER-derived vesicles. pHluorin, a pH-sensitive green fluorescent protein (GFP) variant, is commonly used to analyze the cytosolic pH or the pH of subcellular organelles. In this thesis, it was shown that pHluorin has very low fluorescence intensity and pH sensitivity in the ER and in Zera-induced ER-derived vesicles. Therefore, a superfolder variant of pHluorin was developed which allows reliable pH measurements in these compartments and can be used to analyze whether the organellar or vesicular pH suits a pathway of interest....
The adult mammalian heart is a non-regenerative organ that fails to recover neither functionally nor structurally after insults. Although, reports show that the presences of mitotic nuclei after pathological or physiological cardiac stress in humans, it is widely accepted that the regenerative capacity of the human heart is immensely inadequate to restore the loss of cardiomyocytes (CMs) (Beltrami et al., 2001; Kajstura et al., 1998). Consequently, myocardial infarctions (MIs) are the primary cause of cardiovascular morbidity and mortality. MIs is the irreversible loss of cardiac myocytes due to prolonged myocardial ischemia caused by an imbalance of the metabolic demand of the myocardium and myocardial blood flow (Whelan et al., 2010). Patients with MIs often die prematurely because of heart failure, resulting from irreversible scar formation on the ventricular wall and undermined heart function (Jessup and Brozena, 2003). Despite early intervention and advancements of medical devices for prevention, MIs are still untreatable, unless the heart transplantation approach considered, which is very limited by heart donation (Augoustides and Riha, 2009). Therefore, there is a high demand for standard therapy for heart failure that can restore the loss of CMs, prompt myocardial regeneration, and eventually, reduce morbidity and mortality rate of the disease.
Contrary to the adult mammalian heart, zebrafish display an extraordinary capacity for heart regeneration after the cardiac insult (Poss et al., 2002). This regenerative response relies on the ability of CMs to proliferate and replenish the lost tissue. Zebrafish is indeed one of the most commonly used experimental models for developmental and regenerative biology studies (Gemberling et al., 2013; Gonzalez-Rosa et al., 2017). For decades, the process of cardiac regeneration has been investigated using various cardiac injury models. The most commonly used and well-established injury methods are ventricular apical resection (Poss et al., 2002; Raya et al., 2003), cryoinjury (Chablais et al., 2011; Schnabel et al., 2011), as well as genetic and chemical ablation of heart cells (Curado et al., 2007; Wang et al., 2011). The origin of new cells is one of the most fundamental questions to be addressed during organ regeneration in any regenerative organism, and understanding of such phenomenon is crucial to design effective therapeutic strategies for non-regenerative organisms (Gonzalez-Rosa et al., 2017; Tanaka and Reddien, 2011).
Despite the robust cardiac regenerative potential, to date, only a handful of lineage tracing experiments have been reported in zebrafish heart regeneration. It was proposed that the cellular source of the renewed cardiac tissue might arise from progenitor or stem cells (Lepilina et al., 2006), through CMs dedifferentiation (Jopling et al., 2010; Kikuchi et al., 2010), transdifferentiation from other cell types in the heart tissue, and/or direct proliferation of the existing CMs (Kikuchi and Poss, 2012). Fate-mapping studies using transgenic lines driven by the myl7 promoter have shown that pre-existing CMs contribute to myocardial regeneration. However, myl7 expression is activated at early developmental stages in cardiac progenitor cells and hence precluding the identification of genuinely mature CMs in adult stages. Therefore, the cellular origin of the regenerating CMs remains elusive. Moreover, CM heterogeneity in the developing and adult zebrafish heart has never been explored to provide full insight into the process of regeneration. Therefore, I set out to identify genes exclusively expressed by either immature or mature CMs, generate promoter-driven reporter and CreERT2 lines to characterize the reporters during zebrafish heart development, and regeneration, and eventually to determine the contribution of the immature CMs to the regenerating CMs....
Ubiquitin and the ubiquitin-like protein ATG8 are covalently attached to their respective targets via a coordinated cascade involving E1 activating, E2 conjugating and E3 ligating enzymes. Whereas ubiquitin is conferred to proteins as mono- and/or polymer(s) to alter their stability, localization and/or activity, the ubiquitin-like modifier (UBL) ATG8 is conjugated to the phospholipid phosphatidylethanolamine (PE). The best understood function of ATG8 is during autophagy where ATG8-PE conjugates are incorporated into both layers of incipient autophagosomes and serve as multipurpose docking sites for autophagosomal cargo receptors as well as regulatory factors (termed adaptors) that drive formation and maturation of autophagosomes. Mammalian cells harbor six ATG8 family members that can be subclassified into the LC3- and GABARAP-family and that can all be lipidated. However, it is currently unclear to what extent these proteins are functionally redundant or fulfil unique roles.
Cullin-RING ligase complexes (CRLs) are modular E3 ubiquitin ligases that comprise a RING-finger protein that associates with the ubiquitin-charged E2 enzyme, a substrate recruiting module as well as a cullin scaffold as a linker between RING protein and substrate adaptor. Whereas SCF (SKP1-CUL1-F-box protein) complexes, the most studied CRLs, harbor cullin-1 (CUL1) as scaffold and F-box proteins as substrate binding modules, CUL3-containing CRL complexes employ cullin-3 (CUL3), RING-box protein 1 (RBX1) and BTB proteins as substrate adaptors. Here, the BTB domain serves as binding interface for CUL3 and is usually complemented by an additional protein-protein interaction domain such as MATH or Kelch that mediates binding to the substrate of the E3 ligase complex.
Besides ubiquitylation, guanine nucleotide binding is another common way to regulate protein activity and signaling in cells. Here, small Rho GTPases cycle between active and inactive states by binding of the guanine nucleotides GTP or GDP with the help of regulatory proteins. Whereas GTPase-activating proteins (GAP) render RAC1 inactive by facilitating GTP hydrolysis, guanine exchange factors (GEF) such as T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) activate RAC1 by stimulating the exchange of GDP to GTP. Local control of RAC1 activity is essential to allow a specific cellular response to stimuli such as growth factors or migratory impulses.
This study reports an unexpected link between the GABARAP subfamily of mammalian ATG8 proteins, the ubiquitin proteasome system and RAC1 through the ubiquitylation of the RAC1 GEF TIAM1. The Kelch repeat and BTB domain-containing proteins 6 (KBTBD6) and 7 (KBTBD7) were established as heterodimeric substrate adaptors for CUL3. Interestingly, a thorough proteomic analysis revealed a number of putative substrates but, out of 11 substrate candidates tested, only the RAC1 GEF TIAM1 appeared to be influenced by depletion of CUL3KBTBD6/KBTBD7. Binding studies showed that KBTBD7 binds TIAM1 via the Kelch repeats and that this binding was markedly enhanced when CUL3 activation was abolished upon treatment with the neddylation inhibitor MLN4924. Also, total TIAM1 abundance was increased upon CUL3KBTBD6/KBTBD7 depletion and accumulation of TIAM1 upon proteasome inhibition suggested that TIAM1 is degraded via the proteasome. In vivo ubiquitylation assays and denaturing immunoprecipitations as well as mass spectrometrical analysis confirmed that CUL3KBTBD6/KBTBD7 ubiquitylates TIAM1 at two distinct lysines (K1404 and K1420) close to its C-terminus.
Previously, KBTBD6 and KBTBD7 were found as interactors of several members of the human ATG8 family of proteins in a proteomic study analyzing the human autophagy network. This association was confirmed in the present work. Furthermore, peptide array technology and mutational analysis revealed that KBTBD6 and KBTBD7 employ a classical ATG8-family interacting motif (AIM; also referred to as LC3-interacting region or LIR) as binding interface. The AIMs of KBTBD6 (W-V-R-V) and KBTBD7 (W-V-Q-V) fulfil the consensus AIM sequence motif (F/W/Y1-X2-X3-I/L/V4) and are preceded by several acidic residues and serines. A series of structural and cell biological experiments revealed a binding preference for the GABARAP subfamily of human ATG8 proteins and most importantly, a requirement of the GABARAP-KBTBD6 and -KBTBD7 interaction for TIAM1 ubiquitylation. The finding that TIAM1 binding to KBTBD6 and KBTBD7 AIM mutants was diminished raised the possibility that GABARAP binding mediates the recruitment of CUL3KBTBD6/KBTBD7 to membranes where TIAM1 is localized. Interestingly, colocalization of KBTBD6, GABARAPL1 and TIAM1 in punctuate structures could be observed. Since only a very small fraction of GABARAPL1 colocalized with LC3B, and colocalization between KBTBD6 and LC3B was not observed, these vesicular structures are most likely distinct from autophagosomes. Furthermore, TIAM1 ubiquitylation was reduced when GABARAP, but not LC3B, was depleted or when lipidation of GABARAP was prevented.
Stabilization of TIAM1 upon KBTBD6 and/or KBTBD7 depletion led to elevated TIAM1-dependent RAC1 activity, altered actin morphology with increased cortical actin and loss of vinculin foci. Re-introduction of wild-type KBTBD6 or KBTBD7 but not AIM mutants reverted all these phenotypes. Moreover, depletion of KBTBD6 or KBTBD7 in human breast cancer cells massively increased their invasiveness, whereas TIAM1 knockdown had the opposite outcome. All physiological effects of KBTBD6 and KBTBD7 depletion were inhibited by additional depletion of TIAM1 or RAC1 confirming that the phenotypes observed are indeed mediated by the CUL3KBTBD6/KBTBD7-TIAM1-RAC1 signaling pathway. Intriguingly, KBTBD6 and KBTBD7 were not subject to autophagosomal degradation, thereby establishing a new function for GABARAP proteins beyond autophagosomal degradation in providing a signaling platform for recruitment of the E3 ligase CUL3KBTBD6/KBTBD7 in close proximity to its substrate TIAM1, enabling localized ubiquitylation.
Local restricted control of RAC1 activity by ubiquitylation has been described for TIAM1-RAC1 signaling previously. Examples are HECT, UBA and WWE domain-containing protein 1 (HUWE1)-mediated TIAM1 ubiquitylation that occurs predominantly at cell-cell-junctions in response to hepatocyte growth factor stimulation in MDCKII cells or inhibition of RAC1 activity by the RAC1 GAP protein BCR (breakpoint cluster region) at the leading edge of astrocytes through binding to the TIAM1-Par (polarity) complex. SCFBTRC mediates ubiquitylation of TIAM1 in response to mitogens or DNA damage, though it has not been explored whether this regulation is spatially restricted. Thus, this study adds a novel layer of complexity to the spatial regulation of RAC1 signaling by implicating membrane-bound human ATG8 proteins in this process.
Also, this study is the first report specifically implicating the GABARAP proteins in cellular signaling events. It will be interesting to explore whether the concept of localized signaling mediated by GABARAPs applies to other substrates of CUL3KBTBD6/KBTBD7 and membranerelated signaling processes in which GABARAP proteins are involved. Controlling RAC1 activity at GABARAP-decorated membranes might also be important for trafficking events or autophagy since it was described that RAC1 has an inhibitory function on autophagy. Therefore, spatial restricted ubiquitylation of TIAM1 resulting in specific deactivation of RAC1 could promote the autophagic process when locally needed. Although the catalytic mTOR inhibitor Torin1 and the lysosomal H+ ATPase inhibitor BafilomycinA1 promoted TIAM1 ubiquitylation by increasing the pool of membrane-conjugated GABARAP, but other signals that stimulate GABARAP-KBTBD6/KBTBD7 association and subsequent TIAM1 ubiquitylation are to be identified. Besides, determining the KBTBD6/KBTBD7 binding site in TIAM1 or uncovering a deubiquitylating enzyme (DUB) that locally counteracts the ubiquitylation of TIAM1 will enable a better comprehension of the complete localized signaling cascade.
Impact of F1Fo-ATP-synthase dimer assembly factors on mitochondrial function and organismic aging
(2018)
In aerobic organisms, mitochondrial F1Fo-ATP-synthase is the major site of ATP production. Beside this fundamental role, the protein complex is involved in shaping and maintenance of cristae. Previous electron microscopic studies identified the dissociation of F1Fo-ATP-synthase dimers and oligomers during organismic aging correlating with a massive remodeling of the mitochondrial inner membrane. Here we report results aimed to experimentally proof this impact and to obtain further insights into the control of these processes. We focused on the role of the two dimer assembly factors PaATPE and PaATPG of the aging model Podospora anserina. Ablation of either protein strongly affects mitochondrial function and leads to an accumulation of senescence markers demonstrating that the inhibition of dimer formation negatively influences vital functions and accelerates organismic aging. Our data validate a model that links mitochondrial membrane remodeling to aging and identify specific molecular components triggering this process.
Das Ziel dieser Dissertation war es die biologische Rolle der Ubiquitinierung und die Bedeutung für Alterungsprozesse im filamentösen Ascomyceten Podospora anserina zu untersuchen. Folgende Ergebnisse wurden dabei erzielt:
1. Ubiquitinierte Proteine wurden nachgewiesen und die Deubiquitinierung von Proteinen konnte durch den Einsatz der Inhibitoren Urea, PR-619 und PIC erfolgreich inhibiert werden, was die Anwesenheit aktiver Deubiquitinasen in P. anserina beweist. Zudem wurden erstmalig ubiquitinierte Proteine in P. anserina unter den zur Aufreinigung
gewählten Bedingungen über die Technik der LC-MS/MS identifiziert.
2. Insgesamt wurden 1745 ubiquitinierte Proteine in P. anserina identifiziert, was ca. 16,4 % des gesamten Proteoms darstellt. Somit wurde erstmalig das Ubiquitinom des Ascomyceten P. anserina charakterisiert, welches Proteine aus allen zellulären Kompartimenten enthält.
3. Die erste im Rahmen dieser Arbeit durchgeführte umfassende Studie altersabhängiger Veränderungen im Ubiquitinom eines Organismus zeigt eine Herabregulation der Ubiquitinierung im Alter in der gesamten Zelle. Dem gegenüber steigt die Ubiquitinierung an den Mitochondrien leicht an und unterstreicht die Rolle einer mitochondrialen Qualitätskontrolle durch diesen Prozess.
4. Die Untersuchung der am Ubiquitinierungsprozess beteiligten E3-Ligase PaMUS10 zeigt, dass es sich bei Mus10 um ein essentielles Gen in P. anserina handelt, da die Deletion zu starken mitochondrialen Beeinträchtigungen und dem sofortigen Absterben des Organismus direkt nach der Keimung führt.
5. Zur weiteren Untersuchung von PaMus10 wurde erstmalig und vollständig ein Hygromycin-basiertes „Knockdown“-System in P. anserina etabliert, was die detaillierte Untersuchung eines essentiellen Gens in diesem Organismus ermöglichte. Durch dessen Einsatz konnte eine reduzierte Fertilität, eine verkürzte Lebensspanne sowie Veränderungen der mitochondrialen Morphologie und Funktion als direkte Folge einer PaMus10-Herabregulation nachgewiesen werden.
6. PaMUS10 ist vorwiegend cytosolisch lokalisiert wird aber unter oxidativem Stress oder in gealterten Kulturen an die Mitochondrien rekrutiert, was einen vergleichbaren Mechanismus zur menschlichen E3-Ligase PARKIN darstellt.
7. Der Verlust von PaMUS10 verursacht ein Präseneszenzsyndrom und führt zum vorzeitigen Absterben des Organismus, wohingegen die zusätzliche Expression von PaMus10::Gfp offenbar positive Effekte nach sich zieht, da hier eine deutliche Verlängerung der Lebensspanne beobachtet wurde.
8. Der Vergleich der beiden Ubiquitinome von ∆PaMus10/PaMus10 und Wthph1/2 zeigt eine massive Reduzierung der globalen Ubiquitinierung, welche offenbar durch das Fehlen von PaMUS10 ausgelöst wird und damit dessen Funktion als E3-Ligase untermauert.
9. Im Rahmen der hier durchgeführten Substratanalyse wurden insgesamt 131 Proteine identifiziert, von denen ca. 20 % dem Mitochondrium zugeordnet werden können. Aufgrund der diversen biologischen Prozesse und Funktionen dieser Substrate ist PaMUS10 sowohl in die Ubiquitinierung cytosolischer als auch mitochondrialer Proteine involviert und greift womöglich sogar als zentraler Schalter in die gesamte zelluläre Homöostase ein.
Aufgrund der nicht unerheblichen Zahl der identifizierten ubiquitinierten Proteine (Ubiquitinom) und den fatalen Auswirkungen, die der Verlust einer E3-Ligase in diesem Organismus nach sich zieht, lässt sich folgende grundlegende Erkenntnis formulieren:
Die Ubiquitinierung spielt in P. anserina eine bedeutende Rolle zur Aufrechterhaltung zellulärer Prozesse insbesondere der mitochondrialen Homöostase und beeinflusst dadurch positiv die Entwicklung und Alterung in diesem Organismus.
Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
Wie herzig!
(2019)
Doch Vorsicht – dies ist kein Einblick in die Hirnwindungen eines verliebten Teenagers. Vielmehr handelt es sich hier um einen wissenschaftlichen Blick in die Großhirnrinde einer Maus. Die Forscherinnen und Forscher um Prof. Amparo Acker-Palmer vom Buchmann Institut für Molekulare Lebenswissenschaften und dem Institut für Zellbiologie und Neurowissenschaften der Goethe-Universität haben 2018 in der Zeitschrift "Science" darüber berichtet, dass Blutgefäße bei der Entwicklung neuronaler Zellnetzwerke im Gehirn eine bislang unbekannte Rolle spielen ...
Eine qualitative und quantitative Studie zum Einsatz der virtuellen Mikroskopie in der Schule
(2019)
Das Mikroskop stellt in der Alltagswelt ein Sinnbild für naturwissenschaftliches Arbeiten dar (Coleman 2009, Paulsen 2010). Im Bereich der Lehre eröffnet dieses Laborgerät das Eintauchen in die mikroskopische Dimension und besitzt eine wesentliche Rolle bei der damit verbundenen Erkenntnisgewinnung, insbesondere von funktionsmorphologischen Konzepten (Gropengießer & Kattmann 2008, Kremer 2002). Jedoch wird die Durchführung der klassischen Mikroskopie und damit die aktive Auseinandersetzung mit mikroskopischen Präparaten im schulischen (Biologie-)Unterricht durch verschiedene Faktoren erschwert. Zu den Limitierungen gehören beispielsweise die Verfügbarkeit geeigneter Mikroskope und Dauerpräparate, die aufwendige Vor- und Nachbereitungszeit sowie der zeitliche Aufwand bei der Herstellung hochwertiger mikroskopischer Frischpräparate. Die virtuelle Mikroskopie könnte diese Schwierigkeiten umgehen. Das virtuelle Mikroskop kann als eine Simulation verstanden werden, bei der die bildanalytischen Vorgehensweisen bei mikroskopischen Präparaten analog zur klassischen Mikroskopie nachvollzogen werden können (Gu & Oglivie 2005, Hentschel 2009). Hierbei umfasst das virtuelle Mikroskop ein Akquisitionssystem zum Einscannen und Digitalisieren mikroskopischer Präparate, einen Server zum Speichern und Bereitstellen der entstandenen virtuellen hochauflösenden Aufnahmen (WSI) sowie eine Bildbetrachtungssoftware auf einem Anwendungsrechner (Kalinski et al. 2006). Basierend auf einer Nutzerbefragung wurde eine Betrachtungssoftware programmiert, die hinsicht¬lich ihrer Benutzerfreundlichkeit und ihren Eigenschaften auf den schulischen Einsatz angepasst wurde. Um die Relevanz in diesem Anwendungsfeld zu testen, wurden die Untersuchungen der vorliegenden Arbeit sowohl im Schülerlabor Goethe BioLab als auch in der universitären Lehre der Abteilung für Didaktik der Biowissen¬schaften der Goethe–Universität Frankfurt am Main durchgeführt. Der Schülerlabortag „Blut und das virtuelle Mikroskop“ wurde entwickelt, um die computerbasierte virtuelle Mikroskopie mit Schülern ergänzend zur klassischen Mikroskopie in einem fachlichen Kontext anzuwenden und zu erforschen.
Beruhend auf der Vergleichbarkeit beider Mikroskopiemethoden (Paulsen et al. 2010) lagen die Forschungsschwerpunkte neben der Nutzung der Software durch Schülerinnen und Schülern auf einer gegenüberstellenden Beurteilung beider mikroskopischer Verfahren von Schülern und Lehramtsstudierenden. Es wurden in diesem Zusammenhang drei zentrale Forschungsfragen formuliert.
Die erste Forschungsfrage untersucht das Nutzerverhalten der Schüler (n = 123) bei der virtuellen Mikroskopie mittels automatisch generierter Datensätze während der Anwendung der Bildbetrachtungssoftware. Die Analyse der Anwendungsdaten zeigt, dass das mikroskopische Sehen, insbesondere das Fokussieren auf relevante Bildbereiche, im virtuellen Humanblutausstrich angewandt wurde.
Die zweite Forschungsfrage untersuchte das aktuelle Interesses bei Schülern (n = 293) im direkten Vergleich zwischen virtueller und klassischer Mikroskopie. Dabei wurde das aktuelle Interesse aufgrund des engen Zusammenhangs zum Lernen (vgl. Krapp 1992a) als Indikator der Lernwirksamkeit gewählt. Die Erhebung erfolgte mittels eines Fragebogens. Die Ergebnisse dieser Untersuchung zeigen, dass der Einsatz beider mikroskopischer Verfahren das aktuelle Interesse fördert, das emotionale und das wertbezogene Merkmal sich jedoch zugunsten der klassischen Mikroskopie signifikant unterscheiden.
Im Rahmen der dritten Forschungsfrage erfolgte eine Beurteilung der Vorteile virtueller Mikroskopie gegenüber der klassischen Mikroskopie von Schülern (n = 504) sowie Lehramtsstudierenden (n = 247). Hierbei diente ebenfalls ein Fragebogen als Grundlage der Erhebung. Die Auswertung zeigt, dass sowohl die Schüler als auch die Studierenden die Vorteile der virtuellen Mikroskopie klar erkennen. Es liegen jedoch signifikante Unterschiede zwischen den Versuchsgruppen vor. Die Schüler bewerten die Vorteile betreffend der Förderung von Lernprozessen, des Erkennens von Strukturen und des mikroskopischen Zeichnens höher.
Zusammenfassend bestärken die Ergebnisse dieser Studie die Ansicht, dass das virtuelle Mikroskop nicht als Ersatz, sondern als sinnvolle Ergänzung zu der klassischen Lichtmikroskopie angesehen werden sollte (Bloodgood et al. 2006, Berg et al. 2016, Braun & Kearns 2008, Hufnagl et al. 2012, Mione et al. 2013, Santiago 2018, Scoville & Buskirk 2007). Dabei sollte die vorliegende Arbeit als Einstieg verstanden werden, um bestehende Forschungslücken zu verkleinern, damit ein Transfer der virtuellen Mikroskopie in den schulischen Kontext möglichst lernwirksam erfolgen kann.
Die Neurowissenschaften sind in Forschungsarbeiten für Schüler und Studierende immer wieder als eines der schwierigsten Teilgebiete der Biologie angeführt. Die Inhalte werden überwiegend nicht verstanden. Als mögliche Ursache gelten die seltenen praktischen Zugänge für die Lernenden aufgrund limitierter Ressourcen. Diese Ursache konnte in der vorliegenden Arbeit durch eine Befragung der Lehrkräfte zu ihren Praxisumsetzungen bestätigt werden. 70 % der Lehrkräfte gaben an, dass sie keine Experimente in der Schule zum Thema Nervenzellen anbieten. Experimente zur Verhaltensbiologie führen 65 % der Lehrkräfte nicht durch.
Um Schülern die Möglichkeit zu geben, sich experimentell mit den Themenfeldern der Neuro- und Verhaltensbiologie auseinanderzusetzen, wurden im Rahmen der vorliegenden Arbeit Schülerlabortage auf dem Feld der Neurowissenschaften konzipiert. Die Konzepte wurden schülerorientiert umgesetzt und neurowissenschaftliche Forschung durch den eigenen Umgang mit modernen Forschungsapparaturen erfahrbar gemacht. Die drei Labortage für die Sekundarstufe II wurden wissenschaftlich begleitet: 1) Verhaltensbiologie, 2) systemische Ebene der Elektrophysiologie, 3) elektrophysiologische Forschungsmethoden. Um die Qualität und Wirksamkeit der Labortage beurteilen zu können, wurden sie mit Feedbackerhebungen begleitet. Die drei Labortage wurden sowohl von den Lehrkräften als auch von den Schülern bezüglich ihrer Qualität positiv bewertet. Für die Schüler konnte gezeigt werden, dass die Beurteilung weitgehend unabhängig von einem zugrunde liegenden Interesse an Biologie und Forschung ausfällt. Anhand einer retrospektiven Erhebung wird außerdem gezeigt, dass alle drei Labortage eine höchst signifikante, selbsteingeschätzte Steigerung des „Wissens“, der „Anwendungszuversicht“ und des „Interesses“ bewirken. Schüler mit niedrigen Ausgangswerten zeigen einen besonders hohen Anstieg. Für das Interesse kann weiter gezeigt werden, dass auch Schüler mit hohem Ausgangswert eine große Interessenssteigerung durch den Labortag aufweisen. Das Interesse für den verhaltensbiologischen Labortag liegt etwas niedriger – die Labortage mit elektrophysiologischen Inhalten zeigen dagegen für die Anwendungszuversicht etwas niedrigere Werte.
Der Fokus der fachdidaktischen Forschung lag auf der Betrachtung des experimentellen Zugangs zur Elektrophysiologie über ein entwickeltes „EPhys-Setup“. Dabei handelt es sich um einen quasi-realen Messaufbau. Die Umsetzung kombiniert dazu Komponenten eines realen Elektrophysiologie-Setups (Hands-on Komponenten) mit einer speziell entwickelten schülerfreundlichen Software (Neurosimulation) und einem virtuellen Nervensystem in Form einer Platine. Als Modellnervensystem werden für diese Umsetzung Ganglien von Hirudo medicinalis verwendet – der Neurosimulation liegen originale elektrophysiologische Messspuren des Ganglions zugrunde. Experimentelle Vermittlungsansätze für die Elektrophysiologie finden sich kaum für den Schulbereich. Dem Bedarf einer entsprechenden Beforschung wurde mit verschiedenen Testinstrumenten nachgegangen, um den Vermittlungsansatz mit dem EPhys-Setup bewerten zu können. Dafür fand eine Wirksamkeitsanalyse über die Erhebung der Motivation der Schüler statt (Lab Motivation Scale; Dohn et al. 2016). Von Bedeutung war auch, inwiefern gegenüber der Umsetzung eine Technologieakzeptanz vorliegt (Technology Acceptance Model; Davis 1989), die im Schulkontext ausgehend von der steigenden Einbindung von Technologien einen entsprechenden Forschungsbedarf aufweist. Weiter wurde untersucht, ob sich die Bewertung des EPhys-Setups von der Bewertung einer Kontrollgruppe unterscheidet. Für die Kontrollgruppe wurde die Neurosimulation von den Hands-on Komponenten gelöst und die Schüler arbeiteten ausschließlich PC-basiert. Die Ergebnisse zeigen, dass beide Umsetzungen die Motivation förderten und eine Technologieakzeptanz bei den Schülern aufwiesen. Der Unterschied der Untersuchungsgruppen fällt gering aus. Die Abhängigkeiten, die für die verwendete Simulationsumsetzung gefunden wurden, beziehen sich ausschließlich auf Komponenten der „Freude“. Somit wird der intrinsische Bereich von den Schülern die am EPhys-Setup gearbeitet haben höher bewertet. Zur weiteren Analyse der Testinstrumente wurde auch eine Abhängigkeit der Bewertung vom zugrunde liegenden Biologieinteresse sowie von den Computerfähigkeiten vergleichend betrachtet. Der Einfluss auf die Bewertungen der drei Testskalen ist in vielen Fällen höher als der Einfluss der verwendeten Simulation. Vom individuellen Biologieinteresse der Schüler zeigen alle untersuchten Komponenten eine Abhängigkeit. Die größeren Effekte beziehen sich auf die Komponenten der „Lernwirksamkeit“ oder der „Freude“. Von den individuellen Computerfähigkeiten der Schüler zeigen Komponenten zur „Zuversicht bezüglich der Methoden und der Inhalte“ eine Abhängigkeit.
Acetylcholine (ACh) is the major excitatory neurotransmitter in the insect central nervous system (CNS). However, besides the neuronal expression of ACh receptors (AChR), the existence of non-neuronal AChR in honeybees is plausible. The cholinergic system is a popular target of insecticides because the pharmacology of insect nicotinic acetylcholine receptors (nAChRs) differs substantially from their vertebrate counterparts. Neonicotinoids are agonists of the nAChR and are largely used in crop protection. In contrast to their relatively high safety for humans and livestock, neonicotinoids pose a threat to pollinating insects such as bees. In addition to its effects on behavior, it becomes increasingly evident that neonicotinoids affect developmental processes in bees that appear to be independent of neuronal AChRs. Brood food (royal jelly, worker jelly, or drone jelly) produced in the hypopharyngeal glands of nurse bees contains millimolar concentrations of ACh, which is required for proper larval development. Neonicotinoids reduce the secreted ACh-content in brood food, reduce hypopharyngeal gland size, and lead to developmental impairments within the colony. We assume that potential hazards of neonicotinoids on pollinating bees occur neuronally causing behavioral impairments on adult individuals, and non-neuronally causing developmental disturbances as well as destroying gland functioning.
Pyridine 2,4-dicarboxylic acid is a structural analog of 2-oxoglutarate and is known to inhibit 2-oxoglutare-dependent dioxygenases. The effect of this inhibitor in tomato seedlings grown in MS media supplied with various concentrations of PDCA was investigated, resulting in shorter roots and hypocotyls in a dose-dependent manner. The partial inhibition of growth in roots was more drastic compared to hypocotyls and was attributed to a decrease in the elongation of root and hypocotyl cells. Concentrations of 100 and 250 μM of PDCA decreased hydroxyproline content in roots while only the 250 μM treatment reduced the hydroxyproline content in shoots. Seedlings treated with 100 μM PDCA exhibited enhanced growth of hypocotyl and cotyledon cells and higher hydroxyproline content resulting in cotyledons with greater surface area. However, no alterations in hypocotyl length were observed. Prolyl 4 hydroxylases (P4Hs) are involved in the O-glycosylation of AGPs and were also highly expressed during seedling growth. Moreover PDCA induced a decrease in the accumulation of HRGPs and particularly in AGPs-bound epitopes in a dose dependent-manner while more drastic reduction were observed in roots compared to shoots. In addition, bulged root epidermal cells were observed at the high concentration of 250 μM which is characteristic of root tissues with glycosylation defects. These results indicate that PDCA induced pleiotropic effects during seedling growth while further studies are required to better investigate the physiological significance of this 2-oxoglutarate analog. This pharmacological approach might be used as a tool to better understand the physiological significance of HRGPs and probably P4Hs in various growth and developmental programs in plants.
Die Differenzierung zwischen Teilpopulationen hin zu unterschiedlichen Arten kann nur erfolgen, wenn zwischen diesen Teilpopulationen reproduktive Isolation besteht. Wie die unterschiedlichen Arten von reproduktiver Isolation zusammenwirken und welche Voraussetzungen bestehen müssen, um neue Arten zu bilden, muss in jedem Studiensystem untersucht werden. Ein idealer Ansatzpunkt sind Arten, die sich mehrfach an anspruchsvolle Habitate angepasst haben, deren Artbildung also von ökologischen Habitatparametern bestimmt wird. Dieser Vorgang wird als Ökologische Artbildung bezeichnet. Im Artkomplex Poecilia spec., der im Süden Mexikos mehrere schwefelangepasste Ökotypen ausgebildet hat, wurden erste Hinweise auf eine Korrelation zwischen der Selektionsstärke von natürlicher und sexueller Selektion gefunden, deren Einfluss zusammen die bestehenden reproduktiven Barrieren zwischen Klarwasser- und Schwefelökotyp formen. Wie diese Reproduktionsbarrieren beschaffen sind und wie die Umweltvariable Schwefel auf die Morphologie und das Verhalten der Poeciliiden Einfluss nimmt, wurde in der vorliegenden Arbeit anhand von fünf Fragestellungen untersucht. (1) Die Körperfärbung kann ein aussagekräftiges Signal für die Qualität des potentiellen Partners bei der Fortpflanzung sein. Wie beeinflusst die extreme Umweltvariable Schwefel die Ausbildung von Färbung? (2) Sind die gefundenen Anpassungen der Färbung erblich oder werden sie plastisch entsprechend des Nahrungsangebots ausgebildet? (3) In einem der untersuchten Flusssysteme konnte unvollständige reproduktive Isolation zwischen der Klarwasser- und Schwefelpopulation nachgewiesen werden. Sind in den Mischzonen zwischen diesen beiden Habitaten Hybriden genetisch nachweisbar und bilden diese die Färbungsanpassungen der Klarwasser-, der Schwefelpopulation oder eine intermediäre Form aus? (4) Die Gelbfärbung der Flossen bei Männchen scheint ein geeignetes Merkmal für die Anzeige der Qualität zu sein, da es möglicherweise unabhängig vom Nahrungsangebot ausgebildet wird. Besteht eine weibliche Präferenz für dieses Merkmal? (5) Auch die weibliche Partnerwahlpräferenz wird vom Habitat und dem eigenen Zustand beeinflusst. Wie verändert sich die Präferenz für Männchen mit gutem Ernährungszustand bei Weibchen, die hungrig sind?
Um diese Fragen zu beantworten, wurden in mehreren Jahren Männchen und Weibchen der Arten Poecilia mexicana und Poecilia sulphuraria aus sieben Populationen im Studiengebiet in Südmexiko gefangen und auf ihre Färbung untersucht sowie Laborpopulationen getestet. Es konnten generelle Anpassungen der Färbung an die Umweltvariable Schwefel nachgewiesen werden. Dazu gehören die Aufhellung der Körperregionen, die durch Tarnung (konkret: countershading und background matching) vor Entdeckung durch Prädatoren schützen, und die Reduktion von Gelb- und Rottönen. Diese Anpassung ist vermutlich auf das geringe Angebot an Karotinoiden in den schwefelbelasteten Extremhabitaten zurückzuführen. Außerdem konnten zahlreiche flusssystem¬spezifische Anpassungen beschrieben werden, deren Ursachen in den Unterschieden zwischen den Schwefelhabitaten untereinander begründet sind. Das Flusssystem des Río Tacotalpa stellt hier eine Besonderheit dar, da Männchen eine besonders starke Gelbfärbung der Flossen aufweisen. Wildgefangene und laborgeborene Männchen dieses Flusssystems wurden verglichen, um einen Hinweis auf den Einfluss des Nahrungsangebots auf dieses Merkmal zu untersuchen. Tatsächlich ist die Ausprägung dieses Merkmals, die Gelbfärbung der Flossen, unabhängig vom Angebot an Karotinoiden. Während die hier verwendeten genetischen Analysen nicht geeignet waren, Hybriden aus den Mischzonen zwischen Schwefel- und Klarwasserhabitat nachzuweisen, ergaben die Untersuchungen von Individuen aus den Mischzonen keine eindeutigen Ergebnisse über eine etwaige intermediäre Ausbildung der Färbung. Die Präsentation von Männchen, deren Gelbintensität an den Flossenspitzen künstlich verändert wurde, konnte bei Weibchen keine eindeutige Präferenz für stärker gefärbte Männchen aufzeigen. Vielmehr weist dieses Ergebnis auf eine starke Korrelation zwischen mehreren Merkmalen (z. B. weitere morphologische Merkmale, Verhalten) hin, die für die Beurteilung der männlichen Qualität herangezogen werden. Die weibliche Präferenz für konditionsabhängige Merkmale wird bei schwefelangepassten Weibchen leicht verstärkt, wenn diese hungrig sind. Eine solche flexible Präferenz sollte gerade in Habitaten mit starken Fluktuationen im Nährstoffangebot existieren. Dabei waren Weibchen, denen Videoaufnahmen präsentiert wurden, eher in der Lage, das qualitativ hochwertigere Männchen zu identifizieren, als Weibchen, denen animierte Bilder präsentiert wurden. Auch hier wird davon ausgegangen, dass die Reduktion auf eines oder wenige Merkmale, die für die Partnerwahl zur Verfügung stehen, keine ausreichend starke Reaktion auslösen können. Vielmehr ist der Zugriff auf alle Aspekte der männlichen Erscheinung wichtig, um die Qualität des potentiellen Partners zu beurteilen.
Färbung ist also generell geeignet, den Ökotyp eines Individuums zu bestimmen und ein solches Merkmal kann der Artbestimmung im ersten Schritt der Partnerwahl dienen. Dasjenige männliche Färbungsmerkmal, das über mehrere Generationen gleichbleibend ausgeprägt wurde – die Gelbfärbung der Flossen – reicht jedoch nicht aus, um bei der weiblichen Partnerwahl eine Reaktion auszulösen. Vielmehr deuten die Ergebnisse auf eine enge Korrelation der Färbung mit weiteren Merkmalen in Morphologie und Verhalten eines Individuums hin, die vom wählenden Weibchen stets gemeinsam entsprechend der Multiple-message-Theorie betrachtet werden. Auch der Vergleich zwischen Videoaufnahmen und animierten Fotografien als Stimuli bei der Partnerwahl ergab, dass der Aspekt Verhalten (nur verfügbar mit Videoaufnahmen) für eine Partnerwahlentscheidung von Bedeutung ist.
Meine Arbeit konnte den bestehenden Wissensschatz um die bestehenden reproduktiven Barrieren im Studiensystem um den Aspekt der Färbung erweitern. Meine Ergebnisse zeigen weitere spannende Fragestellungen auf. Je größer das Verständnis der vorliegenden Selektionskräfte und Mechanismen reproduktiver Isolation ist, desto besser kann die Wissenschaft verstehen, welche Umgebungsvariablen welchen Einfluss auf den Prozess der Artbildung haben.
Cardiovascular diseases are a leading cause of morbidity and mortality worldwide. Aging inflicts structural and molecular changes on the heart that oftentimes involve ischemic events, cardiomyocyte apoptosis and cardiac stiffening, which makes it a major risk factor for cardiovascular disease. After being disregarded as transcriptional noise for a long time, long non-coding RNAs have lately emerged as key regulators of many cellular processes in physiology and disease of virtually all tissues and organs, with some of them being differentially regulated during aging.
This study identified a long non-coding transcript antisense to the OXCT1 gene locus, Sarrah, to be downregulated in the heart during aging, after acute myocardial infarction and upon heart failure with preserved ejection fraction. Sarrah is expressed in several cardiac cell types with highest levels in cardiomyocytes, where it is predominantly localized in the nucleus. In mouse and human cardiomyocytes, Sarrah levels are reduced upon exposure to hypoxia or treatment with hypoxiamimetic agents in vitro.
Sarrah exerts an anti-apoptotic function in mouse and human cardiomyocytes as assessed from caspase activity and annexin V staining. Histological stainings of Sarrah-depleted human engineered heart tissue organoids and Sarrah overexpressing infarcted mouse hearts confirmed its anti-apoptotic function. Sarrah also plays a role in cardiomyocyte contractility, which is substantially impaired upon Sarrah silencing in human engineered heart tissue and neonatal rat cardiomyocytes. Additionally, cardiomyocytal Sarrah stimulates endothelial cell proliferation via paracrine effects as observed after Sarrah overexpression in mouse hearts as well as in co-culture settings with human endothelial cells and Sarrah-depleted or Sarrah overexpressing human cardiomyocytes. A microarray analysis revealed that silencing Sarrah in human cardiomyocytes induced apoptosisrelated gene expression. Mechanistically, Sarrah was predicted to form triplexes in human and mouse with promoters of genes downregulated, but not upregulated after Sarrah knockdown, suggesting that Sarrah interacts with target genes to activate their transcription. This interaction was confirmed in vitro using nucleic acid oligonucleotides containing the sequences of the Sarrah triplex motif and the Sarrah binding site of the exemplary target gene GPC6 of both human and mouse. RNA immunoprecipitation experiments in human cells demonstrated that Sarrah is associated with open chromatin, transcription factor CRIP2, transcriptional co-activator p300 and DNA-RNA hybrid structures that also occur in Sarrah target gene promoters, which indicated that Sarrah activates gene expression by triplex formation and recruitment of protein interaction partners. Deleting the triplex motif of endogenous Sarrah in mouse cardiomyocytes augmented apoptosis, showing that triplex formation is of functional relevance for Sarrah action.
Finally, overexpressing Sarrah in an acute myocardial infarction mouse model improved recovery of cardiac contractile function as assessed from ejection fraction, stroke volume, wall motion and wall thickness measured by echocardiography and magnetic resonance imaging. Infarct size was substantially reduced in Sarrah overexpressing mice compared with controls. This in vivo study implies that restoring Sarrah levels in the aged or infarcted heart bears significant therapeutic potential, which can be attributed to the combination of three Sarrah effects: increased cardiomyocytes survival, enhanced contractility of individual cardiomyocytes and paracrine stimulation of endothelial cell proliferation likely contributing to increased angiogenesis and tissue perfusion.
In summary, cardiac lncRNA Sarrah is evolutionary conserved with regard to its genomic locus, function and molecular mechanism. Via triplex formation with gene promoters, it is capable to activate a set of target genes that together mediate the anti-apoptotic and pro-contractile function of Sarrah in cardiomyocytes and that confer angiogenic effects to endothelial cells. A therapeutic utilization of Sarrah in the context of myocardial ischemia is conceivable in the future if Sarrah upregulation proves to be beneficial in further studies.
Background: Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation.
Results: Here we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple μ-wells in an agarose cast. Each μ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR).
Conclusions: We present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of μ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.
The overarching aim of this doctoral research was to examine and quantify the spatiotemporal variability in the movements of nomadic ungulates to better understand the possible drivers and characteristics of such movements as well as to examine the particular conservation challenges associated with nomadic movements.
Cerebellar ataxias are a group of neurodegenerative disorders primarily affecting the cerebellum. Although causative mutations in several genes have been identified there is currently no cure for ataxias.
The first part of this dissertation is focused on Spinocerebellar ataxia type 2 (SCA2). SCA2 is a dominant ataxia caused by repeat expansion mutations in the ATXN2 gene, which encodes the protein Ataxin2 (ATXN2). A polyglutamine (polyQ) tract consisting of CAG repeats interrupted by CAA was identified at exon 1 of ATXN2. Healthy individuals have between 22 and 23 glutamines, while expansions longer than 33 CAG repeats cause SCA2. The most noticeable symptom that SCA2 patients show is ataxic gait; however, they also show cerebellar dysarthria, dysdiadochokinesia, and ocular dysmetria caused by the progressive cerebellar degeneration.
To model the SCA2 disease, we generated a new mouse model where 100 CAG repeats were introduced in the mouse Atxn2 gene via homologous recombination. The characterization of this mouse model, Atxn2-CAG100-KIN, demonstrated that it reproduces the symptomatology observed in SCA2 patients. These animals showed significant loss of weight over time, brain atrophy, and motor deficits.
In addition, ATXN2 intermediate expansions have been linked to the pathology of Amyotrophic lateral sclerosis (ALS) as a risk factor. ALS is a fatal neurodegenerative disease where the motor neurons in the brain and spinal cord degenerate. A hallmark of ALS is the presence of TDP43-positive inclusions in neurons and glia. Further studies of post mortem spinal cord samples from SCA2 patients showed severe and widespread neurodegeneration of the central somatosensory system. Therefore, it was of interest to further investigate the pathology affection of this tissue in the Atxn2-CAG100-KIN line and the relationship between ATXN2 and TDP43. The characterization of the spinal cord pathology via protein quantification, transcript quantification, and immunohistochemistry showed a preferential affection of RNA binding proteins (RBP) in the spinal cord rather than the cerebellum. The ALS-linked factors TDP43 and TIA1 showed time-dependent co-aggregation with ATXN2 in spinal cord sections together with an increase of CASP3 levels. Therefore, this mouse model can help develop new therapies and evaluate their effect in differently affected areas.
A transcriptome data set from Atxn2-CAG100-KIN spinal cord samples at the final disease stage of this mouse model showed a strong up-regulation of RNA toxicity-, immune- and lysosome-implicated factors. These data pointed to a pathological reactivation of the synaptic pruning and phagocytosis in microglia. ATXN2-positive aggregates were found in microglia from spinal cord sections of 14-month-old Atxn2-CAG100-KIN via immunohistochemistry. The characterization of microglial response and the potentially deleterious effects of the expanded ATXN2 in this cell type could lead to therapies to improve patients’ living standards or delay the symptoms’ onset.
The second part of this thesis was focused on an autosomal recessive form of cerebellar ataxia, Ataxia Telangiectasia (A-T), with childhood onset. A-T patients show severe cerebellar atrophy manifesting as ataxia when the child starts to walk. The genetic cause of A-T is loss-of-function-mutations in the Ataxia Telangiectasia Mutated gene (ATM). ATM is a kinase involved in DNA damage response, oxidative stress, insulin resistance, autophagy via mTOR signaling, and synaptic function.
Working with proteome data from cerebrospinal fluid of 12 A-T patients and 12 healthy controls, we aimed to define novel biomarkers that would allow following the neurodegeneration in extracellular fluid. Additional validation efforts with ~2-month-old Atm-knock-out (Atm-/-) cerebellar samples helped us to define a scenario were the deficit of vesicle-associated ATM alters the secretion of ApoB, reelin, and glutamate. As extracellular factors, apolipoproteins and their cargo such as vitamin E may be useful for neuroprotective interventions.
ADAM15, which belongs to the family of the disintegrin and metalloproteinases, is a multi-domain transmembrane protein. A strongly upregulated expression of ADAM15 is found in inflamed synovial membranes from articular joints affected by osteoarthritis and especially rheumatoid arthritis (RA). During the chronic inflammatory process in RA the synovial membrane gets hyperplastic, resulting eventually in the formation of a pannus tissue, which can invade into the adjacent cartilage and bone thereby destroying their integrity. Previously, the expression of ADAM15 in fibroblasts of the RA synovial membrane was found to confer a significant anti-apoptotic response upon triggering of the Fas receptor, which resulted in the activation of two survival kinases, focal adhesion kinase (FAK) and Src. The Fas receptor, also named CD95, belongs to the death receptor family of the tumor necrosis factor receptors and stimulation of Fas/CD95 by its ligand FasL results in the execution of apoptotic cell death in synovial membranes of RA patients. However, the occurrence of apoptotic cell death in vivo in RA synovial tissues is considerably low despite the presence of FasL at high concentrations in the chronically inflamed joint. Accordingly, a general apoptosis resistance is a characteristic of RA-synovial fibroblasts that contributes considerably to the formation the hyperplastic aggressive pannus tissue. The objective of this study was to investigate the mechanisms underlying the capability of ADAM15 to transform FasL-mediated death- inducing signals into pro-survival activation of Src and FAK in rheumatoid arthritis fibroblasts (RASFs).
In the present study, the down-regulation of ADAM15 by RNA interference resulted in a significant increase of caspase 3/7 activity upon stimulation of the Fas receptor in RASFs. Likewise, chondrocytes expressing a deletion mutant of ADAM15 (ΔC), lacking the cytoplasmic domain, revealed increased caspase activities upon Fas ligation in comparison to cells transfected with full-length ADAM15, clearly demonstrating the importance of the cytoplasmic domain for an increased apoptosis resistance. Furthermore, activation of the Fas receptor triggered the phosphorylation of Src at Y416, which results in the active conformation of Src, as well as the phosphorylation of FAK at Y576/577 and Y861 – the target tyrosines phosphorylated by Src - in full-length ADAM15-transfected chondrocytes. However, cells transfected with ADAM15 mutant (ΔC) or with vector control did not exhibit any activation of Src and FAK upon Fas ligation. This suggested the presence of an as yet unknown protein interaction mediating the Fas triggered activation of the two kinases.
In order to identify this mechanism, the application of signal transduction inhibitors interfering with Calcium signaling either by inhibiting calmodulin with trifluoperazine (TFP) or the Calcium release-activated channel (CRAC/Orai1) with BTP-2 efficiently inhibited the phosphorylation of FAK and Src, revealing a role of calmodulin, the major Ca2+ sensor in cells, in ADAM15-dependent and Fas-elicited activation of the two survival kinases. Also, a direct Ca2+ -dependent binding of calmodulin to ADAM15 could be demonstrated by pull-down assays using calmodulin-conjugated sepharose and by protein binding assays using the recombinant cytoplasmic domain of ADAM15 and calmodulin.
Furthermore, it could be demonstrated in living synovial fibroblasts by double immunofluorescence stainings that triggering the Fas receptor by its ligand FasL or a Fas-activating antibody resulted in the recruitment of calmodulin to ADAM15 as well as to the Fas receptor in patch-like structures at the cell membrane. Simultaneously, Src associated with calmodulin was shown to become engaged in an ADAM15 complex, also containing cytoplasmic-bound FAK, by co-immunoprecipitations.
Additional studies were performed to analyze the efficacy of TFP and BTP-2 on apoptosis induction in synovial fibroblasts from 10 RA patients. Using caspase 3/7 and annexin V stainings for determining apoptosis, it could be shown that both inhibitors did not possess any apoptosis inducing capacity. However, when co-incubated with FasL both compounds synergistically enhanced apoptosis rates in the RASFs. Moreover, an additional silencing of ADAM15 revealed a further significant rise in apoptosis rates upon incubation with FasL/TFP or FasL/BTP-2, providing unequivocal evidence for an involvement of ADAM15 in facilitating apoptosis resistance in RASFs.
Taken together, these results demonstrate that ADAM15 provides a scaffold for the formation of calmodulin-dependent pro-survival signaling complexes upon CRAC/Orai1 coactivation by Fas ligation, which provides a new potential therapeutic target to break the apoptosis resistance in RASFs that critically contributes to joint destruction in RA.
Understanding global biodiversity patterns is one of the main objectives of ecology. Spatial variation in species richness can be explained by several environmental factors. The relationships between species richness and environmental factors have been associated with latitudinal, longitudinal and elevational gradients. The number of species is determined by birth, death and migration rates of species in a given area. These rates are affected by abiotic and biotic factors acting at local and regional scales. Climatic seasonal variation may also influence biodiversity, directly through physiological limitations and indirectly through biotic interactions, vegetation structure and food availability. Climate and land use change are the main factors for landscape simplification and biotic homogenization. Thus, the study of community patterns across environmental gradients may help to predict the effect of projected environmental change.
I investigated how abiotic and biotic factors influence different facets of bird diversity across an elevational gradient. My study was conducted along an elevational gradient spanning 2000 m within and around Podocarpus National Park and San Francisco reserve on the southeastern slope of the Andes in Ecuador. The climate is humid tropical montane with a bimodal rain regime. The region is characterized by evergreen premontane forest at low elevations, evergreen lower montane forest at mid elevations and upper montane forest at high elevations. The elevational gradient has natural continuous forests within the protected reserves and fragmented forests surrounding the reserves in a matrix of cattle pastures. To monitor bird diversity, I placed nine 20-m radius point counts within 18 one-hectare plots, in continuous and fragmented forest at 1000, 2000 and 3000 m a.s.l. I recorded and identified all birds for 10 minutes within each point count. Bird communities were sampled eight times per plot, in the most humid season and in the least humid season of 2014 and 2015. To estimate flower and fruit availability, I recorded all plants with open flowers and ripe fruits within each point count. To obtain the relative invertebrate availability, I assessed understory invertebrate fresh biomass using a standardized sweep-netting design along 100-metre borders of each plot. Vertical vegetation heterogeneity was estimated at eight layers above the ground within each point count. Temperature for each plot was obtained using an air temperature regionalization tool and precipitation through remote sensing techniques and meteorological data.
In the first chapter of this thesis, I explored the effects of elevation, climate and vegetation structure on overall bird communities as well as on frugivorous and insectivorous birds. I found that elevation was mostly indirectly associated with bird diversity, jointly mediated via temperature, precipitation and vegetation structure. Additionally, elevation was directly and positively associated with both the overall bird community and with insectivores, but not with frugivores. My findings indicate a reduction of bird diversity due to climatic factors and vegetation structure with increasing elevation. However, the direct, positive effect of elevation suggests that bird diversity was higher than expected towards high elevations, probably due to spatial, biotic and evolutionary settings.
In the second chapter, I analysed the influence of climate and resource availability on temporal variation of bird communities. I found a higher bird diversity in the least humid season than in the most humid season. The seasonality of the bird communities was mainly driven by temperature and precipitation. While temperature had a significant positive effect at high elevations, precipitation had a significant negative effect at low elevations. Resource availability had no significant effect. My findings suggest that the temporal fluctuations in bird communities likely occur due to climate
constraints rather than due to resource limitations.
In the third chapter, I studied the effect of forest fragmentation on taxonomic and functional bird diversity. I found that taxonomic diversity was higher in fragmented compared to continuous forests, while functional diversity was negatively affected by fragmentation, but only at low elevations. The increase of taxonomic diversity in disturbed habitats suggests an increase of habitat generalists, which may compensate the loss of forest specialists. My findings suggest that taxonomic diversity can be uncoupled from functional diversity in diverse communities at low elevations.
My results show the effects of environmental factors on the spatio-temporal patterns of bird communities and the potentially uncoupled responses of taxonomic and functional diversity to forest fragmentation. My findings highlight that bird communities respond differently to abiotic and biotic factors across elevational gradients. Overall, my study helps to better understand the mechanisms that drive species communities in response to complex environmental conditions, which could be an essential contribution for the conservation of bird communities in the tropical Andes.
Investigating the influence of truffle´s microbiome and genotype on the aroma of truffle fungi
(2019)
Truffles (Tuber spp.) are belowground forming fungi that develop in association with roots of various host trees and shrubs. Their fruiting bodies are renowned for their enticing aromas which vary considerably, even within truffles of the same species. This aroma variability might be attributed to factors such as geographical origin, degree of fruiting body maturation, truffle genotype and microbiome (microbial communities that colonise truffle fruiting bodies) which often co-vary. Although the influence of specific factors is highlighted by several studies, discerning the contribution of each factor remains a challenge since it requires an appropriate experimental design. The primary purpose of this thesis was to gain insight into the influence of truffle’s genotype and microbiome on truffle aroma.
This doctoral thesis is comprised of four chapters. Chapter1 (Vahdatzadeh et al., 2018) aimed to exclusively elucidate the influence of truffle genotype on truffle aroma by investigating the aroma of nine mycelial strains of the white truffle Tuber borchii. We also assessed whether strain selection could be employed to improve the human- perceived truffle aroma. Quantitative differences in aroma profiles among strains could be observed upon feeding of amino acids. Considerable aroma variabilities among strains were attributed to important truffle volatiles, many of which might be derived from amino acid catabolism through the Ehrlich pathway. 13 C-labelling experiments confirmed the existence of the Ehrlich pathway in truffles for leucine, isoleucine, methionine, and phenylalanine. Sensory analyses further demonstrated that the human nose can differentiate among strains. Our results illustrated the influence of truffle genotype on truffle aroma and showed how strain selection could be used to improve the human-perceived truffle aroma.
In chapter 2 the existing knowledge on the composition of bacterial community of four truffle species was compiled using meta-analysis approach (Vahdatzadeh et al., 2015). We highlighted the endemic microbiome of truffle as well as similarities and differences in the composition of microbial community within species at various phases of their life cycle. Furthermore, the potential contribution of truffle microbiome in the formation of truffle odorants was studied. Our findings showed that truffle fruiting bodies harbour complex microbial community composed of bacteria, yeasts, filamentous fungi, and viruses with bacteria being the dominant group. Regardless of truffle species, the composition of endemic microbiome of fruiting bodies appeared very similar and was dominated by α-Proteobacteria class. However, striking differences were observed in the bacterial community composition at various stages of the life cycle of truffle.Our analyses further suggested that odorants common to many truffle species might be produced by both truffle fungi and microbes, whereas specific truffle odorants might be derived from microbes only. Nevertheless, disentangling the origin of truffle odorants is very challenging, since acquiring microbe-free fruiting bodies are currently not possible.
Chapter 3 (Splivallo et al., 2019) further characterises truffle-associated bacterial communities of fruiting bodies of the black truffle T. aestivum from two different orchards. It aimed at defining the native microbiome in this truffle species, evaluating the variability of their microbiome across orchards, and assessing factors that shape assemblages of the bacterial communities. The dominant bacterial communities in T. aestivum revealed to be similar in both orchards: although a large portion of fruiting bodies were dominated by the α-Proteobacteria class (Bradyrhizobium genus) similar to other so far-assessed truffle species, in few cases β-Proteobacteria (Polaromonas genus), or Sphingobacteria (Pedobacter genus) were found to be predominant classes. Moreover, factors shaping bacterial communities influenced the two orchards differently, with spatial location within the orchard being the main driver in Swiss orchard and collection season in the French one. Surprisingly, in contrast to other fungi, truffle genotype and the degree of fruiting body maturity seemed not to contribute in shaping the assembly of truffle microbiome. Altogether, our data highlighted the existence of heterogeneous bacterial communities in T. aestivum fruiting bodies which are dominated by either of the three bacterial classes and mainly by the α-Proteobacteria class, irrespective of geographical origin. They further illustrated that determinants driving the assembly of various bacterial communities within truffle fruiting bodies are site-specific. Truffles are highly perishable delicacies with a short shelf life (1-2 weeks), and their aroma changes profoundly upon storage. Since truffle aroma might be at least partially produced by the truffle microbiome, chapter 4 (Vahdatzadeh et al., 2019) focuses on assessing the influence of the truffle microbiome on aroma deterioration of T.aestivum during post harvest storage. Specifically, volatile profile and bacterial communities of fruiting bodies collected from four different regions (three in France and one in Switzerland) were studied over nine days of storage. Our findings demonstrated the gradual replacement of dominant bacterial classes in fresh truffles (α-Proteobacteria, β-Proteobacteria, and Sphingobacteria) by food spoilage bacteria (members of γ- Proteobacteria and Bacilli classes), regardless of the initial diversity of the bacterial classes. This shift in the bacterial community also correlated with changes in volatile profiles, and markers for truffle freshness and spoilage could be identified. Ultimately, network analysis illustrated possible links among those volatile markers and specific bacterial classes. Our data showed that storage deeply influenced the composition of bacterial community as well as aroma of truffle fruiting bodies. They also illustrated the correlation between the shift in truffle microbiome, from commensal to detrimental, and the change of aroma profile, possibly leading to the loss of fresh truffle aroma. Overall, the work undertaken in this thesis demonstrated that truffle genotype and microbiome had a stronger influence on truffle aroma than previously believed.
Role of npas4l and Hif pathway in endothelial cell specification and specialization in vertebrates
(2018)
Cardiovascular development requires two main steps, vasculogenesis and angiogenesis. During vasculogenesis, angioblasts, the precursors of endothelial cells (ECs), specify from the mesoderm and coalesce to form the axial vessels of the vertebrate embryo. Many questions regarding the transcriptional waves initiating and sustaining angioblast specification are still unanswered. The identity of cloche, a gene essential for EC differentiation in zebrafish, was only recently discovered by our group, and very little is known about its upstream regulators or its molecular mechanism of action. I described the molecular players involved in orchestrating npas4l expression, upstream of angioblast specification. By using genetic models and chemical treatments, I identified FGF-Erk axis and BMP signaling to be involved in npas4l regulation. I also showed that eomesa is a potent inducer of npas4l expression. In addition, in vitro experiments indicated that murine Eomes promotes EC specification, acting upstream of Etv2 and Tal1. Using a combination of gain-of-function and loss-of-function models for npas4l, I identified primary and secondary downstream effectors of npas4l. I showed that Npas4l binding sites are present in the promoter of genes involved in hematoendothelial specification, such as tal1, lmo2 and etv2. Importantly, I reported that npas4l is sufficient and necessary to promote the EC specification program. By performing a combined analysis of the developed datasets, I recovered putative genes with a potential role in EC specification. One of the most promising candidates was tspan18b. I generated a mutant allele for tspan18b and observed angiogenic defects in tspan18b-/- embryos, confirming a role for this gene in zebrafish cardiovascular development. I showed that Npas4l binds etv2 promoter in zebrafish. In mammalian embryonic stem cells, however, Etv2 promoter is bound by HIF-1α, a transcription factor homolog to Npas4l. Interestingly, Eomes knockdown in vitro lead to a significant reduction of Hif-1α expression. To test the function of Hif-1α in vivo, I took advantage of a murine loss-of-function model.
Hif-1α mouse mutant embryos exhibit a significant decrease in Etv2 expression, when compared with WT siblings. These data suggest a model where mammals lost npas4l during evolution and HIF-1α acquired a new function, replacing npas4l role in EC specification. I compared the phenotype of Hif-1α mouse mutant with zebrafish hif-1α loss-of- function models. Importantly, zebrafish hif-1α mutant did not show defects in vasculogenesis or EC specification, but in EC specialization, during HSC development. I showed that hypoxia is a potent inducer of HSC formation, and hif-1α as well as hif-2α act upstream of notch1, vegfaa and evi1 in hemogenic endothelial specification.
Conclusions
In this work, I explored the molecular mechanisms underlying EC specification in vertebrates, analyzing the role of bHLH-PAS transcription factors in this biological process. I identified the upstream regulators and the downstream effectors of npas4l, describing a novel role for tspan18b in zebrafish cardiovascular development. Npas4l is a transcription factor necessary and sufficient for angioblast differentiation in zebrafish, but the gene was lost in the mammalian lineage. hif-1α and hif-2α, paralogous genes of npas4l, are involved in the establishment of EC heterogeneity and specifically in the specification of hemogenic endothelium in zebrafish. Murine Hif-1α, however, is responsible for Etv2 regulation, indicating a role for hypoxia inducible factor in initiating the EC specification program in mouse, similarly to npas4l function in zebrafish.
Biodiversity is threatened worldwide because of ongoing habitat loss and fragmentation, overexploitation, pollution, biological invasions and a changing global climate. Due to the major importance of biological diversity for modern human living, efficient conservation and management strategies are required to protect endangered habitats and species. For this purpose, ambitious multilateral agreements on regional and global scale were declared to prevent biodiversity loss.
Efficient biomonitoring methods are required to adequately implement these biodiversity conventions. Species monitoring as a core activity in biodiversity research is an effective tool to assess the status of species and trends within habitats. Data collection can be obtained with visual, electronic or genetic surveys. Still, these monitoring programs can be expensive, laborious and inefficient for accurate species assessments. New techniques based on environmental DNA (eDNA) allows for the detection of DNA traces in environmental samples (soil, sediment, water and air samples) and open up new possibilities for species monitoring. The eDNA methodology enables detection of single species in a qualitative (presence/absence) or (semi-) quantitative way. eDNA metabarcoding approaches can be an effective community structure assessment method.
This thesis, located at the interface between experimental and applied research, illustrates the suitability of the eDNA methodology in applied biomonitoring using the example of the water-borne crayfish plague pathogen Aphanomyces astaci (Schikora 1906). The obtained results provide new insights into A. astaci sporulation dynamics in natural water courses. A. astaci sporulation is influenced by seasonal variation of water temperatures and life history traits (molting, activity, mating) of infected crayfish. The results also imply a high transmission risk of A. astaci spores during the complete year. This thesis compares two eDNA methods, which are successfully and consistently detecting A. astaci spores. Each approach is suitable for different biomonitoring tasks due to the method-specific requirements. The obtained results also reveal spatial variation in A. astaci occurance in the tested water bodies. A. astaci spore estimates are positively correlated with population density and pathogen loads of captured A. astaci- positive crayfish. eDNA results show a downstream zoospore transport of up to three kilometres distance from a distribution hot spot area of A. astaci-infected crayfish. The eDNA methodology is helpful in gaining reliable information on A. astaci occurrence in large water bodies. This information is urgently needed to initiate efficient management decisions for the conservation of European crayfish species.
eDNA-based methods such as for A. astaci detection are a useful complement for conventional monitoring and should have a strong impact on conservation policy. eDNA methodology will be helpful for the practical implementation of the main aims of key conservation agreements and thus will make important contributions to biodiversity protection.
Aims: The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues.
Materials and results: Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL.
Conclusion: In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.
Visual aesthetic experiences unfold over time, yet most of our understanding of such experiences comes from experiments using static visual stimuli and measuring static responses. Here, we investigated the temporal dynamics of subjective aesthetic experience using temporally extended stimuli (movie clips) in combination with continuous behavioral ratings. Two groups of participants, a rate group (n = 25) and a view group (n = 25), watched 30-second video clips of landscapes and dance performances in test and retest blocks. The rate group reported continuous ratings while watching the videos, with an overall aesthetic judgment at the end of each video, in both test and retest blocks. The view group, however, passively watched the videos in the test block, reporting only an overall aesthetic judgment at the end of each clip. In the retest block, the view group reported both continuous and overall judgments. When comparing the two groups, we found that the task of making continuous ratings did not influence overall ratings or agreement across participants. In addition, the degree of temporal variation in continuous ratings over time differed substantially by observer (from slower "integrators" to "fast responders"), but less so by video. Reliability of continuous ratings across repeated exposures was in general high, but also showed notable variance across participants. Together, these results show that temporally extended stimuli produce aesthetic experiences that are not the same from person to person, and that continuous behavioral ratings provide a reliable window into the temporal dynamics of such aesthetic experiences while not materially altering the experiences themselves.
Divergent selection between ecologically dissimilar habitats promotes local adaptation, which can lead to reproductive isolation (RI). Populations in the Poecilia mexicana species complex have independently adapted to toxic hydrogen sulfide and show varying degrees of RI. Here, we examined the variation in the mate choice component of prezygotic RI. Mate choice tests across drainages (with stimulus males from another drainage) suggest that specific features of the males coupled with a general female preference for yellow color patterns explain the observed variation. Analyses of male body coloration identified the intensity of yellow fin coloration as a strong candidate to explain this pattern, and common-garden rearing suggested heritable population differences. Male sexual ornamentation apparently evolved differently across sulfide-adapted populations, for example because of differences in natural counterselection via predation. The ubiquitous preference for yellow color ornaments in poeciliid females likely undermines the emergence of strong RI, as female discrimination in favor of own males becomes weaker when yellow fin coloration in the respective sulfide ecotype increases. Our study illustrates the complexity of the (partly non-parallel) pathways to divergence among replicated ecological gradients. We suggest that future work should identify the genomic loci involved in the pattern reported here, making use of the increasing genomic and transcriptomic datasets available for our study system.
Cell–matrix adhesion and cell migration are physiologically important processes that also play a major role in cancer spreading. In cultured cells, matrix adhesion depends on integrin-containing contacts such as focal adhesions. Flotillin-1 and flotillin-2 are frequently overexpressed in cancers and are associated with poor survival. Our previous studies have revealed a role for flotillin-2 in cell–matrix adhesion and in the regulation of the actin cytoskeleton. We here show that flotillins are important for cell migration in a wound healing assay and influence the morphology and dynamics of focal adhesions. Furthermore, anchorage-independent growth in soft agar is enhanced by flotillins. In the absence of flotillins, especially flotillin-2, phosphorylation of focal adhesion kinase and extracellularly regulated kinase is diminished. Flotillins interact with α-actinin, a major regulator of focal adhesion dynamics. These findings are important for understanding the molecular mechanisms of how flotillin overexpression in cancers may affect cell migration and, especially, enhance metastasis formation.
Bone remodeling can be mimicked in vitro by co-culture models. Based on bone cells, such co-cultures help to study synergistic morphological changes and the impact of materials and applied substances. Hence, we examined the formation of osteoclasts on bovine bone materials to prove the bone resorption functionality of the osteoclasts in three different co-culture set-ups using human monocytes (hMCs) and (I) human mesenchymal stem cells (hMSCs), (II) osteogenic differentiated hMSCs (hOBs), and (III) hOBs in addition of soluble monocyte-colony stimulating factor (M-CSF) and cytokine receptor activator of NFκB ligand (RANKL). We detected osteoclast-specific actin morphology, as well as the expression of cathepsin K and CD51/61 in single cells in set-up II and in numerous cells in set-up III. Resorption pits on bone material as characteristic proof of functional osteoclasts were not found in set-up I and II, but we detected such resorption pits in set–up III. We conclude in co-culture models without M-CSF and RANKL that monocytes can differentiate into osteoclasts that show the characteristic actin structures and protein expression. However, to receive functional bone resorbing osteoclasts in vitro, the addition of M-CSF and RANKL is needed. Moreover, we suggest the use of bone or bone-like materials for future studies evaluating osteoclastogenesis.
In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity.
Volatile organic compounds are secondary metabolites emitted by all organisms, especially by plants and microbes. Their role as aboveground signals has been established for decades. Recent evidence suggests that they might have a non-negligible role belowground and might be involved in root–root and root–microbial/pest interactions. Our aim here was to make a comprehensive review of belowground volatile diversity using a meta-analysis approach. At first we synthesized current literature knowledge on plant root volatiles and classified them in terms of chemical diversity. In a second step, relying on the mVOC database of microbial volatiles, we classified volatiles based on their emitters (bacteria vs. fungi) and their specific ecological niche (i.e., rhizosphere, soil). Our results highlight similarities and differences among root and microbial volatiles and also suggest that some might be niche specific. We further explored the possibility that volatiles might be involved in intra- and inter-specific root–root communication and discuss the ecological implications of such scenario. Overall this work synthesizes current knowledge on the belowground volatilome and the potential signaling role of its constituents. It also highlights that the total diversity of belowground volatiles might be orders of magnitude larger that the few hundreds of compounds described to date.
Symbiotic nitrogen fixation (SNF) in root nodules of grain legumes such as chickpea is a highly complex process that drastically affects the gene expression patterns of both the prokaryotic as well as eukaryotic interacting cells. A successfully established symbiotic relationship requires mutual signaling mechanisms and a continuous adaptation of the metabolism of the involved cells to varying environmental conditions. Although some of these processes are well understood today many of the molecular mechanisms underlying SNF, especially in chickpea, remain unclear. Here, we reannotated our previously published transcriptome data generated by deepSuperSAGE (Serial Analysis of Gene Expression) to the recently published draft genome of chickpea to assess the root- and nodule-specific transcriptomes of the eukaryotic host cells. The identified gene expression patterns comprise up to 71 significantly differentially expressed genes and the expression of twenty of these was validated by quantitative real-time PCR with the tissues from five independent biological replicates. Many of the differentially expressed transcripts were found to encode proteins implicated in sugar metabolism, antioxidant defense as well as biotic and abiotic stress responses of the host cells, and some of them were already known to contribute to SNF in other legumes. The differentially expressed genes identified in this study represent candidates that can be used for further characterization of the complex molecular mechanisms underlying SNF in chickpea.
Along with barley and rice, maize provides staple food for more than half of the world population. Maize ears are regularly infected with fungal pathogens of the Fusarium genus, which, besides reducing yield, also taint grains with toxic metabolites. In an earlier work, we have shown that maize ears infection with single Fusarium strains was detectable through volatile sensing. In nature, infection most commonly occurs with more than a single fungal strain; hence we tested how the interactions of two strains would modulate volatile emission from infected ears. For this purpose, ears of a hybrid and a dwarf maize variety were simultaneously infected with different strains of Fusarium graminearum and F. verticillioides and, the resulting volatile profiles were compared to the ones of ears infected with single strains. Disease severity, fungal biomass, and the concentration of the oxylipin 9-hydroxy octadecadienoic acid, a signaling molecule involved in plant defense, were monitored and correlated to volatile profiles. Our results demonstrate that in simultaneous infections of hybrid and dwarf maize, the most competitive fungal strains had the largest influence on the volatile profile of infected ears. In both concurrent and single inoculations, volatile profiles reflected disease severity. Additionally, the data further indicate that dwarf maize and hybrid maize might emit common (i.e., sesquiterpenoids) and specific markers upon fungal infection. Overall this suggests that volatile profiles might be a good proxy for disease severity regardless of the fungal competition taking place in maize ears. With the appropriate sensitivity and reliability, volatile sensing thus appears as a promising tool for detecting fungal infection of maize ears under field conditions.