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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.
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.
Cardiac trabeculation is one of the essential processes required for the formation of a competent ventricular wall, whereby clusters of ventricular cardiomyocytes (CMs) from a single layer delaminate and expand into the cardiac jelly to form sheet-like projections in the developing heart (Samsa et al., 2013). Several congenital heart diseases are associated with defects in the formation of these trabeculae and lead to embryonic lethality (Jenni et al., 1999; Zhang et al., 2013, Jenni et al., 2001; Towbin 2010). It has been experimentally shown that lack of Nrg1/ErbB2/ErbB4, Angipoetin1/Tie2, EphrinB2/B4, BMP10, or any component of the Notch signaling pathway can cause defective trabeculation. Moreover, changes in blood flow and/or contractility can also affect trabeculation (Samsa et al., 2013). Together, these observations demonstrate that cardiac trabeculation is a highly dynamic and regulated process.
Trabeculation is a morphogenetic process that requires control over cell shape changes and rearrangements, similar to those observed during EMT. Epithelial cells within an epithelium are polarized and establish cell-cell junctions with the neighboring cells (Ikenouchi et al., 2003; Ferrer-vaquer et al., 2010), thus epithelial cell polarity is an important feature to maintain cell shape and tissue structure. During developmental processes such as cell migration and cell division or in disease states epithelial polarity might be disrupted. As a consequence of this alteration, cells lose their tight cell-cell adhesions, undergo cytoskeletal rearrangements, change their shape and gain migratory properties becoming mesenchymal cells (Micalizzi et al., 2010). In epithelial cells, apicobasal polarity is regulated by a conserved set of core complexes, including the PAR, Scribble and Crumbs complexes (Kemphues et al., 1988; Bilder and Perrimon, 2000; Teppas et al., 1984). The polarity proteins composing these complexes interact in a well organized and coordinated-manner creating molecular asymmetry along the apicobasal axis of the cell. In turn, this crosstalk regulates the maturation and stabilization of the junctions between cells and cytoskeleton in order to strengthen cell polarization (Roignot et al., 2013). Amongst the different polarity complex, Crumbs has been shown to be a key regulator of apicobasal polarity during development in both vertebrates and invertebrates (Tepass et al., 1990; Fan et al., 2004).
Here, taking advantage of zebrafish as a model organism, I study in vivo at single cell resolution changes in CM apicobasal polarity during cardiac trabeculation. Moreover, I show which factors regulate CM apicobasal polarity during this process. In addition, I dissect the role of the polarity complex Crumbs in regulating CM junctional rearrangements and the formation of the trabecular network.
In the 'Golden Age of Antibiotics', between 1940 and 1970, the global pharmaceutical companies discovered many antibiotics, such as cephalosporins, tetracyclines, aminoglycosides, glycopeptides, etc., as well as antifungal and antiparisitic agents. Due to several reasons, e.g. the steady re-discovery of already known NPs and the associated high costs, many pharmaceutical companies have significantly scaled back or totally abandoned their NP discovery programs since the late 20th century. Instead those companies started to focus on drug discovery based on combinatorial synthesis and thereby on the creation of enormous synthetic libraries containing small molecules. Unfortunately, this synthetic approach dealing with the optimization of existing NP or antibiotic has its limitations. As a result, leading pharmaceutical companies are re-conducting NPs research to discover new antimicrobials for the upcoming antimicrobial resistance threat. The Natural Product Center of Excellence, a collaboration between Sanofi-Aventis and Fraunhofer IME, is advancing in this context the discovery and development of novel antimicrobial agents for the treatment of infectious diseases through the testing of Sanofi's microbial extract library and strain collection. The aim of the present PhD thesis was the discovery and isolation of novel antimicrobial compounds with improved activities and/or novel MOAs as potential lead compound for a further drug discovery.
Der DNA-Translokator von T. thermophilus HB27, ebenso wie Typ-IV-Pili (T4P), sind Multiproteinkomplexe, die die Membranen und das Periplasma durchspannen. Sie sind ähnlich aufgebaut und enthalten identische Proteine. Der DNA-Translokator vermittelt Transport von DNA in das Zellinnere während der natürlichen Transformation. T4P sind filamentöse Zellorganellen, die an der inneren Membran assembliert werden und bis zu mehrere Mikrometer aus der Zelle hinausragen. Sie dienen der Anhaftung und Fortbewegung der Zellen auf Oberflächen.
Das Ziel dieser Arbeit war es, die Funktionen einzelner Komponenten der Komplexe und ihrer Proteindomänen bei der natürlichen Transformation, der T4P-Assemblierung und den durch T4P vermittelten Funktionen Adhäsion und „twitching motility“ aufzuklären.
Es sind neun Proteine bekannt, die eine duale Rolle als Komponenten des DNA-Translokators und des T4P spielen. Eines dieser Proteine ist die Assemblierungs-ATPase PilF, die Hexamere bildet. Diese cytoplasmatischen ATPase-Komplexe stellen die Energie für die Assemblierung der T4P bereit, ebenso wie für die Aufnahme freier DNA. Es ist jedoch bisher nicht geklärt, wie die durch PilF bereitgestellte Energie auf die anderen Komponenten des DNA-Translokators/T4P übertragen wird.
In dieser Arbeit konnte gezeigt werden, dass PilF an das cytoplasmatische Protein PilM des T4P und DNA-Translokators bindet. Zudem konnten Proteinkomplexe bestehend aus den Proteinen PilM, PilN und PilO heterolog produziert und aus Zellmembranen koisoliert werden. PilF interagierte mit diesen PilMNO-Komplexen via PilM. Diese Interaktionen führt zur Stimulierung der ATPase-Aktivität von PilF. Dies deutet an, dass PilM ein Kupplungsprotein ist, welches die Assemblierungs-ATPase PilF physisch und funktionell mit dem T4P/DNA-Translokator über den PilMNO-Komplex verbindet.
Neben PilF standen Präpiline von T. thermophilus im Fokus dieser Arbeit. Präpiline sind Vorläuferproteine, die zu Pilinen prozessiert werden und als solche dann die Untereinheiten der Pilus-Strukturen bilden.
Zusammenfassend konnten die Rollen einzelner Präpilin-ähnlicher Proteine bei T4P-assoziierten Funktionen geklärt werden und es konnten erste Analysen zur Charakterisierung des weitestgehend unbekannten Proteins ComZ durchgeführt werden. Desweiteren liefert diese Arbeit Hinweise darauf, dass die membranassoziierten Proteine PilM, PilN und PilO Kupplungsproteine sind, die PilF mit den periplasmatischen Komponenten des T4P/DNA-Translokators verbinden und dadurch die ATPase-Aktivität von PilF stimulieren. Die Rollen einzelner Proteindomänen von PilF und PilM bei der Protein-Protein-Interaktion und der Bindung von Liganden wurden aufgeklärt, sowie ihre Funktionen bei den T4P-vermittelten Funktionen und der natürlichen Transformation.
Autophagy, meaning “self-eating”, is an important cellular waste disposal mechanism. Thereby, damaged proteins, lipids and organelles are enclosed by autophagosomes and subsequently transported to the lysosomes for degradation into basic, cellular building blocks. Under basal conditions autophagy prevents the accumulation of defective and harmful material and generally promotes cell survival. However, several studies reported that hyperactivated autophagy, e.g. during developmental processes in lower eukaryotes, or during chemotherapeutic treatment of cancer cells, can also trigger cell death.
In recent years, autophagic cell death (ACD) has been considered as an alternative cell death pathway for tumor therapy, especially for solid tumors with high apoptosis resistance such as glioblastoma. Glioblastoma (GBM) is a very aggressive, malignant primary brain tumor with a median survival of ~ 15 months despite surgery and chemoradiotherapy. Accordingly, there is a great interest in improving GBM therapy through alternative cell death mechanisms. Interestingly, it has been shown that various substances, e.g. AT 101, cannabinoids and the combination of imipramine and ticlopidine (IM+TIC), induce ACD in GBM cells.
The aim of this project was to identify the underlying mechanisms of stress- and drug-induced ACD and its therapeutic potential for glioblastoma treatment. For detailed investigation of ACD, a CRISPR/Cas9-based approach was used to generate ATG5 and ATG7 knockouts as genetic models of autophagy deficiency. In a previous study of our lab it was demonstrated that administration of AT 101 triggers ACD in glioblastoma cells, which was associated with early mitochondrial fragmentation but no signs of apoptosis. Since mitochondrial fragmentation often precedes mitophagy, the first part of this thesis explored the potential role of mitophagy in AT 101-induced cell death.
ATG5-depleted cells confirmed that AT 101 induces ACD. In addition, treatment with AT 101 resulted in a pronounced mitochondrial depolarization, which was at least partly caused by the opening of the mitochondrial permeability pore. Global proteome analysis of AT 101-treated GBM cells revealed a robust decrease in mitochondrial protein clusters as well as a strong increase in the enzyme heme oxygenase-1 (HMOX1). Subsequent experiments for detailed investigation of mitophagy following AT 101 treatment (western blot, flow cytometric MTG and mt-mKeima, qRT-PCR of mitochondrial vs nuclear DNA) consistently indicated strong mitophagy induction by AT 101, which could be reduced by genetic or pharmacological inhibition of autophagy. Furthermore, siRNA-mediated knockdown experiments revealed that the selective mitophagy receptors BNIP3 and BNIP3L and the HMOX1 enzyme play an essential role in AT 101-induced mitophagy and subsequent cell death. Taken together, these data demonstrate that AT 101-induced mitochondrial dysfunction and HMOX1 induction synergize to promote excessive mitophagy with a lethal outcome in glioma cells.
The second part of this thesis focused on the identification of new substances that cause ACD and the investigation of the underlying cell death pathways. Using a cell death screen of the ENZO Screen-Well™ autophagy library in MZ-54 wild-type vs ATG5 and ATG7-depleted cells, loperamide, pimozide, and STF-62247 were identified as ACD-inducing agents. The increase of the autophagic flux and the induction of ACD by these substances was confirmed by using different ATG5 and ATG7 knockout cell lines and the already established positive control IM+TIC.
In contrast to AT 101, IM+TIC, STF-62247, loperamide and pimozide produced neither mitochondrial dysfunction nor mitophagy. Interestingly, it has been described that imipramine, loperamide and pimozide inhibit the lysosomal enzyme acid sphingomyelinase, which is associated with impaired lipid transport. Global proteome analysis and cholesterol staining confirmed that all four substances, but especially loperamide and pimozide, inhibit cellular lipid transport, leading to massive lipid accumulation in the lysosomes. In the further course of the experiments, the connection between defective lipid transport and autophagy was investigated in more detail. On the one hand, the defective lipid transport contributed to the induction of autophagy, on the other hand the massive accumulation of lipids led to lysosomal membrane damage, inhibition of lysosomal degradation at later time points and finally to a lysosomal cell death. Remarkably, it has been shown that hyperactivated autophagy by IM+TIC, loperamide and pimozide massively promotes lysosomal membrane damage. This result highlights the difficulties of a clear distinction between autophagic and lysosomal cell death.
In summary, two new signaling pathways that induce autophagic cell death in GBM cells and may be relevant for glioblastoma therapy were investigated in this study.
Structured illumination microscopy (SIM) is part of the super-resolution methods developed at the beginning of this century. To produce a super-resolution image SIM requires three things: 1) illumination of the sample with a periodic pattern, 2) acquisition of multiple images per plane under different pattern’s phases and orientations and 3) the processing of these images has to be carried with a reconstruction algorithm. The result of the reconstruction is an image with a resolution gain that is proportional to the frequency of the pattern (po). The typical SIM set-up uses an epi-fluorescence configuration, thus the interference angle of the beams that create the pattern is restricted by the angular aperture of the objective. Under this restriction the maximum value of po is given by the cut-off frequency of the objective lens and sets at 2 the maximum resolution gain of SIM under linear illumination.
In the first part of this thesis we present the implementation and characterization of the 2D-SIM set-up designed by Dr. Bo-Jui Chang (B-J. Chang et al., PNAS 2017), this design exploits the concept introduced by light-sheet microscopy, i.e. separation of illumination and detection paths to obtain resolution gains larger than the usual two-fold (Chapter 3). The set-up is named coherent structured illumination light-sheet based fluorescence microscopy (csiLSFM) and it consists of a triangular array of three objectives, such that two are used for illumination and one for detection. With the independent illumination arms is possible to interfere two coherent light-sheets at angles beyond the angular aperture of the detection lens, attaining the maximum interference angle of 180° when the light-sheets counter-propagate. This condition delivers a pattern with a po 1.4 times larger than the cut-off frequency (ωo), hence our set-up provides generic resolution gains of 2.4.
The extraction of the high spatial frequencies that produce the resolution gain in the csiLSFM is a challenge due to a low pattern modulation. The low modulation inherently arises because the frequency associated to the pattern period lies beyond the cut-off frequency of the detection lens. To overcome this challenge we developed a filtering strategy that facilitates the withdrawal of information from a SIM data set, simultaneously the proposed filtering process optimizes the reconstruction algorithm by reducing the periodic artifacts that are recurrent in SIM images. In this same chapter we also performed an spectral analysis of the artifacts and determined that they originate from irregularities in the power spectrum that occur due to the partial or total lack of certain spatial frequencies (fig.4.2 and 4.3), our reconstruction reduces this information drops and diminishes the artifact occurrence. The relevance of our reconstruction pipeline is that it delivers a standardized process to enhance the SIM image in a current context in which the commonly used reconstruction algorithms employ empirical tuning to improve it (fig.4.13). Moreover, the pipeline is applicable to the csiLSFM data and also to images acquired with any other 2D-/3D-SIM set-up (fig.4.10 and 4.11).
The processing of various image data sets acquired with the csiLSFM exposed us to the question of how low the modulation of the illumination pattern can be before no super-resolution frequencies can be extracted. Answering this question is important to guarantee that the SIM data contains enough spatial frequencies to provide significant resolution gains. Thus in chapter 5 we developed a quantitative metric to indirectly determine the pattern modulation from the SIM data and find its critical value to use it as evaluation criterion. We called this metric the quality factor (Q-factor) and it represents the normalized strength (amplitude) of the extracted frequencies respect to the Gaussian noise contained in the images. Through simulations we estimated that Q=0.11 is a critical value and a SIM data set requires this as minimum value is to deliver a significant resolution gain. Q works then as an assessment tool for classifying SIM data as optimal or sub-optimal, i.e. Q≥0.11 or Q<0.11. We demonstrated such application with data acquired in various SIM commercial set-ups to prove its feasibility in the field (fig.5.6-5.11)
As mentioned at the beginning of this abstract SIM requires a specialized set-up and a processing algorithm to produce super-resolution images. This thesis contributes to these two areas in the following aspects: first, in its linear version a structured illumination microscope is highly associated to a 2-fold resolution gain. Here we demonstrated the possibility of extending this gain to 2.4 using our custom set-up the csiLSFM. Second, a reconstructed SIM image is prone to artifacts due to the mathematical process it undergoes, here we analyzed the artifact sources and identified them with drops of spatial information in the reconstructed spectrum, based on these conclusions we designed a processing pipeline to facilitate the extraction of spatial frequencies and directly reduce artifacts. A third and final outcome of this thesis is the development and practical implementation of a quantitative index to evaluate the quality of SIM data in terms of its relevant information content (Q-factor). Accordingly, the overall contributions of this work were done in the areas of SIM set-up, SIM reconstruction procedure and SIM data evaluation.