Biologische Hochschulschriften (Goethe-Universität)
Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold
Lesley F. Haire
- SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.
Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish
Machteld J. van Amerongen
Felix B. Engel
- The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.
Modelling the impact of future climate and land use change on vegetation patterns, plant diversity and provisioning ecosystem services in West Africa
- Global climate change and land use change will not only alter entire ecosystems and
biodiversity patterns, but also the supply of ecosystem services. A better understanding
of the consequences is particularly needed in under-investigated regions, such as West
Africa. The projected environmental changes suggest negative impacts on nature, thus
representing a threat to the human well-being. However, many effects caused by climate
and land use change are poorly understood so far.
Thus, the main objective of this thesis was to investigate the impact of climate and
land use change on vegetation patterns, plant diversity and important provisioning
ecosystem services in West Africa. The three different aspects are separately explored
and build the chapters of this thesis. The findings help to improve our understanding of
the effects of environmental change on ecosystems and human well-being.
In the first study, the main objectives were to model trends and the extent of
future biome shifts in West Africa that may occur by 2050. Also, I modelled a trend in
West African tree cover change, while accounting for human impact. Additionally,
uncertainty in future climate projections was evaluated to identify regions with reliable
trends and regions where the impacts remain uncertain. The potential future spatial
distributions of desert, grassland, savanna, deciduous and evergreen forest were
modelled in West Africa, using six bioclimatic models. Future tree cover change was
analysed with generalized additive models (GAMs). I used climate data from 17 general
circulation models (GCMs) and included human population density and fire intensity to
model tree cover. Consensus projections were derived via weighted averages to: 1)
reduce inter-model variability, and 2) describe trends extracted from different GCM
projections. The strongest predicted effect of climate change was on desert and
grasslands, where the bioclimatic envelope of grassland is projected to expand into the
Sahara desert by an area of 2 million km2. While savannas are predicted to contract in the
south (by 54 ± 22 × 104 km2), deciduous and evergreen forest biomes are expected to
expand (64 ± 13 × 104 km2 and 77 ± 26 × 104 km2). However, uncertainty due to different
GCMs was particularly high for the grassland and the evergreen forest biome shift.
Increasing tree cover (1–10%) was projected for large parts of Benin, Burkina Faso, Côte d’Ivoire, Ghana and Togo, but a decrease was projected for coastal areas (1–20%).
Furthermore, human impact negatively affected tree cover and partly changed the
direction of the projected climate-driven tendency from increase to decrease.
Considering climate change alone, the model results of potential vegetation (biomes)
showed a ‘greening’ trend by 2050. However, the modelled effects of human impact
suggest future forest degradation. Thus, it is essential to consider both climate change
and human impact in order to generate realistic future projections on woody cover.
The second study focused on the impact and the interplay of future (2050) climate
and land use change on the plant diversity of the West African country Burkina Faso.
Synergistic forecasts for this country are lacking to date. Burkina Faso covers a broad
bioclimatic gradient which causes a similar gradient in plant diversity. Thus, the impact of
climate and land use change can be investigated in regions with different levels of species
richness. The LandSHIFT model from the Centre of Environmental System research CESR
(Kassel, Germany) was adapted for this study to derive novel regional, spatially explicit
future (2050) land use simulations for Burkina Faso. Additionally, the simulations include
different assumptions on the technological developments in the agricultural sector. Oneclass
support vector machines (SVMs), a machine learning method, were performed with
these land use simulations together with current and future (2050) climate projections at
a 0.1° resolution (cell: ~ 10 × 10 km). The modelling results showed that the flora of
Burkina Faso will be primarily negatively impacted by future climate and land use
changes. The species richness will be significantly reduced by 2050 (P < 0.001, paired
Wilcoxon signed-rank test). However, contrasting latitudinal patterns were found.
Although climate change is predicted to cause species loss in the more humid regions in
Southern Burkina Faso (~ 200 species per cell), the model projects an increase of species
richness in the Sahel. However, land use change is expected to suppress this increase to
the current species diversity level, depending on the technological developments. Climate
change is a more important threat to the plant diversity than land use change under the
assumption of technological stagnation in the agricultural sector.
Overall, the study highlights the impact and interplay of future climate and land
use change on plant diversity along a broad bioclimatic gradient in West Africa.
Furthermore, the results suggest that plant diversity in dry and humid regions of the tropics might generally respond differently to climate and land use change. This pattern
has not been detected by global studies so far.
Several of the plant species in West Africa significantly contribute to the
livelihoods of the population. The plants provide so-called non-timber forest products
(NTFPs), which are important provisioning ecosystem services. However, these services
are also threatened by environmental change. Thus, the third study aimed at developing a
novel approach to assess the impacts of climate and land use change on the economic
benefits derived from NTFPs. This project was carried out in cooperation with Katja
Heubach (BiK-F) who provided data on household economics. These data include 60
interviews that were conducted in Northern Benin on annual quantities and revenues of
collected NTFPs from the three most important savanna tree species: Adansonia digitata,
Parkia biglobosa and Vitellaria paradoxa. The current market prices of the NTFPs were
derived from respective local markets. To assess current and future (2050) occurrence
probabilities of the three species, I calibrated niche-based models with climate data (from
Miroc3.2medres) and land use data (LandSHIFT) at a 0.1° resolution (cell: ~ 10 × 10 km).
Land use simulations were taken from the previous study on plant diversity. Three
different niche-based models were used: 1) generalized additive models (regression
method), 2) generalized boosting models (machine learning method), and 3) flexible
discriminant analysis (classification method). The three model simulations were averaged
(ensemble forecasting) to increase the robustness of the predictions. To assess future
economic gains and losses, respectively, the modelled species’ occurrence probabilities
were linked with the spatially assigned monetary values. Highest current annual benefits
are obtained from V. paradoxa (54,111 ± 28,126 US$/cell), followed by P. biglobosa
(32,246 ± 16,526 US$/cell) and A. digitata (9,514 ± 6,243 US$/cell). However, in the
prediction large areas will lose up to 50% of their current economic value by 2050.
Vitellaria paradoxa and Parkia biglobosa, which currently reveal the highest economic
benefits, are heavily affected. Adansonia digitata is negatively affected less strongly by
environmental change and might regionally even supply increasing economic benefits, in
particular in the west and east of the investigation area. We conclude that adaptive
strategies are needed to create alternative income opportunities, in particular for women
that are responsible for collecting the NTFPs. The findings provide a benchmark for local policy-makers to economically compare different land use options and adjust existing
management strategies for the near future.
Overall, this thesis improves our understanding of the impacts of climate and land
use changes on West African vegetation patterns, plant diversity and provisioning
ecosystem services. Climate change had spatially varying impacts (positive and negative
effects) on the vegetation cover and plant diversity, while predominantly negative effects
resulted from human pressure. Regional contrasting impacts of environmental change
were also found considering the provisioning ecosystem services.
Optical mapping as a routine tool for bacterial genome sequence finishing
Barry S. Goldman
Helge Björn Bode
- Background: In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.
Results: Whole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished.
Conclusion: Our experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.
Impact of tumour microenvironmental factors on dendritic cell differentiation and function
- Um der Erkennung durch das körpereigene Immunsystem entkommen, weisen Tumore
Modifikationen in ihrer Mikroumgebung auf. Zu diesen gehören u. a. veränderte
Sauerstoffkonzentrationen im Tumorkern und die Freisetzung biochemischer Faktoren aus
Tumorzellen, welche die Funktion von Tumor-assoziierten Phagozyten, wie z.B.
Dendritischen Zellen (DC) beeinflussen. DC sind professionelle Antigen-präsentierende
Zellen, die eine Spezialisierung in verschiedene funktionale Subtypen aufweisen. Myeloische
DC (mDC) sind besonders effizient in Hinsicht auf die Präsentation von Antigenen,
wohingegen plasmazytoide DC (pDC) regulatorisch auf das Immunsystem einwirken. Beide
Subtypen spielen eine wichtige Rolle bei der Karzinogenese.
Während humane mDC, zur therapeutischen Verwendung, ex vivo aus Monozyten
hergestellt werden können, war dies für humane pDC bisher nicht möglich. Ein war deshalb
ein erstes Ziel dieser Arbeit, ein Protokoll zur Generierung humaner pDC aus humanen
Monozyten zu entwickeln. Diese wurden mittels des Wachstumsfaktors Fms-related tyrosine
kinase 3 ligand (Flt3-L) zu pDC-Äquivalenten differenziert, welche als monocyte-derived pDC
(mo-pDC) bezeichnet wurden. In der Tat zeigten mo-pDC ein für humane pDC
charakteristisches Oberflächenmarkerprofil und wiesen, im Vergleich zu mDC, eine geringe
Kapazität zur Induktion der Proliferation autologer T Zellen und zur Phagozytose
apoptotischer Zellen auf. Mo-pDC erwarben im Verlauf ihrer Differenzierung aus Monozyten
eine kontinuierlich erhöhte Expression des pDC-spezifischen Transkriptionfaktors E2-2 und
seiner spezifischen Zielgene. Der wichtigste funktionale Parameter von pDC ist die
Produktion großer Mengen von Interferon-α (IFN-α). Mo-pDC sezernierten, nach vorheriger
Aktivierung mit Tumornekrosefaktor-α (TNF-α) oder wenn zu ihrer Differenzierung neben
Flt3-L auch Vitamin D3 oder all-trans-Retinolsäure verwendet wurde, ebenfalls große
Wurden mo-pDC unter Hypoxie, einem prominenten Faktor der Tumormikroumgebung,
generiert, so waren die Expression des spezifischen Transkriptionsfaktors E2-2 und die
Freisetzung von IFN-α stark vermindert. Diese Daten zeigten zunächst, dass mo-pDC für das
Studium von Differenzierung und Funktion humaner pDC eingesetzt werden können.
Weiterhin lieferten sie Hinweise auf eine veränderte Differenzierung humaner pDC unter
Hypoxie. In einem nächsten Schritt wurde folglich untersucht, ob Hypoxie auch die
Differenzierung von pDC aus deren physiologischen Vorläufern beeinflusst. Wurden
Knochenmarkszellen der Maus mit Flt3-L unter Normoxie oder Hypoxie kultiviert, so war die
Differenzierung zu pDC unter Hypoxie in der Tat unterdrückt. Dies war abhängig von der
Hypoxie-induzierten Aktivität des Hypoxie-induzierten Faktors 1 (HIF-1), da die Flt3-Linduzierte
Differenzierung von murinen Knochenmarkszellen, in denen die Expression von
HIF-1 in pDC-Vorläuferzellen ausgeschaltet war, unter Hypoxie normal verlief.
Zusammenfassend kann also gesagt werden, dass Hypoxie, durch Aktivierung von HIF-1,
Differenzierung und Funktion von pDC unterdrückt. Dieser Mechanismus könnte zu ihrer
beschriebenen Dysfunktion in humanen Tumoren beitragen.
Neben Hypoxie sind viele andere Faktoren an der Immunsuppression in Tumoren beteiligt.
Eine Komponente der Mikroumgebung in Tumoren ist das Vorhandensein apoptotischer
Tumorzellen. Apoptose von Tumorzellen findet, im Kontrast zur generellen Sicht von
Tumoren als Apoptose-resistente Entitäten, auch in unbehandelten Tumoren im Überfluss
statt. Apoptotische körpereigene Zellen unterdrücken unter physiologischen Bedingungen
das Immunsystem. Deshalb könnte das Freisetzen von apoptotischem Material oder die
Sekretion von Faktoren aus sterbenden Tumorzellen einen starken Einfluss auf die Funktion
von Tumor-assoziierten DC und die damit verbundene Aktivierung von tumoriziden
Lymphozyten haben. Eine diesbezügliche Studie war das zweite Ziel der vorliegenden
Arbeit. Humane mDC wurden zu diesem Zweck mit Überständen lebender, apoptotischer
oder nekrotischer humaner Brustkrebszellen aktiviert und anschließend mit autologen T
Zellen ko-kultiviert. Danach wurde das zytotoxische Potential der ko-kultivierten T Zellen
analysiert. Interessanterweise unterdrückte die Aktivierung mit Überständen apoptotischer
Tumorzellen die DC-vermittelte Generierung tumorizider T Zellen durch die Ausprägung
einer Population von regulatorischen T Zellen (Treg), die durch die gleichzeitige Expression
der Oberflächenmoleküle CD39 und CD69 charakterisiert war. Die Ausprägung der CD39-
und CD69-exprimierenden Treg Zell-Population war abhängig von der Freisetzung des
bioaktiven Lipids Sphingosin-1-Phosphat (S1P) aus apoptotischen Zellen, welches durch den
S1P-Rezeptor 4 zur Freisetzung des immunregulatorischen Zytokins IL-27 aus mDC führte.
Neutralisierung von IL-27 in AC-aktivierten Ko-Kulturen von mDC und T Zellen blockierte die
Generierung von CD39- und CD69-exprimierenden Treg Zellen und resultierte folglich in der
Aktivierung zytotoxischer T Zellen. Weiterhin war die Bildung von Adenosin in den Ko-
Kulturen für die Unterdrückung zytotoxischer T Zellen vonnöten. Erste Experimente lieferten
Hinweise auf eine direkte Interaktion von CD69- und CD39-exprimierenden Treg Zellen mit
CD73-exprimierenden zytotoxischen T Zellen. CD39 und CD73 werden für die Bildung von
Adenosin aus ATP benötigt, weswegen die Interaktion von Treg Zellen und zytotoxischen T
Zellen die Adenosin-Produktion fördern könnte.
Zusammenfassend zeigen die hier präsentierten Befunde wie Faktoren der
Tumormikroumgebung die Funktion von humanen DC Subtypen beeinflussen können. Ein
Verständnis der zugrundeliegenden Mechanismen kann wertvolle Informationen für die Wahl
effektiver Immuntherapien oder Chemotherapien liefern und so die Therapie humaner
Coordinated optimization of visual cortical maps (I) symmetry-based analysis
Justin C. Crowley
- In the primary visual cortex of primates and carnivores, functional architecture can be characterized by maps of various stimulus features such as orientation preference (OP), ocular dominance (OD), and spatial frequency. It is a long-standing question in theoretical neuroscience whether the observed maps should be interpreted as optima of a specific energy functional that summarizes the design principles of cortical functional architecture. A rigorous evaluation of this optimization hypothesis is particularly demanded by recent evidence that the functional architecture of orientation columns precisely follows species invariant quantitative laws. Because it would be desirable to infer the form of such an optimization principle from the biological data, the optimization approach to explain cortical functional architecture raises the following questions: i) What are the genuine ground states of candidate energy functionals and how can they be calculated with precision and rigor? ii) How do differences in candidate optimization principles impact on the predicted map structure and conversely what can be learned about a hypothetical underlying optimization principle from observations on map structure? iii) Is there a way to analyze the coordinated organization of cortical maps predicted by optimization principles in general? To answer these questions we developed a general dynamical systems approach to the combined optimization of visual cortical maps of OP and another scalar feature such as OD or spatial frequency preference. From basic symmetry assumptions we obtain a comprehensive phenomenological classification of possible inter-map coupling energies and examine representative examples. We show that each individual coupling energy leads to a different class of OP solutions with different correlations among the maps such that inferences about the optimization principle from map layout appear viable. We systematically assess whether quantitative laws resembling experimental observations can result from the coordinated optimization of orientation columns with other feature maps.
Species distribution modelling of stream macroinvertebrates under climate change scenarios
- There is increasing evidence that climate change will have a severe impact on species’ distributions by altering the climatic conditions within their present ranges. Especially species inhabiting stream ecosystems are expected to be strongly affected due to warming temperatures and changes in precipitation patterns. The aim of this thesis was to
investigate how distributions of aquatic insects, i.e., benthic stream macroinvertebrates would be impacted by warming climates. The methods comprised of an ensemble forecasting technique based on species distribution models (SDMs) and climate change scenarios of the Intergovernmental Panel on Climate Change of the year 2080. Future model projections were generated for a wide variety of species from a number of taxonomic orders for two spatial scales: a stream network within the lower mountain ranges of Germany, and the entire territory across Europe. In addition, the effect of the modelling technique on habitat suitability projections was investigated by modifying the choice of study area (continuous area vs. stream network) and the choice of predictors (standard vs. corrected set).
Projections of future habitat suitability showed that potential climate-change impacts would be dependent on species’ thermal preferences, and with a similar pattern for both spatial scales. Future habitat suitability was projected to remain for most or all of the modelled species, and species were projected to track their climatically suitable conditions by shifting uphill along the river continuum within the lower mountain ranges, and into a north-easterly direction across Europe. Cold-adapted headwater and high-latitude species were projected to lose suitable habitats, whereas gains would be expected for warm-adapted river and low-latitude species along the river continuum and across Europe, respectively. Additionally, habitat specialist species in terms of endemics of the Iberian Peninsula were identified as potential climate-change losers, highlighting their restricted habitat availability and therefore vulnerability to warming climates.
The main findings of this thesis underline the high susceptibility of stream macroinvertebrates to ongoing climate change, and give insights into patterns of possible consequences due to changes in species’ habitat suitability. Concerning the methodology, a clear recommendation can be given for future modelling approaches of stream macroinvertebrates by building models within a stream network and with a careful choice of environmental predictors, to reduce uncertainties and thus to improve model projections.
Secondary successions after shifting cultivation in a dense tropical forest of southern Cameroon (Central Africa)
- The ongoing debate on deforestation in the tropics usually points out agriculture and logging as the main causes. The two activities are often linked and the trails created by logging com-panies with their heavy machines are afterwards used by farmers to penetrate deep into the forest and cultivate. Shifting cultivation is a widespread agricultural practice in the tropics and its sustainability is often a matter of controversy. It is necessary to investigate forest recovery after shifting cultivation, analyze its succession stages for comparison with regeneration after natural disturbance, and evaluate its role for discussing the hazards of deforestation.
Identification and characterization of genes and signaling pathways involved in proliferation and differentiation of mammary epithelial cells
- The mammary gland is a perfect system to study the pathways regulating organogenesis during development of an individual. The proper development of the mammary gland requires a tight coordination of expression of many genes involved in proliferation and differentiation. The aim of this work was to identify novel genes and pathways involved in the development of the mammary gland and to find possible correlations between the signaling pathways and their downstream targets that are activated during proliferation and functional differentiation of mammary epithelial cells. In this study rapamycin has been used to inhibit the mTOR protein to analyze its role during mammary gland development. Further a genomic approach was used to identify genes differently expressed during this process. The analysis of the effects caused by the inhibition of the mTOR signaling pathway by using rapamycin on mammary epithelial cells for the first time demonstrate that mTOR plays central role in the coordination of pathways governing the proliferation and differentiation of epithelial cells during mammary gland development. More detailed analysis led to the identification of Id1 and Id2 as two major downstream effectors of the mTOR signaling pathway regulating proliferation and differentiation respectively. The genomics analysis revealed several interesting genes involved in the regulation of a proliferative or secretory phenotype of normal epithelial cells in vitro. Various genes identified by microarray analysis are of high interest and to determine their role in mammary gland development. Among the identified genes some contribute to process of proliferation like Nol5 and Kpna2, whereas other genes are required for proper functional differentiation such as Nkd2 and Cited4. Importantly, the mentioned candidate genes are also interesting regarding cancer development, since deregulation of their expression might contribute to tumor formation. The findings described in this work clearly contribute to our better understanding of the mTOR signaling pathway regulating expression of the genes involved in the development of mammary gland. In addition, the presented results should allow broadening our view of the events that contribute to breast cancer development and help to design better anticancer therapies in the future.
Analyses of diurnal rhythms in human post-mortem tissues
- Rhythmic changes in environmental lighting conditions have ever been the most reliable environmental cue for life on earth. Nature has therefore selected a genetically encrypted endogenous clock very early in evolution, as it provided cells and subsequently organisms with the ability to anticipate persevering periods of light and darkness. Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. The mammalian endogenous master clock, which synchronizes the body to environmental time, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. As an integral part of the time-coding system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic nocturnal synthesis and immediate release of the hormone melatonin. In contrast to the transcriptional regulation of melatonin synthesis in rodents, a post-translational shaping is indicated in the human pineal gland. Another important mediator of circadian time and seasonality to the body is the pituitary gland. The aim of this work was to elucidate regulation of melatonin synthesis in the human pineal gland. Furthermore, presence and regulation of clock genes in the human pineal and pituitary gland, and in the SCN were analyzed. Therefore, human tissue, taken from regular autopsies, was analyzed simultaneously for different parameters involved in melatonin biosynthesis and circadian rhythm generation. Presented data demonstrate that post-mortem brain tissue can be used to detect the remnant profile of pre-mortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland. Together with data obtained for clock genes and their protein products in the pituitary, data presented here offer 1) a new working hypothesis for post-translational regulation of melatonin biosynthesis in the human pineal gland, and 2) a novel twist in the molecular competence of clock gene proteins, achieved by nucleo-cytoplasmic shuttling in neuronal and neuroendocrine human tissue. Furthermore, in this study, oscillations in abundance of clock gene proteins were demonstrated for the first time in the human SCN.