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Many hominin species are best physically represented and understood by the sum of their dental morphologies. Generally, taxonomic affinities and evolutionary trends in development (ontogeny) and morphology (phylogeny) can be deduced from dental analyses. More specifically, the study of dental remains can yield a wealth of information on many facets of hominin evolution, life history, physiology and ecological adaptation; in short, the organisms paleobiomics. Functionally, teeth present information about dietary preferences, that is, the dietary niche in ecological context and, in turn, masticatory function. As the amount and types of information that can be gleaned from 2-dimensional tooth measurement exhaust themselves, 3-dimensional microscopic modeling and analysis presents a largely fertile ground for reexamination and reinterpretation of dental characteristics (Bromage et al., 2005). As such, a novel, non-destructive approach has been developed which combines the work of two established technologies (confocal microscopy and 3D modeling) adapted specifically for the purpose of mineralized tissue imaging. Through this method, 3D functional masticatory and therefore occlusal molar microwear is able to be visualized, quantified and comparatively analyzed to assess dietary preference in Javanese Homo erectus. This method differs from other microwear investigative techniques (defining 'pits'- vs- 'scratches', microtexture analysis etc.) in that it defines a molars masticatory microwear functional interactions in 3-dimensions as its baseline dataset for further interpretations and analyses. Due to poor specimen collection techniques employed during the first half of the 20th century, the very complex geologic nature of the Sangiran Dome and disagreements over its chronostratigraphy, only very few scientific works have addressed the Sangiran 7 (S7) Homo erectus molar collection (n=25) (e.g. Grine and Franzen, 1994; Kaifu, 2006). Grine and Franzen's (1994) work was a predominantly qualitative initial assessment of the specimens and identified five specimens that might better be ascribed to a fossil pongid rather than H. erectus. They also noted several molars to which tooth position (M1 or M2) was unable to be ascribed (Grine and Franzen, 1994). Kaifu (2006) comparatively examined crown sizes in several S7 molars.
The Sangiran 7 collection originates from two distinct geologic horizons: ten from the older Sangiran Formation (S7a, ~1.7 to 1.0mya) and fifteen from the younger, overlying Bapang Formation (S7b, ~1.0 to .7mya). During this million year period, Java was connected to the mainland during various glacio-eustatic low-stands in sea level. These mainland connections varied in size, extent, climatic condition and therefore in faunal and floral composition. As the S7 sample may be representative of the earliest Homo erectus migrants into Java and spans long durations of occupation, its investigation yields potential to understand the various influences climatic and ecogeographic fluctuations had on these populations. Since the sample consists only of teeth, an ecodietary approach has been deemed the most logical and appropriate investigative approach. Questions regarding the intra- and inter- S7 sample
relationships will also be addressed.
By comparing various aspects of the H. erectus dentition against that of hunter/ gatherer's (H/G) whose diet is known, functional dietary similarity can be directly correlated. Thus a comparative molar sample consisting of the below historic hunter/ gather's (n=63) has been included in order to assess H. erectus's diet in ecological context: Inuit (n=9), Pacific Northwest Tribes (n=11), Fuegians (n=11), Australian Aborigines (n=12) and Bushman (n=20). Methodologically, this approach produces a 3D facet microwear vector (fmv) signature for each molar which can then be compared for statistical similarity.
Microwear (and, as such, the fmv signatures) was defined by the regular, parallel striations found on specific cusp facets known to arise from patterned, directional masticatory movements. This differs significantly from post-mortem or taphonomic microwear which produces striations at irregular angles on multiple, non-masticatory surfaces (Peuch et al.1985, Teaford, 1988). A 'match value' is produced to determine the similarity of two molars fmv's. The 'match values' are ranked (high to low) and these rankings are used to statistically analyze and infer dietary preference: between Sangiran 7 (as an entire sample) compared against that of the historic hunter/ gatherer H. sapiens whose diet and ecogeography is known; within S7a and S7b and then among the S7 sample (eg. S7a-vs-S7b); whether the purported Pongo molars actually affiliate well with H. erectus, the hunter-gatherer's or if they demonstrate distinctly different fmv signatures altogether; whether fmv signatures are useful in distinguishing molars whose tooth position is in doubt (eg. M1 or M2).
When compared against individual H/G molars, the results show that Sangiran 7 H. erectus most closely correlates with Bushmen across all areas of fmv signature analysis. However, within broader dietary categories (yearly reliant on proteinaceous foods; seasonally reliant on proteinaceous foods; not reliant on proteinaceous foods), it was found that H. erectus most closely allied with the two hunter/ gatherer subpopulations associated with the 'Seasonally reliant on proteinaceous foods' (Australian Aboriginals and Pacific Northwest Tribes). There was also evidence for dietary change or specialization over time. As the environment changed during occupation by the earlier Sangiran to the later Bapang individuals, the dietary preference shifted from a focus on vegetative foods to a diet much more inclusive of proteinaceous resources.
These results are considered logical within the larger ecogeographic and chronostratigraphic context of the Sangiran Dome during the Pleistocene. However, a larger sample would be needed to confirm this. Although general dietary preferences can be drawn from this method, it is not possible at present to define specific foods consumed on a daily basis (eg. tubers or tortoise meat).
Out of the five specimens possibly allied with Pongo, S7-14 matched at the 'high' designation with a hunter/ gatherer, S7-62 matched 'moderately', S7-20 matched 'low' while the remaining two were not able to be matched with any other teeth for various reasons. Although designation to Pongo cannot be ruled on at this time using this method, it does demonstrate that at least two of the teeth correlate well with various hunter/ gatherer's who do not share dietary similarity with Pongo. This suggests their designation as Pongo should be more closely reevaluated. As for the four specimens whose tooth position was unsure, S7-14 matched 'highly' with 1st molars, S7-62 and S7-78 matched 'moderately' with 2nd and 1st molars respectively while S7-20 only matched at the 'low' designation. Although this approach is still exploratory, it adds another analytical tool for use in defining tooth position.
In sum, this method has demonstrated its usefulness in defining and functionally analyzing a novel 3D molar microwear dataset to interpret dietary preference. Future work would include a pan- H. erectus molar sample in order to illuminate broader populational, taxonomic and dietary correlations within and amoung all H. erectus specimens. A larger, more heterogenous historic H/G sample would also be included in order to provide a wider dietary comparative population. This method can be further extended to include and compare any and all hominins as well as any organism which produces micro wear upon it molars. Also, the data obtained and resultant fmv signature diagrams have the potential to be incorporated into 3D VR reconstructions of mandibular movement thus recreating mastication in extinct organisms and leading to more robust anatomical and physiological investigations especially when viewed in the context of larger environmental conditions or changes.
Das geographische Verbreitungsgebiet von Arten ist ein fundamentales Struktur gebendes Merkmal der biologischen Welt. Warum Arten so verteilt sind, wie sie sind ist seit langem eine der zentralen Fragen in Ökologie, Biogeographie und Evolution. Gegenwärtig verändern sich, im Wesentlichen als unbeabsichtigtes Nebenprodukt menschlicher ökonomischen Aktivitäten und Populationsdynamik, die geographischen Verbreitungsgebiete von Arten mit entscheidender Bedeutung für Land- und Forstwirtschaft, als Krankheitsvektoren oder als Teil der biologischen Systeme, die Ökosystemfunktionen bereitstellen. Daher ist es dringend notwendig, dass wir unser Verständnis über die Dynamiken, aus denen die geographische Verbreitung von Arten erwachsen, verbessern. Mit dieser Doktorarbeit versuche ich, in drei Untersuchungen zur Dynamik der Verbreitungsgebiete von Singvögeln einen Beitrag zu unserem in Entwicklung begriffenen Verständnis der multiplen Faktoren die Artverbreitungsgebiete beeinflussen, zu leisten.
1) Zu einem mechanistischeren Verständnis von Artmerkmalen und Verbreitungsgebietsgrößen: Ein wichtiger, ungelöster Fragenkomplex in der Makroökologie ist, die immense interspezifische Variation in der Größe geographischer Verbreitungsgebiete zu verstehen. Während man davon ausgeht, dass Artmerkmale wie Fekundität und Körpergröße einen Effekt auf Verbreitungsgebietsgrößen haben, fehlt ein allgemeines Verständnis davon, wie Verbreitungsgebietsgrößen von mehreren Merkmalen gemeinsam beeinflusst werden. Hier beurteilen wir den Effekt von Lebensgeschichtsmerkmalen (Fekundität, Ausbreitungsfähigkeit), ökologischen Merkmalen (Habitatnische, Nahrungsnische, Zugverhalten, Flexibilität im Zugverhalten) und morphologischen Merkmalen (Körpergröße) auf die globale Verbreitungsgebietsgröße von 165 europäischen Singvögeln. Wir identifizieren Hypothesen zur Beziehung von Artmerkmalen und Verbreitungsgebietsgrößen aus der Literatur und verwenden die Methodik der Pfadanalyse, um sie zu testen. Die Größe der globalen geographischen Verbreitungsgebiete europäischer Singvögel wurde von Lebensgeschichtsmerkmalen (Fekundidtät und Ausbreitungsfähigkeit), ökologischen Merkmalen (Habitatnischenbreite, Nahrungsnischenposition und Zugverhalten) und von Körpergröße beeinflusst. Artmerkmale beeinflussten Verbreitungsgebietsgrößen auf direktem und indirektem Weg. Insbesondere der Einfluss von Körpergröße war komplex mit positiven und negativen Effekten über verschiedene Pfade. Die Größe von Verbreitungsgebieten ist sehr wahrscheinlich auch von anderen Faktoren als von Artmerkmalen abhängig. Wir zeigen, dass es notwendig ist, den direkten und indirekten Einfluss einer Vielzahl von Merkmalen zu entwirren, um die Mechanismen, die makroökologische Beziehungen generieren, aufzuklären.
2) Konkurrenz und Ausbreitungsfähigkeit interagieren bei der Bestimmung der geographischen Verbreitung von Vögeln: Es ist weiterhin eine Herausforderung für Ökologie und Evolutionsbiologie, die Faktoren zu verstehen , die die geographische Verbreitung von Arten beeinflussen. Wir untersuchen wie Konkurrenz, Ausbreitungsfähigkeit, das Alter eines Taxons und Habitatverschiebungen seit dem letzten glazialen Maximum das Ausmaß beeinflussen, in dem Arten der Vogelgattung Sylvia in allen Gegenden mit geeigneten Umweltbedingungen vorkommen (d.h. range filling).
Wir haben range filling in der Vogelgattung Sylvia (Grasmücken) unter Verwendung von Boosted Regression Trees und Ridge-Regression quantifiziert. Mittels multipler Regression haben wir für die Effekte von intragenerischer Konkurrenz, Ausbreitungfähigkeit, Alter des Taxons und Habitatverschiebung seit dem letzten glazialen Maximum auf range filling getestet.
Grasmücken mit hoher Ausbreitungsfähigkeit zeigten höheres range filling, aber nur wenn Konkurrenz in Gebieten mit weniger geeignetem Habitat innerhalb ihres potentiellen Verbreitungsgebietes niedrig war. Das Alter eines Taxon und Habitatverschiebung seit dem letzten glazialen Maximum hatten keinen konsistenten Effekt. Wir zeigen, dass die Verbreitungsgebiete von Grasmücken mit hoher Wahrscheinlichkeit durch den simultanen, interaktiven Effekt von Konkurrenz und Ausbreitungsfähigkeit geformt werden. Wenn biotische Interaktionen wie Konkurrenz generell die Fähigkeit von Arten beeinflussen auf der kontinentalen Skala neue Gebiete zu kolonisieren, wird es eine Herausforderung sein, den Effekt von Klimawandel auf Biodiversität vorherzusagen.
3) Nischenverfügbarkeit in Zeit und Raum: Vogelzug der Grasmücken: Im Kontext neuer Fortschritte in der ökologischen Nischenmodellierung sind sowohl die Umwelt als auch die ökologische Nische einer Art als statische Entitäten behandelt und quantifiziert worden. In der Realität sind aber die Umwelt und die Nischenanforderungen einer Art auf einer Vielzahl von Skalen dynamisch. Wir schlagen ein konzeptionelles System vor das berücksichtigt, wie die realisierte Nische und geographische Verbreitung von Arten durch die entkoppelte raumzeitliche Verfügbarkeit unterschiedlicher Umweltbedingungen und durch Veränderungen der Nischenanforderungen über die Lebenszeit eines Organismus geformt werden. Das Testen von aus dem konzeptionellen System abgeleiteten Vorhersagen am Beispiel des Vogelzugs der Grasmücken ergab neue Erkenntnisse: Das Verfolgen der Klimanische im geographischen Raum war höchstwahrscheinlich nicht die treibende Kraft für Migration in der Gattung und steht potentiell im Konflikt mit dem Verfolgen der Landnutzungsnische. Die Nischen der Grasmücken waren während der Brutsaison schmaler, was zeigt, dass Nischenanforderungen zeitlich dynamisch sein können. Wir legen nahe, dass die Berücksichtigung dynamischer Umwelten und Nischenanforderungen zu einer entscheidenden Verbessserung unseres Verständnisses der treibenden Faktoren hinter der Bewegung von Organismen im Raum und der Dynamik ihrer Nischen und Verbreitungsgebiete führt.
Diatoms contribute largely to the total primary production of the ecosphere and are key players in global biogeochemical cycles. Their chloroplasts are surrounded by four membranes owing to their secondary endosymbiotic origin. Their thylakoids are arranged into three parallel bands and differentiation of thylakoid membranes into grana or stroma is not observed. The fucoxanthin chlorophyll a/c binding proteins act as the light harvesting proteins and play a role in photoprotection during excess light as well. The diatom genome encodes three different families of antenna proteins. Family I are the classical light harvesting proteins called "Lhcf". Family II are the red algae related Lhca-R1/2 proteins called "Lhcr" and family III are the photoprotective LI818 related proteins called "Lhcx".
All known Fcps have a molecular weight in the range of 17-23 kDa. They are membrane proteins and have shorter loops and termini compared to LHCs of higher plants and are therefore extremely hydrophobic. This makes the isolation of single specific Fcps using routine protein purification techniques difficult.
The purification of a specific Fcp containing complex has not been achieved so far and until this is done several questions concerning light harvesting antenna systems of diatoms cannot be answered. For e.g. Which proteins interact specifically? Are various Fcps differently pigmented? Which pigments interact with each other and how? Which proteins contribute to photosystem specific antenna systems? Can pure Fcps be reconstituted into crystals like LHCII proteins? In order to answer these questions specific Fcp containing complexes have to be purified. ...
Climate and subsequent environmental changes are regarded as one driver of species evolution. Against this background the present study investigates the evolutionary history of the mammalian family Bovidae (Cetartiodactyla, Mammalia), today the most species-rich family of large herbivores on the African continent. Temporal and spatial patterns in that group’s evolution are the focus of the present study and were investigated using methods and data deriving from multiple disciplines (palaeontology, genetics, climatology, conservation biology). The results serve as a validation of macroevolutionary hypotheses of species evolution.
A major proportion of African mammalian fossils can be assigned to that family. Due to their morphological adaptations, bovid species are highly indicative of their habitats. Hence, bovids are of great importance for paleontology. However, a strong taphonomic bias is present in the fossil record of bovids, favoring large and arid- adapted species. Molecular phylogenies of extant species and species distribution modelling combined with climate reconstructions can help to overcome these limitations.
A molecular phylogeny, based on the cytochrome b gene of 136 bovid species served as basis for analysis of temporal patterns. Divergence events were dated using the relaxed molecular clock approach. The tree was time calibrated at 30 nodes using information inferred from the fossil record. Lineage-Through-Time plots and the respective statistical analyses reveal detailed temporal patterns in the evolutionary history of tribes and groups combining arid- and humid-adapted tribes. The resulting pattern shows three distinct phases. Phase 1 (P1) is dominated by speciation events within the humid group, while the second phase (P2) is marked by a dominance of speciation within the arid group. The switch in diversification rates (BDS) from P1 to P2 is dated to 2.8 million years ago. The third phase (P3) shows low diversification rates for all groups, starting around 1.4 million year ago and culminates in a significantly reduced diversification rate for the complete family at 0.8 million years ago. Both transitions are contemporaneous with global climate changes and turnover events in fossil faunal communities.
To investigate the impact of climate changes onto the habitat availability within the last 3 million years and its putative influence on diversification rates, the species distribution modeling method was applied. For 85 African species and subspecies the climate niches were established and grouped into 5 climate-groups based on their climate preferences. For each group the available habitat for the period before and after the BDS was calculated on continental scale using reconstructed climate scenarios. To evaluate the modeled habitat distributions, regional analyses were performed in test areas surrounding well studied fossil sites (Laetoli, Olduvai, Chiwondo Beds, Lothagam, Koobi Fora, West Turkana, Swartkrans, Sterkfontain und Toros-Menalla). Habitat profiles (HP) permitted the comparison of the model based habitat reconstruction with the interpretations of classic paleontological reconstruction. The validity of the habitat modeling has been shown in particular for East African test areas. The reconstructions for the northern and southern fossil sites does not support the modeled habitats in these areas. Yet, the method of habitat- profiling may serve as suitable tool for environmental reconstruction of areas lacking sufficient paleontological material. A comparison of habitat availability before and after the BDS on continental scale identified a significant loss of habitat for humid adapted groups (7-22%) and habitat gain for arid adapted groups (19-173%). The climatically intermediate group experiences a tremendous gain of habitat (3366%). The greatest environmental change was modeled for East Africa, initiated by a progressive regional aridification.
In addition to the distribution modeling for past climate conditions, the geographical distribution was modeled for the future, i.e. for climate scenarios representing the years 2050 and 2080 under a putative climate change scenario (global surface warming). It was shown that in particular the arid groups have to expect a remarkable loss of habitat (41-76%), while a gain of available habitat can be expected for the humid adapted groups (114-577%). The climatically intermediate group suffers the strongest habitat loss (85%). Regions with locally stable climate conditions were detected and may serve as potential refugia and are already today known as Africa’s hot spots of biodiversity.
The results show a positive correlation of high diversification rates and increasing habitat availability. None of the tested speciation hypotheses taken alone explains the observations (e.g., Turnover-pulse Hypothesis, Relay Model). A major element in these hypotheses is the passive fragmentation of populations induced by unfavorable climate changes. In contrast, the Periodic Model (Grubb 1999) considers natural, periodically recurring climate changes and moreover, the active dispersal of individuals and resulting founder events. I added the effect of a superimposed directed climate trend – like the progressive aridification since the late Pliocene in Africa – which leads to a bias in the proportion and probability towards leading edge effects. This Directed Periodic Model explains the patterns found in the evolution of Bovidae.
The combination of a molecular phylogeny and species distribution modeling, together with information inferred from the fossil record, reveals remarkable temporal and spatial patterns in the evolution of bovids, and helps overcome the limitations of the fossil record. The present study highlights the importance of active dispersal and founder populations in speciation processes. A point widely unattended in speciation hypotheses. The fully dated molecular phylogeny is the most densely sampled tree for the family Bovidae to date and may serve as a framework for a connection of present and future population studies, permitting the connection of medium-scale with long- term effects induced by climate and environmental changes.
In the first part of this work, the development of a novel two-dimensional native gel electrophoretic system (2-D BN/hrCNE) is described. This new system simplifies proteomics and biochemical analysis of mega protein complexes that are dissociated into the constituent complexes during 2-D electrophoresis, thereby reducing the complexity of the system considerably. This technique is exceptionally well suited for the in-gel detection of fluorescence-labeled proteins and the identification of individual enzymes and protein complexes by specific in-gel assays on native gels.
In the second part, a new technique for the native immunoblotting of blue native gels (NIBN) was developed. This new technique allows for the identification of conformation-specific antibodies and the discrimination of antibodies recognizing linear epitopes of denatured proteins. Identification of conformation-specific antibodies is becoming increasingly important not only for the electron microscopic identification of native proteins but also for structural investigations in general. For this purpose, a commonly used protocol for Western blotting of blue native gels was modified in such a way that the native state of proteins and protein complexes was retained throughout the complete protocol. Instead of using the denaturing methanol in Western blotting protocols, mild detergents such as Tween 20, digitonin and Brij 35 were used for the obligatory removal of protein bound Coomassie-dye.
The detection of respiratory complex I by activity staining on the blot membrane demonstrated that all three non-ionic detergents preserved the native state of complex I. The native state of the enzyme on the blot membrane was also monitored and confirmed with the help of a set of conformation-specific antibodies. NIBN can be used as a simple alternative method to the demanding native ELISA to screen for conformation-specific antibodies for structural studies. Unlike the time consuming native ELISA, NIBN does not require introduction of appropriate affinity tags and purification of the target protein by chromatography. Thus, the NIBN technique is especially useful for microscale projects and for proteins not easily accessible to genetic manipulation.
The third part aimed at identification of the immediate protein interaction partners of Cox26, a hydrophobic protein that has been identified by our group as a novel component of yeast respiratory supercomplex. Multi-dimensional electrophoretic techniques were applied to identify non-covalent and covalent protein-protein interactions of Cox26. Three-dimensional electrophoresis (BNE/BNE/SDS-PAGE) gave both qualitative and quantitative information on covalent and non-covalent interactions of Cox26 and subunits of cytochrome c oxidase (complex IV), and showed that most of the Cox26 protein was non-covalently bound to the complex IV moiety of the respirasomes. Four-dimensional electrophoresis (BNE/BNE/SDS/SDS-PAGE) applying reducing and non-reducing conditions revealed that a minor fraction of Cox26 used a single cysteine residue in the center of a predicted transmembrane helix to form a disulfide bond with the Cox2 subunit of complex IV. A structural role of Cox26 protein in the assembly/stability of respiratory strings or patches has been suggested.
The last part of this work focused on the isolation and characterization of native and morphologically intact nucleoids from bovine heart mitochondria, since only a few studies on nucleoid organization and composition have been carried out on mammalian tissues. The nucleoids appeared as distinct bands (apparent mass around 30-36 MDa) in blue native-PAGE on large pore gels. The moderate variation in particle size seems to reflect variations in the binding of loosely nucleoid-associated components like respiratory chain complexes. The estimated 30-36 MDa mass of nucleoids on native gels suggested that each nucleoid contains one mtDNA molecule provided that nucleoids contains equal amounts of DNA, protein and RNA (Miyakawa et al., 1987).
Electron microscopic analysis of native nucleoids, which was performed by Dr. Karen Davies from the Max-Planck-Institute of Biophysics, Department of Structural Biology, Frankfurt, showed homogenous pool of particles with dimensions in 85x100 nm (in negative stain) and 100x150 nm (in cryo-tomography). Some of the nucleoids showed dumbbell-shape indicating dimerization of nucleoids. Recent EM and high-resolution light microscopy analysis of mammalian nucleoids have reported that nucleoids have a size of 70 nm in average. We also observed the same size of 70 nm in cryo-tomogramms when we applied harsher treatment of the native nucleoid particles with dimensions 100x150 nm. This observation is in agreement with published nucleoid sizes from both EM and high-resolution light microscopy, if we assume that native nucleoids have been dissociated under harsher treatment.
The protein composition of bovine heart mt-nucleoids was analyzed by a number of complementary approaches to identify low and highly abundant, easily dissociating and tightly bound proteins, and to rank the 90 most abundant mt-nucleoid proteins. Native and denaturing gel electrophoresis techniques were coupled to LC-MS/MS to achieve a comprehensive protein component analysis. Qualitative MS analysis of highly purified nucleoids identified more than 400 proteins, including well known nucleoid proteins such as mitochondrial transcription factor and mtDNA-binding protein (TFAM), mitochondrial single-stranded DNA-binding protein (mtSSB), mitochondrial DNA polymerase subunit gamma-2 (POLG2) and mitochondrial helicase C26H10ORF2 protein (Twinkle). These proteins were ranked according to Mascot scores, and sorted according to presumed functional properties. A large group of proteins involved in protein synthesis comprised an almost complete set of subunits of mitochondrial ribosomes suggesting that the nucleoids contained significant amounts of mitochondrial ribosomes. Identification of sixty six proteins from the oxidative phosphorylation (OXPHOS) system comprising around 100 proteins in total suggested that OXPHOS proteins are also associated with mt-nucleoids.
Interestingly, TFAM, described as a main mtDNA packaging factor in human and other mammalian cells, was not confirmed here as a major nucleoid component from bovine heart mitochondria. Fluorescence staining of protein spots on 2-D IEF/SDS gels clearly identified TFAM, but according to the stain intensity, this protein did not rank in the list of the 90 most abundant nucleoid proteins. Western blot analysis of sucrose gradient fractions revealed an enrichment of putative TFAM isoform in nucleoid fractions. Unexpectedly, the uncharacterized mitochondrial protein Es1 was identified as the most abundant nucleoid protein in bovine heart nucleoids instead. This implicates that nucleoid organization may differ between species and tissues. A functional characterization of Es1 is required to clarify its role in mammalian nucleoids.
Einfluss des Transkriptionsfaktors Tal1 auf die Osteoklastogenese durch Regulation von DC-STAMP
(2012)
Das menschliche Knochengewebe unterliegt einem ständigen Auf- und Abbau. Der Knochenumbau, die so genannte Knochenremodellierung, findet stetig statt und etwa 10 % des gesamten Knochengewebes werden innerhalb eines Jahres erneuert (Lerner UH, 2006). Während der Knochenremodellierung befindet sich die Zellaktivität der Knochenaufbauenden Osteoblasten und der Knochen-abbauenden Osteoklasten in einem empfindlichen Gleichgewicht (Karsenty G und Wagner EF, 2002; Teitelbaum SL, 2000).
Durch Störung des Gleichgewichts zwischen Osteoblasten und Osteoklasten kann es zu Knochen-assoziierten Krankheiten wie Osteoporose oder Osteopetrose kommen (Helfrich MH, 2003; Sambrook P und Cooper C, 2006). Osteoklasten sind multinukleäre Zellen, die in der Lage sind die Knochenmatrix zu resorbieren (Teitelbaum SL, 2000). Sie entstehen aus pluripotenten, hämatopoetischen Stammzellen durch Differenzierung und Zellfusion von Monozyten/Makrophagen-Vorläuferzellen (Menaa C et al., 2000, Yavropoulou MP und Yovos JG, 2008). Die Osteoklasten-Differenzierung wird hauptsächlich durch die Zytokine M-CSF (macrophage colony stimulating factor) und RANKL (receptor activator of nuclear factor k b ligand) induziert. Sie initiieren ein spezifisches Expressionsmuster Osteoklasten-spezifischer Gene und aktivieren die Zellfusion in Osteoklasten-Vorläuferzellen zur Bildung reifer Osteoklasten (Boyle WJ et al., 2003; Asagiri M und Takayanagi H, 2007). Die RANKL-vermittelte Induktion der Osteoklastogenese beruht auf der Initiierung eines streng regulierten Netzwerks aus Transkriptionsfaktoren (Yang X und Karsenty G, 2002). Einige Transkriptionsfaktoren, die während der Osteoklasten-Differenzierung induziert und exprimiert werden, sind nicht auf Osteoklasten beschränkt. Sie erfüllen auch Aufgaben in anderen hämatopoetischen Differenzierungsprozessen (Engel I und Murre C et al., 1999), so dass vermutlich die Kombination der Transkriptionsfaktoren entscheidend für die Osteoklastogenese ist.
Der basic helix-loop-helix-Transkriptionsfaktor Tal1 (T-cell acute lymphocytic leukemia 1, auch Scl1, stem cell leukemia 1) ist ein entscheidender Faktor in der primitiven und der definitiven Hämatopoese (Bloor AJ et al., 2002; Shivdasani RA et al., 1996). Die Expression von Tal1 konnte bisher in verschiedenen hämatopoetischen Zelllinien gezeigt werden, u.a. in monozytischen Zellen (Elefanty AG et al., 1998; Green AR et al., 1992; Pulford K et al., 1995; Dey S et al., 2010).
In der vorliegenden Arbeit wurde der Einfluss des Transkriptionsfaktors Tal1 in Monozyten und reifen Osteoklasten, vor allem in Bezug auf genregulatorische Prozesse während der Osteoklasten-Differenzierung, untersucht. Der Transkriptionsfaktor Tal1 wird in vitro und in vivo in Osteoklasten-Vorläuferzellen und reifen Osteoklasten exprimiert. Die Proteinexpression von Tal1 wird durch die Inkubation der Zellen mit RANKL induziert, jedoch wurde dies in Bezug auf die mRNA-Expression von Tal1 nicht beobachtet, so dass vermutlich eine posttranskriptionelle Regulation von Tal1 vorliegt.
Die Überexpression von Tal1 sorgte für eine Blockade der Differenzierung von Osteoklasten-Vorläuferzellen in reife Osteoklasten. Der Verlust von Tal1 in primären Monozyten/Makrophagen-Zellen führte zur veränderten Expression von über 1200 Genen, wobei jeweils etwa 600 Gene herauf- bzw. herabreguliert waren. Dies verdeutlicht, dass Tal1 sowohl an der Aktivierung als auch an der Reprimierung der Genexpression in Osteoklasten-Vorläuferzellen beteiligt ist. Die Liste der herabregulierten Gene beinhaltete u.a. das Osteoklasten-spezifische Enzym Acp5 (auch TRAP, tartrate resistant acid phosphatase), die Liste der herauf regulierten Gene beinhaltete u.a. DC-STAMP (dendritic cell specific transmembrane protein) und ATP6V0D2 (d2 isoform of vascuolar ATPase V0 domain), beide werden im Zusammenhang mit der Zellfusion während der Osteoklasten-Differenzierung beschrieben (Kim K et al., 2008; Kim T et al., 2010; Yagi M et al., 2005). Der Promotor von DC-STAMP beinhaltet mehrere potentielle Bindestellen für Tal1 und Osteoklastenspezifische Transkriptionsfaktoren. Es konnte gezeigt werden, dass Tal1, PU.1 und MITF im Bereich um 343 bp vor dem Transkriptionsstartpunkt des DC-STAMP-Promotors binden und dass Tal1 mit den Osteoklasten-spezifischen Transkriptionsfaktoren PU.1 und MITF interagiert. Der inhibitorische Effekt von Tal1 auf die Osteoklasten-Differenzierung kommt durch die Reprimierung der Aktivität der Osteoklasten-spezifischen Transkriptionsfaktoren PU.1 und MITF auf dem DC-STAMP-Promotor in Osteoklasten-Vorläuferzellen zustande. Während der Osteoklastogenese kommt es zu einer verringerten Tal1-Bindung auf dem DCSTAMP-Promotor, wodurch die Tal1-vermittelte Inhibierung der Expression aufgehoben wird.
Die Bindung von PU.1 und MITF auf dem Promotor von DC-STAMP nimmt während der Osteoklasten-Differenzierung zu. Die Expression von DC-STAMP wird im Verlauf der Osteoklastogenese induziert, wodurch es zur Zell-Zell-Fusion kommt.
Die Analyse des transkriptionellen Netzwerks, das die Fusion mononukleärer Zellen in reife Osteoklasten reguliert, vertieft das molekulare Verständnis der Osteoklasten-Differenzierung und kann zur Entwicklung neuer therapeutischer Ansätze beitragen, die in der Behandlung von Osteoporose, Riesenzelltumoren und anderen Osteoklastenassoziierten Krankheiten verwendet werden können.
Background: The genome of the carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii, Order: Dasyuromorphia), was sequenced in the hopes of finding a cure for or gaining a better understanding of the contagious devil facial tumor disease that is threatening the species’ survival. To better understand the Tasmanian devil genome, we screened it for transposable elements and investigated the dynamics of short interspersed element (SINE) retroposons.
Results: The temporal history of Tasmanian devil SINEs, elucidated using a transposition in transposition analysis, indicates that WSINE1, a CORE-SINE present in around 200,000 copies, is the most recently active element. Moreover, we discovered a new subtype of WSINE1 (WSINE1b) that comprises at least 90% of all Tasmanian devil WSINE1s. The frequencies of WSINE1 subtypes differ in the genomes of two of the other Australian marsupial orders. A co-segregation analysis indicated that at least 66 subfamilies of WSINE1 evolved during the evolution of Dasyuromorphia. Using a substitution rate derived from WSINE1 insertions, the ages of the subfamilies were estimated and correlated with a newly established phylogeny of Dasyuromorphia. Phylogenetic analyses and divergence time estimates of mitochondrial genome data indicate a rapid radiation of the Tasmanian devil and the closest relative the quolls (Dasyurus) around 14 million years ago.
Conclusions: The radiation and abundance of CORE-SINEs in marsupial genomes indicates that they may be a major player in the evolution of marsupials. It is evident that the early phases of evolution of the carnivorous marsupial order Dasyuromorphia was characterized by a burst of SINE activity. A correlation between a speciation event and a major burst of retroposon activity is for the first time shown in a marsupial genome.
Introduction: The involvement of platelets in various diseases has been increasingly recognized in the recent decades. This contribution is believed to involve platelet secretion and formation of reactive microparticles. Platelets contain two functionally important forms of vesicles, alpha and dense granules, which are secreted upon activation of platelets. Alpha granules incorporate larger molecules such as adhesive proteins, e.g. P-selectin, vWF and fibrinogen; chemokines like PF4 and RANTES and growth hormones like VEGF and PDGF are among the most important proteins attributed to the involvement of platelets in pathological conditions. In contrast, dense granules contain small molecules like ADP, ATP, serotonin and histamine, and they are more rapidly and completely secreted than alpha granules. Like in all secreting cells, regulated exocytosis in platelets is mediated by “zippering” of three different classes of SNARE proteins. The subtypes of these proteins found to be involved in platelet secretion are SNAP-23, syntaxin-2 and -4 and VAMP-3 and -8. Apart from SNARE proteins, other conserved proteins influencing exocytosis by e.g. acting on SNARE proteins have been described, one of the most important ones being Munc13. Platelets contribute to the progression of atherosclerosis by local deposition of inflammatory mediators like PF4, RANTES and CD40L, which leads to enhanced leukocyte recruitment and plaque formation. In 1865, Armand Trousseau first described the correlation between cancer and thrombotic events. Since the 1960s, an increasing number of studies have found an involvement of platelets also in the progression of cancer, especially in the formation of metastases. Platelets bind to circulating tumor cells and may shield them from NK cell attacks and shear stress. Platelets may also facilitate the interaction of tumor cells with other cell types and the vessel wall. Lastly, they may secrete molecules that influence the tumor cell phenotype and invasiveness.
Aims of this study: We sought to generate and describe genetically modified mouse lines with defective platelet secretion and to employ these mouse lines in murine models of atherosclerosis and tumor progression to study the role of platelet secretion under pathological in vivo conditions.
Results: Clostridial toxins cleave members of the SNARE protein family and can thus completely block exocytosis of neuronal and other cells. We generated three transgenic mouse lines expressing tetanus, botulinum-E or -C light chains and two transgenic mouse lines with dominant-negative mutations of SNAP-23 under the control of the platelet-specific PF4 promotor. None of these constructs was able to interfere with platelet secretion despite expression of the transgene. A functional null mutant of the only Munc13 isoform expressed in platelets, Munc13-4, showed complete lack of dense granule secretion, measured by ATP release, while alpha granule release as determined by PF4 and vWF secretion, was unaltered. Morphology, composition and adhesion of these platelets were also normal. Aggregation in response to U46619 and collagen and formation of large aggregates in flow chamber assays was attenuated. Munc13-4-deficient mice showed a severe defect in bleeding time and no formation of stable aggregates in FeCl3 thrombosis model. In response to B16 melanoma and LLC1 carcinoma cells, Munc13-4 KO platelets also showed complete abrogation of dense granule secretion, whereas alpha granule secretion and binding of platelets to tumor cells was unchanged. Interestingly, wild-type platelets, but not Munc13-4 KO platelets, enhanced transmigration of B16 and LLC1 cells through an endothelial cell layer. Exogenous ATP was able to mimic the effect of wild-type platelets and the ATP-degrading enzyme apyrase blocked platelet-mediated tumor cell transmigration. Platelets incubated with tumor cells secreted large amounts of ATP. Murine endothelial cells showed perturbed adherens junctions identified by irregular VE-cadherin staining and gap formation when incubated with supernatants from tumor cell-activated platelets as well as increased permeability under the same conditions. Addition of apyrase preserved normal endothelial morphology and function. In vivo, primary tumor growth and weight was comparable in wild-type and Munc13-4 KO mice upon B16 or LLC1 flank injection but formation of lung metastases was strongly reduced. Number, but not size of metastases was also reduced upon i.v. injection of B16 and LLC1 cells. We found P2Y2 and P2X4 receptors to be the most abundantly expressed endothelial metabotropic and ionotropic ATP receptors, respectively. Neither knock-down nor inhibition of P2X4 in endothelial cells influenced platelet-mediated transendothelial migration of B16 cells, but knock-down of P2Y2, for which no specific antagonist is available, strongly reduced plateletdependent tumor cell transmigration. When B16 melanoma cells were injected i.v. shortly after FITC-dextran (70 kDa) into wild-type mice, prominent leakage of FITC-dextran was observed three hours post-injection at extraluminal sites in the lung. In contrast, leakage into the lung parenchyma was at basal levels in Munc13-4 KO and P2Y2 KO mice after B16 cell injection. Marginal vascular leakage in Munc13-4 KO mice lacking platelet ATP secretion and in P2Y2 KO mice lacking the main endothelial ATP receptor correlated with strongly reduced extravasation of CFSE-labeled B16 melanoma cells 6 hours post-injection in these mice. Consistently, P2Y2 KO mice showed strongly reduced formation of metastases in the lung after i.v. injection of B16 or LLC1 tumor cells. Bone marrow-transplanted LDLR KO mice reconstituted with Munc13-4-deficient or wildtype bone marrow and subjected to 16 weeks of high fat diet showed no significant difference in atherosclerotic plaque formation in the aorta.
Discussion: We hereby provide a thorough analysis of a mouse line with an exclusive defect in platelet dense granule secretion, thus representing a unique genetic tool to study the role of dense granule secretion in various contexts without interfering with other platelet functions. We also provide evidence how extravasation of circulating tumor cells is facilitated by tumor cell-induced ATP release from platelets. This ATP release destabilizes endothelial barriers and facilitates tumor cell extravasation and formation of metastases in the target organ. Since metastasis is the leading cause of cancer death, pharmacological interference with endothelial P2Y2 receptor function may represent a promising therapeutic strategy.
Zelluläre Immuntherapien mit hochaufgereinigten allogenen NK-Zellen sind eine mögliche Therapieoption um den GvL/T-Effekt nach haploidenter SZT bei pädiatrischen Hochrisikopatienten mit malignen Erkrankungen zu verstärken. Im Rahmen der in Frankfurt a. M. laufenden klinischen Phase I/II NK-Zell-Studie wurden 16 pädiatrische Patienten sowohl mit unstimulierten (NK-DLI(unstim), 9 Patienten) als auch mit zehn Tage ex vivo IL-2 (1000 U/ml) stimulierten Spender-NK-Zellen (NK DLI(IL-2 stim), 9 Patienten) an den Tagen (+3), +40 und +100 nach haploidenter SZT behandelt. Bisher gibt es kaum Daten über den Verbleib der transfundierten Zellen und den Einfluss der NK-DLI Immuntherapie auf sowohl zelluläre als auch humorale Komponenten des Immunsystems der Patienten. Da die Patienten zudem nach haploidenter SZT zur GvHD-Prophylaxe das immunsuppressive Medikament Mycophenolat-Mofetil (MMF) erhalten, sind Untersuchungen zum Einfluss von MMF auf die Funktionalität der Spender-NK-Zellen von großem Interesse. In der vorliegenden Arbeit wurde mit Hilfe eines studienbegleitenden, nichtinvasiven in vivo Immunmonitorings erstmalig ein unterschiedlicher Einfluss von NK-DLI(unstim) im Vergleich zu NK-DLI(IL-2 stim) auf zelluläre Bestandteile und auf die Zytokin/Chemokin-Spiegel des peripheren Blutes der Patienten vor und 10 min, 1 h, 4 h und 24 h nach der Zelltherapie beschrieben. Mittels durchflusszytometrischen single platform Analysen konnten sowohl Spender-NK-Zellen als auch patienteneigene NK-Zellen phänotypisch und funktionell charakterisiert und unterschieden werden. So wurden neben einer gesteigerten zytotoxischen Aktivität IL-2 stimulierter NK-Zellen auch Unterschiede hinsichtlich der Expression des Oberflächenmoleküls CD56, des Aktivierungsmarkers CD69, des Natürlichen Zytotoxizitäts-Rezeptors (NCR) NKp44 und des Lymphknoten Homing Moleküls CD62L beobachtet. Des Weiteren führte die Applikation von NK-DLI(IL-2 stim) zu einer signifikanten Zellmigration in der peripheren Blutzirkulation der Patienten. Während sich die Zahl der neutrophilen Granulozyten innerhalb von 4 h im peripheren Blut vervierfachte wurde unmittelbar 10 min nach NK-DLI(IL-2 stim) ein massiver Zellverlust von eosinophilen Granulozyten, Monozyten, dendritischen Zellen und vor allem der NK-Zellen beobachtet. Eine ausgeprägtere Reduktion der immunregulatorischen CD56(bright)CD16(dim/-) NK-Zellen hatte zudem eine Verschiebung in der prozentualen Verteilung der NK-Zell-Subpopulationen zur Folge. In den folgenden 24 h normalisierten sich alle Zellzahlen wieder zu den Werten vor NK-DLI. Anhand der beschriebenen phänotypischen Unterscheidungsmerkmale konnte gezeigt werden, dass dabei nur patienteneigene NK-Zellen in die periphere Blutbahn zurückkehrten. Die Zellmigration war zudem in vivo von einem signifikanten Anstieg der proinflammatorisch- und chemotaktisch-wirkenden Zytokine/Chemokine IL-2, IL-6, IL-8, IFN-γ, MCP-1 und MIP-1β im peripheren Blut der Patienten 10 min nach NK-DLI(IL-2 stim) Applikation begleitet. Die Applikationen von NK-DLI(unstim) zeigten im Gegensatz dazu keine vergleichbaren Effekte. Ein weiteres Ziel dieser Arbeit war herauszufinden, ob eine Therapie mit dem Immunsuppressivum MMF nach haploidenter SZT die Funktionalität dieser hochaktivierten Spender-NK-Zellen beeinträchtigt und somit die Effektivität der Immuntherapie gefährdet. Es konnten Einschränkungen in der ex vivo Funktionalität der NK-Zellen durch therapeutisch relevante Konzentrationen (1 bis 10 µM) des aktiven Metaboliten Mycophenolsäure (MPA) gezeigt werden. Die MPA-Inkubation führte zu einer dosisabhängigen aber reversiblen Inhibition der IL-2 bedingten ex vivo NK-Zell-Proliferation. Auch die während des ex vivo Expansionsprozesses induzierte Zytokin/Chemokin-Sekretion wurde signifikant gehemmt. Eine 24-stündige MPA-Inkubation der IL-2 stimulierten NK-Zellen führte zudem zu einer eingeschränkten Mobilität der NK-Zellen. Dies korrelierte mit einer signifikant reduzierten zytotoxischen Aktivität gegen K-562 Tumorzellen, was jedoch nicht mit einer Oberflächenreduktion der NCRs und des NKG2D Rezeptors assoziiert war. Auch die durch die IL-2 Stimulation verursachte Hochregulierung der in Aktivierung und Migration involvierten Oberflächenmoleküle CD25, LFA-1, ICAM-1, CCR5, CXCR7, DNAM-1 und CD62L wurde durch MPA inhibiert. Im Gegensatz dazu hatte eine 24-stündige oder 4-tägige MPA-Behandlung bereits vorstimulierter NK-Zellen keinen Einfluss. Zudem konnte im Rahmen dieser Arbeit erstmalig gezeigt werden, dass die IL-2 induzierte intrazelluläre Signaltransduktion durch MPA beeinflusst wird. Dies äußerte sich in einer partiell bis vollständig inhibierten Phosphorylierung zentraler Signalmoleküle wie STAT-3, -4 und -5, der MAP-Kinase ERK1/2 und der Proteinkinase AKT. Dadurch wurden die durch IL-2 geförderten Zellprozesse wie das Überleben, die Proliferation und die zytotoxische Aktivität der NK-Zellen drastisch gehemmt. Dies steht vermutlich im Zusammenhang mit einer reduzierten Expression der IL-2R Untereinheit (CD25), wodurch die Ausbildung des hochaffinen IL-2-Rezeptors auf der Zelloberfläche verhindert wurde. Demzufolge scheint die eingeschränkte NK-Zell-Effektorfunktion Ursache einer durch MPA gestörten IL-2 Signaltransduktion zu sein. Zusammenfassend scheinen NK-DLI(IL-2 stim) durch die gesteigerte Zytotoxizität, die geringere Sensitivität gegenüber der MPA-Exposition sowie durch die mutmaßliche Extravasation der stimulierten NK-Zellen aus der peripheren Blutbahn einen Vorteil gegenüber NK-DLI(unstim) zu haben. Ob dies auch in einer Steigerung der Effektivität der Immuntherapie resultiert und damit die Prognose der Patienten verbessert werden kann, werden jedoch erst zukünftige Studien mit größeren Patientenkohorten abschließend zeigen können.
Savanna regions in West Africa are valuable cultural landscapes and provide a wide range of ecosystem services for human well-being and are frequently affected by human-induced disturbances. Aside from agricultural activities (crop production and animal husbandry), the harvesting of timber and non-timber forest products is crucial for household income, alimentation and medicinal purposes. Most indigenous woody species have undergone increasing anthropogenic pressure as social and economic conditions have changed dramatically during recent decades, resulting in further habitat fragmentation and increased disturbance severity. Human land use activities influence growth conditions for plants by altering various abiotic factors, such as light, nutrient availability and water supply. They are found to alter demographic parameters (e.g., germination, seedling and sapling growth, survival and mortality rates) of woody plant individuals and alter the structure and stability of populations. The degree of anthropogenic disturbance varies between land-cover types, distance to settlements, and protection status. In the context of land-use change, there is an urgent need to better understand and evaluate the impact of land-use on savanna vegetation, particularly on the population biology of common savanna woody species. A major conclusion to be drawn from this thesis is that land use influences savanna vegetation in a complex way and does not necessarily lead to a decline or loss of tree populations and species. It is rather that in a constantly changing landscape, as a result of human-induced disturbances, populations of ubiquitous and some common species can be stable over time. The abundance of some species tends to decline consistently, whereas others benefit from human disturbance. Moreover, the study provides an insight into the structure and dynamics of common, dominant and less dominant savanna woody plants in a communal and a protected area. There is a need for further basic studies to assess the impact of land use and ecological preferences of all species, including repeated density studies that look at survivorship and transition probabilities over a number of seasons as well as longterm in-situ experiments in settlement areas in order to better understand woody plant populations in settlement areas as the few remaining semi-natural sites are likely to decrease in the future. A challenge will be the development of strategies to protect species within a landscape under cultivation.