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Signal-dependent regulation of actin dynamics is essential for many cellular processes, including directional cell migration. In particular, cell migration is initiated by lamellipodia, actin-based protrusions of the plasma membrane. The formation of these protruding structures require incessant assembly and disassembly of actin filaments. The Arp2/3 complex and WAVE proteins are essential for both lamellipodium formation and its dynamics. WAVEs mediate the activation of the Arp2/3 complex downstream of the small GTPase Rac, thus being critical for Rac- and RTK-induced actin polymerization and cell migration. The WAVE-family proteins are always found associated with multiprotein complexes. The most abundant WAVE-based complex is referred to as the WANP (WAVE2-Abi-1-Nap1-PIR121) complex. IQGAP1 is a huge scaffolding protein with multiple protein-interacting domains. IQGAP1 participates in many fundamental activities, including regulation of the actin cytoskeleton, mitogenic, adhesive and migratory responses, as well as in cell polarity and cellular trafficking. IQGAP1 binds to N-WASP, thus raising the possibility that it might control actin nucleation by the Arp2/3 complex. In this study, IQGAP1 was found co-immunoprecipitated not only with WAVE, but also with the endogenous WANP-complex subunits. Correspondingly, IQGAP1 associated to both anti-WAVE and anti-Abi-1 immuno-complexes. Pull-down experiments proved that IQGAP1 binds directly to the WANP-complex subunits. Physical interaction between IQGAP1 and the reconstituted WANP complex could also be demonstrated. Together, these data indicate that IQGAP1 is an accessory component of the WANP complex. Interestingly, the IQGAP-WANP complex disassembled after either EGF stimulation or transfection with constitutively active Cdc42 and Rac1. HeLa cells devoid of IQGAP1 showed diminished and less persistent ruffling upon EGF, but not HGF, stimulation in comparison with the control. This phenotype was accompanied by a strong reduction in chemotaxis towards both growth factors, which was as dramatic as in WANP-complex knockdown (KD) cells. Moreover, GM130 and Giantin showed a polarized and flat ribbon-like pattern in control cells, as it is expected for cis- and cis/medial-Golgi markers. Conversely, small and dispersed vesicular structures were found in both IQGAP1 KD and WANP-complex KD cells. Importantly, Arp2/3-complex silencing resulted in the same phenotypes. Consistently, Brefeldin A-induced disassembly of the Golgi strongly inhibited the IQGAP1-WANP-complex interaction and chemotaxis towards EGF in wild-type cells. The re-expression of an RNAi-resistant wild-type IQGAP1 in IQGAP1 KD cells fully rescued both the ruffling abilities and Golgi structure. A constitutively active mutant, unable to bind to neither Rac1 /Cdc42 nor the WANP complex, could reconstitute only the former defect. Hence, this study shows that actin dynamics regulated by the IQGAP1-WANP complex controls Golgi-apparatus architecture and its contribution to cell chemotaxis. The working model here proposes that at the Golgi apparatus, recruitment of the WANP complex by IQGAP1 leads to the assembly of actin filaments required to maintain the appropriated Golgi morphology. The dissociation of the complex may be required to allow the remodeling of the Golgi membranes in order to respond following a chemoattractant gradient.
Succinate:quinone oxidoreductases (SQORs) are integral membrane protein complexes, which couple the two-electron oxidation of succinate to fumarate (succinate → fumarate + 2H+ + 2e-) to the two-electron reduction of quinone to quinol (quinone + 2H+ + 2e- → quinol) as well as catalyzing the opposite reaction, the reduction of fumarate by quinol. In mitochondria and some aerobic bacteria, succinate:ubiquinone reductase, also known as complex II of the aerobic respiratory chain or as succinate dehydrogenase from the tricarboxylic acid (TCA or Krebs) cycle, catalyzes the oxidation of succinate by ubiquinone, which is mildly exergonic under standart conditions and not directly associated with energy storage in the form of a transmembrane electrochemical proton potential (Δp). Gram-positive bacteria do not contain ubiquinone but rather menaquinone, a quinone with significantly lower oxidation-reduction (“redox”) midpoint potential. In these cases, the catalyzed oxidation of succinate by quinone is endergonic under standard conditions. Consequently, these bacteria face a thermodynamic problem in supporting the catalysis of this reaction in vivo. Based on experimental evidence obtained on whole cells and purified membranes, it had previously been proposed that the SQR from Gram-positive bacteria supports this reaction at the expense of the protonmotive force, Δp. Nonetheless, it has been argued that the observed Δp dependence is not associated specifically with the activity of SQR because the occurrence of artifacts in experiments with bacterial membranes and whole cells can not be fully excluded. Clearly, definitive insight into the mechanism of catalysis of this intriguing reaction required a corresponding functional characterization of an isolated, membranebound SQR from a Gram-positive bacterium. The first aim of the present work addresses the question if the general feasibility of the energetically uphill electron transfer from succinate to menaquinone is associated specifically to a single enzyme complex, the SQR. The prerequisite to achieve this goal was stable preparation of this enzyme.
In dieser Arbeit wurden die physiologische Funktion innerhalb der Ribosomenbiogenese und die physikalischen Interaktionen des nukleolären, essentiellen Proteins Nep1p in der Hefe Saccharomyces cerevisiae untersucht. Durch Hefe-Zwei-Hybrid-Experimente und biochemische Analysen konnte eine Homodimerisierung des Proteins festgestellt sowie eine strukturabgeleitete Dimerisierungsmutante identifiziert werden. Ebenfalls aus der Struktur des Nep1p-Homologs aus Methanocaldococcus jannaschii konnte eine Nop14p-Bindungsregion auf der der Dimerkontaktfläche abgewandten Seite des Hefeproteins vorhergesagt und nach in vitro-Mutagenese bestätigt werden. Innerhalb des Nop14-Proteins wurden zwei Domänen charakterisiert, die im Zwei-Hybrid-System mit Nep1p interagieren. Aus Strukturdaten in Kombination mit Hefe-Drei-Hybrid-Experimenten konnte die RNA-Bindungsregion an der Dimerkontaktfläche des Nep1-Proteins lokalisiert werden. In Drei-Hybrid-Selektionen wurden RNA-Sequenzen mit hoher Affinität zu dem M. jannaschii Nep1p identifiziert, die auf eine Bindung des Proteins bei Helix 35 der 16S rRNA schließen lassen. Aufgrund der hohen Konservierung dieser rRNA-Region ist eine Bindung des Hefeproteins an die 18S rRNA-Schleife von Nukleotid 1189-1196 sehr wahrscheinlich. Da Nep1p eine große Ähnlichkeit zu Proteinen der SPOUTFamilie von Methyltransferasen aufweist, war von einer rRNA-Methylierung im Verlauf der Ribosomenbiogenese als katalytische Funktion des Proteins auszugehen. Aus verschiedenen Drei-Hybrid-Experimenten zur RNA-Bindungungsspezifität ergab sich als mögliche Reaktion die N1-Methylierung des Nukleotids 1-Methyl-3-(3-Amino-3-Carboxypropyl)-Pseudouridin (m1acp3Y) 1191 der 18S rRNA. Durch eine spezifische radioaktive Markierung der acp-Gruppe konnte gezeigt werden, dass Nep1p keinen Einfluss auf die spätere Aminocarboxypropylmodifizierung hat. Diese findet auch bei einer Deletion der snoRNA35 statt, also auch an einem Uridin, und ist unabhängig von dem cytoplasmatischen Protein Tma20p. In RP-HPLC-Experimenten konnte nachgewiesen werden, dass die 18S rRNA einer Dnep1Dnop6-Doppelmutante ein Aminocarboxypropyl-modifiziertes Nukleosid enthält, dass sich in seinem Retensionsverhalten von dem m1acp3Y eines Wildtyps unterscheidet. Bei dem in diesem Stamm detektierten acp-modifizierten Nukleosid handelt es sich vermutlich um ein nicht-methyliertes acpY, was eine Funktion von Nep1p als N1-Methyltransferase des Nukleotids Y1191 der 18S rRNA höchst wahrscheinlich macht. Diese katalytische Funktion konnte in Zusammenarbeit mit Prof. Wöhnert auch für das M. jannaschii Nep1p gezeigt werden. Dass sowohl eine snr35- Deletion als auch eine 18S rRNA-Mutation des Nukleotids 1191 nicht letal sind, machte deutlich, dass die N1-Methylierung nicht die essentielle Funktion von Nep1p darstellen kann. Weiterhin konnte nachgewiesen werden, dass die Suppression der nep1-1ts-Mutante durch S-Adenosylmethionin nicht auf der Unterstützung der Methyltransferase-Aktivität des Proteins, sondern vermutlich eher auf einer generellen Stabilisierung des temperatursensitiven Proteins beruht. Sowohl im Hefe-Nep1p als auch im humanen Homolog wurden durch biochemische und genetische Experimente mehrere Phänotypen der Bowen-Conradi-Mutation (Aspartat 90 zu Glycin in ScNep1p) nachgewiesen. Diese lassen auf eine Aggregation des mutierten Proteins sowie eine dadurch bedingte Fehllokalisation innerhalb der Zelle schließen. Zusätzlich ist aber auch ein RNA-Bindungsdefekt durch den Aminosäureaustausch wahrscheinlich. Nichtsdestotrotz liegt offensichtlich ausreichend Nep1p-Protein vor, dass seine essentielle Funktion erfüllen kann, da die Mutation selbst zu keinem Wachstumsphänotyp führt. Erst bei einer partiellen Translationsrepression des mutierten Proteins unter Verwendung des artifiziellen Tetrazyklin-Aptamer-Systems ist ein verlangsamtes Wachstum von Hefezellen zu beobachten, was dieses System geeignet zur Analyse von möglichen Therapeutika macht.
The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGF/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK-binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGF1 as shown by ELISA and by the inhibitory effects of a neutralizing TGF antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGF type II receptor TGF-RII) we furthermore demonstrate a functional involvement of both types of TGF receptors. However, rapamycin did not compete with TGFfor TGF-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in Dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFSmad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin. Keywords: FK506 binding protein; p38 MAP kinase; rapamycin; renal fibrosis; Smads; TGFβ
The documentation of life on Earth, that is, the inventorization of nature and the naming and classification of organisms found therein, is a major task for biologists today and a fundamental precondition for nature conservation efforts. This study aimed at contributing to the inventory of amphibians and reptiles in selected, previously understudied ecoregions of Bolivia. I strove to document diversity patterns and seek possible ecological and historical reasons for these patterns. Special attention was paid to the Chiquitano Region situated in the eastern lowlands of Bolivia in a climatic transition zone between the humid evergreen Amazon Forests and the deciduous thorn-scrub vegetation of the Gran Chaco. In congruence with its location in the transition zone, the Chiquitano Region displays a mosaic of habitats: The vegetation is dominated by the endemic Chiquitano Dry Forest, which is probably the largest extant patch of Seasonal Dry Tropical Forest, with enclaves of savanna, the western outliers of the Cerrado biome of central Brazil. Taxonomic revisions: The taxonomic data in this study are used as a tool to measure biodiversity, to assess biogeographic relationships, and to evaluate conservation needs. Since all is predicated on the taxonomic decisions made, an adequate taxonomy is essential, and taxonomy can be regarded as the foundation of this study. The methodology encompassed a variety of herpetological field techniques, such as different survey methods, preparation and documentation of voucher specimens, recording of frog calls, and herpetological laboratory techniques, such as morphology, molecular procedures with mtDNA, phylogenetic analyses, and bioacoustic analysis and descriptions of frog calls. A total of 1251 specimens belonging to 200 species were obtained during this study, including 87 amphibian and 123 reptile species. This constitutes about 36% of the herpetofauna currently known for Bolivia, about 34% of the amphibians currently known for Bolivia and about 40% of the reptiles, respectively. In the course of this study, a new species of frog was described from the study site Caparu in the eastern lowlands of Bolivia; this species, Hydrolaetare caparu Jansen, Gonzales & G. Köhler 2007, differs from the other two congeners in external morphology (e.g., lateral fringes and relative length of fingers, size of palmar tubercle, webbing of toes, and colouration) and advertisement call. Two new colubrid snake species were also described from the study site San Sebastián. Thus far, both are known only from the Chiquitano Region, Provincia Ñuflo de Chávez. Phalotris sansebastiani Jansen & G. Köhler 2008 differs from all the other species in the genus in having a triangular projection of the red snout colouration reaching onto the parietals. Xenopholis werdingorum Jansen, Gonzales & G. Köhler 2009 can be identified as a member of the genus Xenopholis by its vertebral morphology. It differs from the other two species of Xenopholis in having a unique uniform dorsal colour pattern, and from X. scalaris in having two prefrontals and a narrow septum within the neural spine and perpendicular to its long axis as evident in the x-ray images. A review of a small collection of pitvipers from different lowland localities and from the Inter-Andean dry valleys of the region of Pampagrande revealed one new species of Bothrops and one of Bothrocophias (both to be formally described elsewhere). The two pitviper species differ morphologically and genetically from their congeners. The results of a brief review of a small collection of frogs of the genus Scinax (Anura: Hylidae) from different localities in the lowlands, together with analyses of their bioacoustics, suggest an unknown cryptic diversity in Bolivian species of Scinax cf. fuscomarginatus and allies. However, further studies are necessary to clarify the taxonomic status of these populations. In addition, this study provides new data on the morphology (e.g., pholidosis) of snakes, many of them previously known only from few museum specimens. Keys to the Bolivian lizard species of Cercosaura and the Bolivian snake species of Chironius, Clelia, Liophis, Lystrophis, Phalotris, and Xenodon are presented here for the first time. New information on distribution includes many range extensions of amphibian and reptile species, such as five new country records (one frog species, four snake species) and six new departmental records (two frog species, four snake species). Observations on ecology and natural history: Several observations on ecology and natural history were made during field work. Visual signaling, an aspect of territorial behavior that was already known for several species of the genus Phyllomedusa, could be described for the first time for Phyllomedusa boliviana (Jansen & J. Köhler 2007). Furthermore, during audio surveys of an anuran community at the study site San Sebastián from 2005 to 2007, a decline of certain amphibian populations was observed in the rainy season 2006/2007 (Jansen et al., in press). This is possibly related to an extreme drought in the dry season of 2006 where 158 consecutive days without rainfall were recorded. In addition, a new method for measuring intensity of anuran choruses by means of a continuous sound pressure metre was developed (Jansen 2009). The method was suitable to detect calling phenology (during one night), as well as differences in calling activity (between two nights). Biodiversity and biogeographical relationships: Species lists were compiled at the six study sites Pampagrande, Los Volcanes, San Sebastián, Caparú, El Espinal und El Corbalan. The total amphibian and reptile species numbers observed ranged from 37 to 101 with the highest species numbers in San Sebastián (101) and Caparú (89) and the lowest in Los Volcanes (37) and El Espinal (41). A preliminary species list of the herpetofauna of the Chiquitano Region was presented, including 60 amphibian and 84 reptile species. The majority of the amphibians of the Chiquitano Region are classified predominantly as inhabitants of open formations (41 species, 68.3%). Interestingly, even the majority of species recorded from the Chiquitano Dry Forest (32 species) are usually associated with open formations (22 species, 66.7%), followed by the number of species associated with open and forest formations (8 species, 24.4%). Only two of the observed species (6.0%) are predominant forest dwellers. The amphibian assemblage of the Chiquitano Region is most similar in composition to that of the Cerrado biome: 46 species (76.7%) occur in the Cerrado as well, and three species are regarded as Cerrado endemics (5.0%). The Chiquitano Region shares considerably fewer amphibian species with the other biomes (Amazon: 22 species, 36.7%; Gran Chaco: 13 species, 21.7%; Caatinga: 16 species, 26.7%). The reptile assemblage also has significant affinities to the Cerrado, which can be seen in the high proportion of reptile species distributed in that biome (68 species; 81.0%). Affinities to the other biomes are as follows: Amazon (48 species, 57.1%), Chaco (37 species, 40.1%), and Caatinga (30 species, 35.7%). When arranged in mutually exclusive biome categories, reptiles and amphibians showed similar patterns so that the majority of both amphibians and reptiles of the Chiquitano Region can be regarded as widespread. The high proportion of reptile species probably endemic to this region (5 species, 6.0%) is remarkable (i.e. Tropidurus xanthochilus, Apostolepis phillipsi, Phalotris sansebastiani, Xenopholis werdingorum, and Micrurus diana). In an analysis of the biodiversity patterns and biogeographical relationships of the herpetofauna of the study sites, these sites were compared with literature data from 37 localities and included in a presence/absence matrix with a total of 657 amphibian and reptile species in the surrounding South American biomes Amazon, Cerrado and Gran Chaco. The biogeographic relationships between these sites were evaluated using the Coefficient of Biogeographic Resemblance (CBR), cluster analysis, and multidimensional scaling (MDS) of sites. The analyses were first conducted on amphibians and reptiles combined, and than group-specific each for amphibians, reptiles, lizards, and snakes, separately. A “bias-reduced analysis” was developed for a better understanding of the affinities of the amphibians. In this analysis, e.g., the distinct habitat types of the Chiquitano Region, the Chiquitano Dry Forest and the Cerrado were taken into account. Analyses of the biodiversity patterns revealed that the sites in the Amazon comprise highest species numbers, as expected, followed successively by the sites in the Cerrado biome and sites in-between the two biomes. Within the eastern lowlands of Bolivia, the Chiquitano Region is the most rich in species. Comparing it with the other South American sites, the Chiquitano Region has a surprisingly high alpha diversity, especially in amphibians. The microgeographic variation in species composition (beta diversity) in the Chiquitano Region is also remarkably high and obviously related to the mosaic character of the vegetation and habitats. However, the bias-reduced analysis revealed that the amphibian fauna of the open areas and savannas at Hacienda San Sebastián (with 36 species in the Cerrado and pastureland) was one of the most species-rich savanna sites known for amphibians in South America. Considering that the Hacienda San Sebastián site is only ca. 3300 ha (= 1.29 amphibian species per km2), this outcome is particularly suprising. The results of the analyses of the biogeographical relationships suggest that the herpetofauna of Bolivia’s lowlands, including the Beni, the Pantanal and the Chiquitano Region, is as distinct from the herpetofauna of the Gran Chaco, Amazon, and Cerrado as these biomes are from each other. The Chiquitano herpetofauna in particular represents a unique and well-defined herpetofaunal assemblage when compared to all surrounding localities and biomes. This is supported by high CBR-values, findings from the cluster analysis, as well as a clear separation of the Chiquitano sites in the MDS. Biogeographic relations exist in all the surrounding biomes, but are strongest to Cerrado, followed by the Amazon. This study strongly suggests that the Chiquitano herpetofauna is composite and has multiple affinities. This is congruent with a well-defined Chiquitano flora, avifauna and mammalian fauna, suggesting a similar history. The bias-reduced analysis revealed a more detailed picture of the biogeographic relations of the Chiquitano Region, especially the Chiquitano Dry Forest. I argue here that the Chiquitano Dry Forest herpetofauna is a “young”, and “former savanna herpetofauna”. Whereas the Chiquitano Dry Forest is rather poor in amphibian and reptile species, and endemics are lacking from this forest type, the isolated Cerrado enclaves are especially diverse in species and probably contain locally endemic species, such as Phalotris sansebastiani and Xenopholis werdingorum. The colonization of the young Chiquitano Dry Forest may have taken place from savannas by mainly open area species, and only briefly through the Amazon. The results emphasise the importance of bias-reduction in studies of biogeography, e.g., by using group-specific analyses or by taking into account criterias as area size and heterogeneity of compared sites. The different biogeographic patterns of reptiles and amphibians of the Andean valleys indicate a different history of these two groups. In regard to reptiles, dispersals and withdrawals into the valleys in warm humid and dry cool periods in the Pleistocene seem likely, supported by a relation between the valleys and the dry lowland (e.g., Chaco). However, it is more plausible that, during these climatic fluctuations, amphibians migrated to adjacent, more humid regions, such as Yungas. The study verified the known patterns of sister-species pairs in the Inter-Andean Dry Forest and the lowlands. Additionally, pairs of populations with slight differences in morphology were found in the valleys and in the lowlands (Cercosaura parkeri and Xenodon rhapdocephalus). Further studies must test the taxonomic status of these populations. The discovery of new species of Bothrops and Bothrocophias from the Andean valleys has several implications, and possible reasons for the high endemism in the dry valleys are discussed. Conservation and outlook: The high local alpha and beta diversity of the Chiquitano herpetofauna shows that this is a region of complex faunal interaction, which reflects the present heterogeneity of the region, but which is possibly also related to a complex geological and environmental history. The Chiquitano Region can be assessed as a region of distinct regional herpetofaunal diversity charaterised by small scale diversity patterns. It therefore merits recognition as a unique ecoregion, and conservation effort should be increased. Further research is necessary to solve the taxonomic problems addressed in this study. Moreover, future work should be directed towards the development and institution of longterm monitoring programs to evaluate the effects of climate change and changes in land-use on biodiversity, especially that of the Chiquitano Region.
Orthopoxviruses are large DNA viruses that replicate within the cytoplasm of infected cells encoding over a hundred different proteins. The orthopoxviral 68k ankyrin‐like protein (68k‐ank) is highly conserved among orthopoxviruses, and this study aimed at elucidating the function of 68k‐ank. The 68k‐ank protein is composed of four ankyrin repeats (ANK) and an F‐box‐like domain; both motifs are known proteinprotein interaction domains. The F‐box is found in cellular F‐box proteins (FBP), crucial components of cellular E3 ubiquitin (Ub) ligases. With yeast‐two‐hybrid screens and subsequent co‐immunoprecipitation analyses, it was possible to identify S‐phase kinase‐associated protein 1a (Skp1a) as a cellular counterpart of 68k‐ank via binding to the F‐box‐like domain. Additionally, Cullin‐1 was co‐precipitated, suggesting the formation of a viral‐cellular SCF E3 Ub ligase complex. Modified Vaccinia virus Ankara (MVA) ‐ being attenuated and unable to replicate in most mammalian cell lines due to a block in morphogenesis – nevertheless, expresses its complete genetic information attributing to its properties as promising vector vaccine. Conservation of 68k‐ank as the only ANK protein encoded by MVA implied a substantial role of this viral factor. Hence, its function in the viral life cycle was assessed by studying a 68k‐ank knock‐out MVA. A mutant phenotype manifested in nonpermissive mammalian cells characterized by a block succeeding viral early gene expression and by a reduced ability of the virus to shutoff host protein synthesis. Studies with MVA encoding a 68k‐ank F‐box‐like domain truncated protein revealed that viral‐cellular SCF complex formation and maintenance of viral gene expression are two distinct, unrelated functions fulfilled by 68k‐ank. Moreover, K1, a well‐described VACV host range factor of the ANK protein family, is able to complement 68k‐ank function. This suggests that gene expression of MVA putatively depends on the ANKs encoded in 68k‐ank. In addition to the important findings in vitro, first virulence studies with the mouse pox agent, ectromelia virus (ECTV) deleted of the 68k‐ank ortholog (C11) suggested that this factor contributes to ECTV virulence in vivo.
Gepaarte assoziative Magnetstimulation (PAS) kann im primären menschlichen Motorkortex (M1) sowohl langzeitpotenzierungs- (LTP) als auch langzeitdepressionsähnliche (LTD) Erregbarkeitsveränderungen hervorrufen. Dies kann durch die Untersuchung magnetisch evozierter Potentiale (MEP) erfasst werden. Dagegen ist wenig über die Auswirkungen von PAS auf willkürliche Aktivität des motorischen Kortex bekannt. Im ersten Experiment haben wir bewegungsabhängige kortikale Potentiale (MRCP) bei zehn gesunden Probanden im EEG registriert, um die willkürliche Aktivität im Motorkortex während der Vorbereitung zweier motorischer Aufgaben zu erfassen. Die Probanden mussten dabei entweder den Daumen abduzieren (Hauptmuskel: Musculus abductor pollicis brevis, APB) oder das Handgelenk strecken (Hauptmuskel: Musculus extensor carpi radialis, ECR). Die Amplituden der motorisch evozierten Potentiale im APB wurden dabei durch PASLTP gesteigert, durch PASLTD vermindert und blieben bei PAScontrol unverändert. Im Gegensatz dazu wurden sie im ECR durch keine PAS-Bedingungen verändert. PASLTP verminderte die Negativität der MRCP während des späten Bereitschaftspotentials (-500 bis 0 ms vor Bewegungsbeginn) nur in der APB-Aufgabe. Diese Veränderungen zeigten sich hauptsächlich über zentralen Elektroden kontralateral zur bewegten Hand. Dieser Effekt korrelierte negativ mit dem durch PASLTP induzierten MEP-Anstieg im APB. PASLTD und PAScontrol hatten dagegen keinen Einfluss auf die MRCP Amplituden. Unsere Ergebnisse deuten auf eine spezifische Wechselwirkung von PAS mit willkürlicher Aktivität im Motorkortex während der Vorbereitung motorischer Aufgaben hin. Dies könnte durch ein Zusammenspiel aus erhöhter Exzitabilität von M1 und einer unterbrochenen effektiven Konnektivität zwischen prämotorischen Arealen und M1 erklärt werden. Die Modulation des dorsolateralen prämotorischen Kortex (PMd) durch repetitive transkranielle Magnetstimulation (rTMS) verändert die kortikospinale Erregbarkeit in M1. Die Auswirkungen von PMd-rTMS auf vorbereitende Prozesse für willkürliche Bewegungsabläufe sind jedoch unklar. Contingent negative variation (CNV) repräsentiert im EEG kortikale Vorbereitungsprozesse äußerlich getriggerter Bewegungen während das Bereitschaftspotential (BP) Vorbereitungsprozesse intern getriggerter Bewegungen repräsentiert. Im zweiten Experiment wurden CNV und BP jeweils vor und nach PMd-rTMS untersucht. Das Experiment bestand aus drei CNV-Versuchsblöcken mit insgesamt 243 Durchgängen. Dabei mussten die Probanden auf visuelle Anweisung hin eine zwei-Item Finger-Bewegungssequenz durchführen. RTMS wurde sowohl mit 1 Hz als auch mit 5 Hz bei einer Intensität von 110% der aktiven motorischen Schwelle (AMT) unter individueller MR-Navigation appliziert. Die Erfassung des BP erfolgte während der Durchführung derselben motorischen Aufgaben, allerdings bekamen die Probanden keine Anweisungen. Die Durchschnittsamplituden der frühen und späten Komponente von CNV (CNV1:1500-500 ms vor dem Startsignal (S2); CNV: 500-0 ms vor S2) und der frühen und späten Komponente des BP (BP1: 1500-500 ms vor EMG Beginn; BP2: 500-0 ms vor EMG-Beginn) wurden quantitativ für 25 zentrale Elektrodenpositionen verglichen. CNV2 zeigte eine signifikante Bahnung über dem frontal-zentralen Bereich nach 1 Hz PMd-rTMS, blieb aber unverändert nach 5 Hz PMd-rTMS. CNV1, BP1 und BP2 blieben durch 1 Hz und 5 Hz PMd-rTMS unbeeinflusst. Diese Ergebnisse lassen vermuten, dass der dominante PMd eine wichtigere Rolle in der Vorbereitung extern getriggerter Bewegungen spielt, als dies bei intern getriggerten Bewegungen der Fall zu sein scheint. Die CNV2-Antwort könnte eine intensive Interaktion innerhalb des menschlichen motorischen Kontrollnetzwerks anzeigen, die möglicherweise auf kompensationsähnlichen Mechanismen beruht.
Paleoecology is the study of organismal interactions with the environment in the geological past. Organisms are influenced in their distribution and abundance by abiotic factors such as temperature and precipitation. A change in these factors, for example by major climatic shifts, would then affect the communities of organisms. Studying this hypothesized causal link between climatic and faunal change is especially interesting for the Plio-Pleistocene of East Africa due to the fact that our own ancestors also inhabited these regions. Both the Turkana basin in Kenya and the Lake Albert region in Uganda offer unique opportunities to investigate these paleoecological issues. Their late Miocene through Pleistocene deposits provide a very good record of climatic, vegetation and faunal change in East Africa (Pickford et al. 1993, Leakey et al. 1995, 1998, McDougall & Feibel 2003, Wynn 2004). This study focuses on the mammal family Bovidae as they are good indicator of vegetation and environment (e.g. Vrba 1980, 1995, Shipman & Harris 1988, Bobe & Eck 2001, Bobe & Behrensmeyer 2004, Bobe et al. 2007). Bovidae are quite species-rich and inhabit a wide range of habitats from tropical rain forests to deserts which predicates their array of morphological adaptations (ecovariables) to these environments. Diet is the ecovariable that is most to climate and thus habitat change. Therefore, the fossil Bovidae are especially suitable for reconstructing past environments. The objective of this thesis is to test the hypothesis that, from the late Miocene through the Holocene, Africa has experienced an overall increase in aridity and concomitant pulses of habitat change. The hypothesis predicts that increasing aridity causes a likewise growth in the abundance of taxa adapted to open arid environments. In particular, an increase in bovid grazers should be observed in combination with a decrease of bovid browsers. To test this hypothesis, I examine the fossil bovid communities from each stratigraphic member of Lake Turkana (Lothagam, Kanapoi, West Turkana and Koobi Fora) and Lake Albert (Nkondo-Kaiso region) and through a taxonomic and a functional perspective reconstruct the paleoenvironments and -climates from approximately 8 to 0.6 Ma. This study is the first to use taxonomic and ecomorphological data together to reconstruct the paleoenvironments of the Turkana basin and the Nkondo-Kaiso region of Lake Albert. In a first analysis, mesowear, as introduced by Fortelius & Solounias (2000), is used to gather information about the diet of bovids. As a result of my preliminary investigations on upper vs. lower molars of recent species, the sample of fossil bovid specimens from the Turkana basin and Lake Albert were found to be unsuitable to reveal a meaningful diet reconstruction. Therefore, the bovids are assigned to diet categories based on literature. For each member of the time period from 8.0 to 0.6 Ma, I provide a detailed characterization of the bovid fauna in terms of α- and β- diversity both on tribe and diet level based on presence-absence as well as for the Turkana basin on abundance data. Statistical comparisons between the fossil bovid communities and those in modern protected areas with known vegetation and climatic conditions have yielded modern analogues for each stratigraphic member. Following that I provide paleoclimatic conditions such as assumed mean annual temperature for each member. Based on abundance of diet categories in the bovid communities, the paleoclimate of the Turkana basin was in general cooler and considerably more humid during the late Miocene to the Pleistocene than today. The mean annual temperature at Lothagam is assumed as 22.2 °C, the annual precipitation as 685 mm for 8.0 – 6.54 Ma and 4.9 – 3.4 Ma. The intervening time period is characterized by a slightly lower mean annual temperature and precipitation (20.3 °C, 583 mm). From 4.17 to 4.07 Ma Kanapoi faced 21.3 °C and 592 mm rainfall. In the eastern part of the basin the climate was warmer and more humid (3.4 – 2.68 Ma: 26.2, 961 mm; 2.68 – 1.3 Ma: 27.1 °C, 935 mm) from 3.4 to 1.3 Ma than in the preceeding eras. In the western part, the climate became warmer and more humid ~500,000 years later and was more variable than that in the eastern basin. From 2.94 to 2.52 Ma the mean annual temperature was 26.2 °C and the annual precipitation 961 mm. Between 2.34 and 1.6 Ma the climate again cooled and became drier as before 2.94 Ma. A second shift to higher temperature and precipitation occurred after 1.6 Ma (27.1 °C, 935 mm) lasted until 1.34 Ma. The results of the bovid community analyses do not support the hypothesis of increasing aridity in Eastern Africa during the late Mio- to Pleistocene. Instead, the results show that the bovid communities differed much over time and on a relatively small spatial scale. Regional paleovegetation and paleoclimate exhibit fluctuations through the studied time period at western Turkana and differences between the western and eastern part of the Turkana basin. This is indicative of a patchy habitat distribution both on temporal and spatial levels. Increased climate variability predicts an increase in landscape complexity as proposed by the ‘variability selection hypothesis’ (Potts 1998a+b). Therefore, this thesis research supports the hypothesis of increased landscape complexity on the spatial level. This study has important implications for future research. First, an analysis based on ecovariable characteristics such as diet may be preferred to a taxonomic analysis. Second, abundance data should be used for an ecovariable analysis because the results then provide more precise information on the paleovegetation and –climate than just the presence of these adaptations in the faunal community. Lastly, as this study is based on one mammal family, further studies on other mammal groups should be conducted to increase the database of exploited resource by the entire faunal community. Most significantly this study provides a basis for new interpretations of faunal community distributions. It also raises the question whether small scale spatial community variability is also to be expected at other fossil sites. If so then this methodology has important implications for reconstructions of paleovegetation and paleoclimate.
Die Brustdrüse (glandula mammaris) bietet ein einzigartiges Modellsystem zum Studium der adulten Stammzellen und der molekularen Signalwege, welche die Selbsterneuerung dieserZellen sowie die Proliferation und Differenzierung der Vorläuferzellen kontrollieren. Die Brustdrüse besteht aus dem Brustepithel und dem Stroma, das zum größten Teil aus dem Fettgewebe gebaut ist. Es enthält auch andere Zelltypen z. B. Fibroblasten und Makrophagen. Die Entwicklung der Brustdrüse findet hauptsächlich nach der Geburt, während der Pubertät, Schwangerschaft und Laktation statt. Ein funktionelles Brustepithel wird während der aufeinander folgenden Zyklen von Schwangerschaft, Laktation und Abstillen auf- und wieder abgebaut. Diese Regenerations-Kapazität kann für die Organrekonstitution genutzt werden. Die Transplantation der kleinen Anzahl von Brustepithelzellen oder des Drüsenfragments in das Fettgewebe einer Empfängermaus, deren eigenes Brustepithel entfernt wurde (cleared fat pad), führt zur vollständigen Epithelregeneration. Die zyklische Entwicklung und Regenerations-Fähigkeit des Epithelgewebes lässt auf die Existenz von Stammzellen schließen, die im Verbund der Epithelzellen überdauern. Diese gewebespezifischen Stammzellen sind in der Lage sich durch asymmetrische Zellteilung zu erneuern (self-renewal) und gleichzeitig die differenzierenden Vorläuferzellen zu bilden. Die während der Pubertät und Schwangerschaft erhöhten systemischen Hormone, lokalen Wachstumsfaktoren und Zytokine kontrollieren die Stammzellen-Proliferation und die Differenzierung der Vorläuferzellen in den verschiedenen Brustepithel-Zelllinien: Myoepithel-, Luminal- und Alveolarzellen. Aufgrund der Tatsache, dass die Entstehung von Brustkrebs mit aberranten Proliferations- und Differenzierungsprogrammen in malignanten Stamm-/ Vorläuferzellen (cancer stem cells) einhergeht, ist die Identifizierung der Signalwege, die diese Prozesse regulieren, für die Stammzellen- und Krebs-Forschung sehr bedeutend. Basierend auf diesen Erkenntnissen wurden im Rahmen des vorliegenden Projektes die Methoden zur genetischen Manipulation von nicht-angereicherten Brustdrüsen-Stammzellen entwickelt. Durch effiziente lentivirale Transduktion von adhärenten Primärzellen wurden nahezu 90% der Zellen, einschließlich der Stammzellen, transduziert. Diese Optimierung erfolgte durch 1) die Anwendung von konzentrierten Lentiviren mit hoher Qualität, 2) Passagierung der Primärzellen und Entfernung von Gewebeklumpen von den VIII Primärzellkulturen, und besonders 3) durch die Reduzierung der Zelldichte während der viralen Transduktion. Für Brust-Stammzellen sind keine spezifischen Oberflächen-Marker bekannt und daher ist ihre Isolierung deutlich erschwert. Man konnte sie bis jetzt nur anhand der moderaten Expression von CD24 (hitzestabilen Antigen) und hoher Expression von CD49f oder CD29 (α6- oder β1-Integrin) ungefähr 10-fach anreichern. Allerdings haben andere Studien gezeigt, dass die Transplantation der FACS-sortierten Stammzellen zu einer Schädigung der Stammzellen und folglich zu einer Reduktion der Repopulation-Frequenz führen kann. Aus diesem Grund wurden die genetisch modifizierten Stammzellen nicht sortiert. Durch die Transplantationen der transduzierten Primärzellen wurde ihr Stammzellen-Anteil in ihrer natürlichen Nische (cleared fat pad) selektiert. Die transplantierten Stammzellen sind in der Lage duktale Auswüchse zu entwickeln. Mit dieser Strategie konnten Transplantate mit homogener Expression von Fluoreszenz-Markergenen, wie z. B. GFP, erzielt werden. FACS Analysen der Zellen, die aus Transplantaten isoliert werden, haben gezeigt, dass alle drei Brustepithelzell-Populationen, nämlich Luminal-, Basal- und Stammzellen, transduziert waren und GFP exprimierten und daher aus transduzierten Zellen hervor gingen. Die Transplantationen einer Mischung der unterschiedlich fluoreszenzmarkierten Stammzellen ergaben einzelne verzweigte Auswüchse, in denen jeweils nur ein Fluoreszenz-Markerprotein exprimiert wurde. Sie stammen sehr wahrscheinlich von einzelnen transduzierten Stammzellen ab und wachsen jeweils in einem begrenzten Bereich des Brustfettgewebes aus. Die Immun-Antwort der Empfängermäuse gegen Fluoreszenz- Markerproteine könnte das Auswachsen der Transplantate inhibieren. Brustepithelium-Rekonstitutionen waren daher in den Rag2-/-γc-/- Empfängermäusen mit geschwächtem Immunsystem besonders effiziert. Die lentivirale Manipulation von Stammzellen und deren Einsatz in Brustepithelium-Rekonstitutionen kann als alternative Methode zur gewebsspezifischen Knockout-Technik angesehen werden. Für die Etablierung dieser Methode wurde im Rahmen dieser Arbeit ein zentraler Transkriptionsfaktor in der Brustentwicklung, signal transducer and activator of transcription 5 (Stat5), untersucht. (...)
Bioaktive Sphingolipide spielen eine wichtige Rolle bei der Regulation vieler entscheidender Prozesse in Endothelzellen. Speziell dem S1P wird eine Schlüsselfunktion in der Regulation der Proliferation und Migration von Endothelzellen beigemessen. Diese Prozesse sind notwendige Teilschritte in der Angiogenese, welche eine wichtige biologische und medizinische Bedeutung hat. So ist die umorangiogenese eine Voraussetzung für die adäquate Versorgung des Tumors mit Sauerstoff und Nährstoffen und trägt des Weiteren zu dessen Aggressivität bei. Ein weiteres Merkmal vieler solider Tumoren ist das Vorkommen hypoxischer Bereiche und erhöhter NO-Spiegel, die zudem, je nach Konzentration, als proangiogene Faktoren wirken können. Das Hauptziel dieser Arbeit lag in der Untersuchung des Einflusses hypoxischer Bedingungen und des Signalmoleküls NO auf das Sphingolipidgleichgewicht in der humanen Endothelzelllinie EA.hy 926. Ein spezieller Fokus wurde dabei auf die Regulation der S1P synthetisierenden Sphingosinkinasen (SK) und der daraus resultierenden Beeinflussung angiogener Zellantworten gelegt. In dieser Arbeit konnte gezeigt werden, dass sowohl Hypoxie als auch NO die Aktivität der SK-1, jedoch nicht der SK-2, langanhaltend erhöhten. Dieser Aktivitätsanstieg wurde durch eine Aktivierung des SK-1-Promotors mit einer nachfolgenden verstärkten mRNA- und Proteinexpression vermittelt. Dabei war unter Hypoxie der Transkriptionsfaktor HIF-1α der entscheidende SK-1 regulierende Faktor. Zusätzlich führte die durch Hypoxie ausgelöste Hochregulation der SK-1 zu einem Anstieg der zellulären S1P-Spiegel und zu einer Reduktion der Sphingosinkonzentration. Mechanistische Untersuchungen des Effekts von NO auf die SK-1 zeigten, dass dieser unabhängig von erhöhten cGMP-Konzentrationen aufgrund einer Aktivierung der löslichen Guanylatzyklase (sGC) durch NO war. Zum einen hatte die Verwendung des cGMP-Analogons 8-Bromo-cGMP und des Aktivators der sGC YC-1 keinen Einfluss auf die SK-1-Proteinexpression, und zum anderen hatte die Kostimulation mit dem sGC-Inhibitor ODQ keinen hemmenden Effekt auf die durch NO ausgelöste Hochregulation der SK-1. Demgegenüber ist die klassische MAPK-Kaskade essenziell für die Wirkung von NO auf die SK-1, da der MEK-Inhibitor U0126 die erhöhte Proteinexpression der SK-1 verhindern konnte. Die Aktivierung des vorgeschalteten kleinen GTP-bindenden Proteins p21ras ist ein möglicher Mechanismus, über den NO die Aktivierung der MAPKKaskade verursachen könnte (Lander et al, 1995). In einem weiteren Teil der Arbeit wurde die funktionelle Konsequenz einer Regulation der SK-1 durch Hypoxie und NO in EA.hy 926 Zellen untersucht. Sowohl Hypoxie als auch NO wurden als pro-angiogene Faktoren beschrieben. Daher wurde anhand von Migrationsassays und in-vitro-Angiogeneseassays untersucht, ob die SK-1 bei diesen Zellantworten eine Rolle spielt. Die Depletion der SK-1 durch die Verwendung einer spezifischen siRNA oder mit Hilfe von lentiviralen shRNA-Konstrukten zeigte, dass die Hochregulation der SK-1 unter Hypoxie und NO essenziell für die erhöhte Migration der Endothelzellen ist. Zusammenfassend zeigen die Daten, dass hypoxische Bedingungen und NO zu einer Hochregulation der SK-1 in EA.hy 926 Zellen führten und eine erhöhte Migrationsrate dieser Zellen zur Folge hatten. Somit ist die SK-1 ein interessanter therapeutischer Ansatzpunkt zur Behandlung von Krankheiten, die mit einer pathologischen Angiogenese einhergehen, wie es im Tumorgeschehen der Fall ist.