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SYCE3, ein neues Synaptonemalkomplexprotein: Expression, funktionelle Analyse und Bindungspartner
(2011)
Der Synaptonemalkomplex ist eine evolutionär hoch konservierte Struktur. Er wird spezifisch während der Prophase I der Meiose ausgebildet und ist essentiell für die Segregation der homologen Chromosomen während der Meiose und auch für die Entstehung genetischer Vielfalt. Der Synaptonemalkomplex ist eine proteinöse Struktur, deren Aufbau dem einer Leiter ähnelt. Dabei werden die Leiterholme als Lateralelemente bezeichnet. Sie bestehen unter anderem aus den Proteinen SYCP2 und SYCP3 und assoziieren mit dem Chromatin der homologen Chromosomen. Die Stufen der Leiter bestehen hingegen aus Transversalfilamenten, deren Hauptkomponente parallele Homodimere des meiosespezifische Proteins SYCP1 sind. Dabei wird ein SYCP1 Dimer mit seinem C-Terminus in den Lateralelementen verankert und kann über seine N-terminale Domäne eine schwache Interaktion mit der N-terminalen Domäne eines gegenüberliegenden SYCP1 Dimers eingehen. Um diese Bindung zu stabilisieren werden Proteine des Zentralelements des Synaptonemalkomplexes benötigt: Während SYCE1 durch seine Interaktion mit SYCP1 die N-terminale Assoziation zweier gegenüberliegender SYCP1 Dimere stabilisiert, verknüpfen die zwei anderen zentralelementspezifischen Proteine SYCE2 und Tex12 lateral benachbarte SYCP1 Filamente und breiten so das SYCP1 Netzwerk entlang der chromosomalen Achsen aus. Dieser Prozess wird als Synapse bezeichnet und stellt eines der Schlüsselereignisse der Meiose dar. Fehler während dieses Prozesses führen meist zu Aneuploidie der entstehenden Gameten oder zum Abbruch der Meiose und somit zu Infertilität des betroffenen Organismus. In dieser Arbeit wurde mit SYCE3 ein neues Protein des murinen Synaptonemalkomplexes charakterisiert. Es konnte gezeigt werden, dass SYCE3 meiosespezifisch in Männchen und Weibchen exprimiert wird und Bestandteil des Zentralelements des Synaptonemalkomplexes ist. Hierbei zeigt es dasselbe Verteilungsmuster wie SYCP1 und SYCE1 und kann mit beiden Proteinen interagieren. Eine zusätzliche Interaktion konnte zwischen SYCE3 und SYCE2 nachgewiesen werden. Durch Untersuchungen an entsprechenden Knockout Mausmodellen konnte in dieser Arbeit außerdem gezeigt werden, dass SYCE3 in Abwesenheit von SYCP1 nicht an die chromosomalen Achsen rekrutiert werden kann. Die Ausbildung der Lateralelemente und auch die Anwesenheit der anderen zentralelementspezifischen Proteine SYCE1 und SYCE2 sind hingegen für die Anlagerung von SYCE3 an die chromosomalen Achsen nicht essentiell. Somit steht SYCE3 hinsichtlich seiner Bedeutung für die Paarung und die Synapse der homologen Chromosomen hierarchisch offenbar über den bisher beschriebenen Zentralelementproteinen SYCE1, SYCE2 und Tex12. Die funktionelle Bedeutung von SYCE3 für die Synapse der homologen Chromosomen und für den korrekten Ablauf der homologen Rekombination wurde im Rahmen dieser Arbeit durch die Herstellung und die Charakterisierung einer Syce3-/- Maus detailliert untersucht: Dabei führte der Knockout von SYCE3 zur Infertilität in beiden Geschlechtern, die gleichzeitig mit einer signifikanten Reduktion der Größe der entsprechenden Hoden und Ovarien im Vergleich zum Wildtyp einherging. Weitere Untersuchungen ergaben zudem, dass es in Syce3 defizienten Tieren zu einem Abbruch der Meiose kommt. Dabei hatte das Fehlen von SYCE3 keinen Einfluss auf die Ausbildung der Axialelemente. Die Initiation der Synapse hingegen war sowohl in Oocyten als auch in Spermatocyten in Abwesenheit von SYCE3 stark gestört. Darüber hinaus konnte in der vorliegenden Arbeit nachgewiesen werden, dass das Fehlen von SYCE3 Einfluss auf die homologe Rekombination nimmt: Zwar können sich frühe (DNA Doppelstrangbrüche) und intermediäre (Transitionsknoten) Rekombinationsereignisse in der Abwesenheit von SYCE3 ausbilden, die Prozessierung zu späten Rekombinationsstrukturen (Rekombinationsknoten) und die damit einhergehende Ausbildung von Crossing-over Strukturen fand jedoch nicht statt. Zusammengefasst wurde in dieser Arbeit gezeigt, dass das neue Synaptonemalkomplexprotein SYCE3 essentiell für die Fertilität von Mäusen ist. Durch den Knockout von Syce3 kann die Synapse zwischen den Homoligen nicht initiiert werden und es findet kein Crossing-over statt. Im Assembly Prozess des Synaptonemalkomplexes agiert SYCE3 oberhalb der anderen zentralelementspezifischen Proteine und unterhalb von SYCP1.
There is more and more evidence for the cancer stem cell hypothesis which believes that cancers are driven by a cellular subcomponent that has stem cell properties which is self-renewal, tumorigenicity and multilineage differentiation capacity. Cancer stem cells have been connected to the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. This hypothesis changes our conceptual approach of oncogenesis and shall have implications in breast cancer prevention, detection and treatment, especially in metastatic breast cancer for which no curative treatment exists. Given the specific stem cell features, novel therapeutic pathways can be targeted. Since the value of vaccinia virus as a vaccination virus against smallpox was discovered by E. Jenner at 18th century, it plays an important role in human medicine and molecular biology. After smallpox was successfully eradicated, vaccinia virus is mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The outstanding capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes which encode therapeutic or diagnostic proteins to be expressed when a tumor is infected. The emphasis in this study was the establishment of methods for the enrichment of human breast cancer stem-like cells from cancer cell lines and characterization of those cancer stem-like cells in vitro and in vivo. Furthermore, by using the Genelux Corporation vaccinia virus strain GLV-1h68, the isolated cancer stem-like cells can be targeted not only in vitro but also in vivo more efficiently. Side-population (SP) cells within cancers and cell lines are rare cell populations known to be enriched cancer stem-like cells. In this study, we used Hoechst 33342 staining and flow cytometry to identify SP cells from the human breast cancer cell lines MCF-7 and GI-101A as models for cancer stem-like cells. Considering the cytotoxicity of Hoechst dye and the restriction of instrument, we did not carry out further studies by this method. Utilizing in vitro and in vivo experimental systems, we showed that human breast cancer cell line GI-101A with aldehyde dehydrogenase activity (ALDH) have stemlike properties. Higher ALDH activity identifies the tumorigenic cell fraction which is capable of self-renewal and of generating tumors that could recapitulate the heterogeneity of the parental tumor. Furthermore, the cells with higher ALDH activity display significant resistance to chemotherapy and ionizing radiation, which proves their stem-like properties again. The cells which have higher ALDH activity also are more invasive compared to cells which have lower ALDH activity, which connects the cancer stem-like cells with cancer metastases. By analyzing the popular human breast cancer stem cells surface markers CD44, CD49f and CD24, it was discovered that the cells with higher ALDH activity have stronger CD44 and CD49f expression than in those cells with lower ALDH activity, which further confirms their stem-like properties. Finally, the cells with higher ALDH activity and lower ALDH activity were infected in vitro and used in virotherapy in a mouse xenograft model was performed. The results indicated that the vaccinia virus GLV-1h68 can replicate in cells with higher ALDH activity more efficiently than cells with lower ALDH activity. GLV-1h68 also can selectively target and eradicate the xenograft tumors which were derived from cells with higher ALDH activity. The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastases. EMT was induced in immortalized human mammary epithelial cells (HMLEs) and in GI-101A cells, which results in the acquisition of mesenchymal traits and in the expression of stem cell markers. Furthermore, the EMT-induced GI-101A cells showed resistance to chemotherapy and invasion capacity. CD44+/CD24- cells were enriched during the EMT induction. Following flow cytometry sorting by using CD44, CD24 and ESA surface marker, the sorted cells were tested in a mouse model regarding tumorigenicity. Unexpectedly, we found that CD44+/CD24+/ESA+ cells could initiate tumors more efficiently rather than CD44+/CD24-/ESA+ and other fractions in EMTinduced GI-101A cells. We also infected the CD44+/CD24+/ESA+ and CD44+/CD24- /ESA+ cells in vitro and performed virotherapy in a mouse xenograft model. The results indicated that the vaccinia virus GLV-1h68 is able to replicate in CD44+/CD24+/ESA+ cells more efficiently than in CD44+/CD24-/ESA+ cells. GLV-1h68 was also capable to selectively target and eradicate the xenograft tumors which derived from CD44+/CD24+/ESA+ cells. Moreover, CD44- cells have much lower tumorigenicity in the mouse model and CD44- cells derived-tumors are not responsive to vaccinia virotherapy. In summary, we have successfully established an in vitro and in vivo system for the identification, characterization and isolation of cancer stem-like cells from the human breast cancer cell line GI-101A by using the ALDEFLUOR assay. The vaccinia virus GLV-1h68 was able to efficiently target and eradicate the higher ALDH activity cells and tumors derived from those cells. Although contrary to the current assumption, CD44+/CD24+/ESA+ cells in the EMT-induced GI-101A cell line showed stem-like properties and GLV-1h68 was able to efficiently target and eradicate the CD44+/CD24+/ESA+ cells and tumors which derived from those cells. Finally, improved understanding of cancer stem cells may have tremendous relevance for how cancer should be treated. It is menacing that cancer stem cells are resistant to almost all anti-tumor approaches which have already been established for the treatment of metastatic diseases such as ionizing radiation, hormonal therapy, chemotherapy, and small molecular inhibitors. Therefore, it is promising that our results suggest that these cancer stem cells may be susceptible to treatment with oncolytic vaccinia virus.
Die Kernhülle umgibt als geschlossenes Membransystem einen jeden Zellkern und ist damit ein gemeinsames Merkmal aller eukaryotischen Zellen. Sie besteht aus einer inneren und einer äußeren Kernmembran sowie der nukleoplasmatischen Kernlamina, die aufgrund zahlreicher assoziierter Proteine in enger Wechselbeziehung mit der inneren Kernmembran steht. Neben der rein räumlichen Trennung nukleärer und zytoplasmatischer Strukturen hat die Kernhülle bedeutenden regulatorischen Einfluss auf die gesamte Zelle. So ist sie unter anderem an der Steuerung der genomischen Aktivität, an der nukleo- und zytoplasmatischen Signalübertragung und in hohem Maße an der Positionierung und Formerhaltung des Zellkerns beteiligt. Es mehren sich die Hinweise, dass die Kernhülle auch während der Gametogenese, der Differenzierung befruchtungsfähiger Keimzellen, eine zentrale Rolle einnimmt und folglich auch mit bislang ungeklärten Ursachen humaner Infertilität in Kontext stehen könnte. Um die Bedeutung der Kernhülle für die Keimbahn der Säuger generell besser verstehen zu können, wurden in dieser Arbeit ausgewählte Bestandteile der Keimzellkernhülle untersucht. Dadurch sollte der Kenntnisstand erweitert werden, in welcher Weise die Kernhülle dynamische, morphologische und vor allem für die Keimbahn essentielle Prozesse beeinflusst; insbesondere während der meiotischen und der postmeiotischen Differenzierungsphase bei männlichen Mäusen. Im Mittelpunkt stand dabei einerseits Lamin C2, ein meiosespezifisches A-Typ Lamin, dessen Verlust zu einer schwer geschädigten Meiose und infolgedessen zu vollständiger männlicher Infertilität führt. Es zeigte sich, dass Lamin C2-defiziente männliche Mäuse schwerwiegende Defekte bei der Paarung und Synapsis der homologen Chromosomen in der meiotischen Prophase I aufweisen und aufgrund apoptotischer Spermatocyten keine reifen Spermien bilden können. Es wird angenommen, dass die Assoziation homologer Chromosomen bzw. die Abstoßung nicht-homologer durch gerichtete Telomerbewegungen entlang der Kernhüllenperipherie vorangetrieben bzw. verhindert wird. Da Lamin C2 seinerseits diese Wanderung der Telomere durch eine Flexibilisierung der Spermatocytenkernhülle vereinfachen soll, ist es durchaus vorstellbar, dass sein Verlust verlangsamte Telomerbewegungen, eine gestörte Homologenfindung und folglich Fehlpaarungen zur Folge hat. Ein weiteres zentrales Thema war die Erforschung potentieller LINC-Komplexe während der Differenzierungs- und morphologischen Umgestaltungsphase postmeiotischer Keimzellen. LINC-Komplexe sind kernhüllendurchspannende Proteingebilde aus SUN-Proteinen in der inneren und Nesprinen in der äußeren Kernmembran, die nukleäre Strukturen an das Zytoskelett binden. Da sie aufgrund dieser strukturellen Eigenschaft die Kernmorphologie beeinflussen können, erscheinen sie als äußerst geeignet, an der Formierung des Spermienkopfes beteiligt zu sein. Die detaillierte Untersuchung spermiogeneserelevanter LINC-Komplex-Bestandteile ergab, dass während der Spermiogenese tatsächlich zwei neue, strukturell einzigartige LINC-Komplexe gebildet werden, die darüber hinaus auf den entgegengesetzten Seiten differenzierender Spermatiden polarisieren. Da sie den Kern dort an jeweils spezielle Zytoskelettelemente binden könnten, wurde in dieser Arbeit das Modell der LINC-Komplex vermittelten Umformung des Spermienkopfes aufgestellt. Insgesamt trägt diese Arbeit durch die funktionelle Analyse von Lamin C2 und die Identifizierung neuer LINC-Komplexe dazu bei, die Wichtigkeit der Kernhülle für die Spermatogenese zu vertiefen und auszuweiten.
Molecular modelling and simulation are powerful methods in providing important in-formation on different biological systems to elucidate their structural and functional proper-ties, which cannot be determined in experiment. These methods are applied to analyse versa-tile biological systems: lipid membrane bilayers stabilized by an intercalated single wall carbon nanotube and retroviral proteins such as HIV protease and integrase. HIV-1 integrase has nuclear localization signals (NLS) which play a crucial role in nuclear import of viral preintegration complex (PIC). However, the detailed mechanisms of PIC formation and its nuclear transport are not known. Previously it was shown that NLSs bind to the cell transport machinery e.g. proteins of nuclear pore complex such as transportins. I investigated the interaction of this viral protein HIV-1 integrase with proteins of the nuclear pore complex such as transportin-SR2 (Shityakov et al., 2010). I showed that the transportin-SR2 in nuclear import is required due to its interaction with the HIV-1 integrase. I analyzed key domain interaction, and hydrogen bond formation in transportin-SR2. These results were discussed in comparison to other retroviral species such as foamy viruses to better understand this specific and efficient retroviral trafficking route. The retroviral nuclear import was next analyzed in experiments regarding the retroviral ability to infect nondividing cells. To accomplish the gene transfer task successfully, ret-roviruses must efficiently transduce different cell cultures at different phases of cell cycle. However, promising and safe foamy viral vectors used for gene transfer are unable to effi-ciently infect quiescent cells. This drawback was due to their inability to create a preintegra-tion complex (PIC) for nuclear import of retroviral DNA. On the contrary, the lentiviral vec-tors are not dependant on cell cycle. In the course of reverse transcription the polypurine tract (PPT) is believed to be crucial for PIC formation. In this thesis, I compared the transduction frequencies of PPT modified FV vectors with lentiviral vectors in nondividing and dividing alveolar basal epithelial cells from human adenocarcinoma (A549) by using molecular cloning, transfection and transduction techniques and several other methods. In contrast to lentiviral vectors, FV vectors were not able to effi-ciently transduce nondividing cell (Shityakov and Rethwilm, unpublished data). Despite the findings, which support the use of FV vectors as a safe and efficient alternative to lentiviral vectors, major limitation in terms of foamy-based retroviral vector gene transfer in quiescent cells still remains. Many attempts have been made recently to search for the potential molecules as pos-sible drug candidates to treat HIV infection for over decades now. These molecules can be retrieved from chemical libraries or can be designed on a computer screen and then synthe-sized in a laboratory. Most notably, one could use the computerized structure as a reference to determine the types of molecules that might block the enzyme. Such structure-based drug design strategies have the potential to save off years and millions of dollars compared to a more traditional trial-and-error drug development process. After the crystal structure of the HIV-encoded protease enzyme had been elucidated, computer-aided drug design played a pivotal role in the development of new compounds that inhibit this enzyme which is responsible for HIV maturation and infectivity. Promising repre-sentatives of these compounds have recently found their way to patients. Protease inhibitors show a powerful sustained suppression of HIV-1 replication, especially when used in combi-nation therapy regimens. However, these drugs are becoming less effective to more resistant HIV strains due to multiple mutations in the retroviral proteases. In computational drug design I used molecular modelling methods such as lead ex-pansion algorithm (Tripos®) to create a virtual library of compounds with different binding affinities to protease binding site. In addition, I heavily applied computer assisted combinato-rial chemistry approaches to design and optimize virtual libraries of protease inhibitors and performed in silico screening and pharmacophore-similarity scoring of these drug candidates. Further computational analyses revealed one unique compound with different protease bind-ing ability from the initial hit and its role for possible new class of protease inhibitors is dis-cussed (Shityakov and Dandekar, 2009). A number of atomistic models were developed to elucidate the nanotube behaviour in lipid bilayers. However, none of them provided useful information for CNT effect upon the lipid membrane bilayer for implementing all-atom models that will allow us to calculate the deviations of lipid molecules from CNT with atomistic precision. Unfortunately, the direct experimental investigation of nanotube behaviour in lipid bilayer remains quite a tricky prob-lem opening the door before the molecular simulation techniques. In this regard, more de-tailed multi-scale simulations are needed to clearly understand the stabilization characteristics of CNTs in hydrophobic environment. The phenomenon of an intercalated single-wall carbon nanotube in the center of lipid membrane was extensively studied and analyzed. The root mean square deviation and root mean square fluctuation functions were calculated in order to measure stability of lipid mem-branes. The results indicated that an intercalated carbon nanotube restrains the conformational freedom of adjacent lipids and hence has an impact on the membrane stabilization dynamics (Shityakov and Dandekar, 2011). On the other hand, different lipid membranes may have dissimilarities due to the differing abilities to create a bridge formation between the adherent lipid molecules. The results derived from this thesis will help to develop stable nanobiocom-posites for construction of novel biomaterials and delivery of various biomolecules for medi-cine and biology.
This work delves into the recently developed ‘Whedo’-aquaculture-system in the rural community of Malanville (North Benin)and aims on providing a closer insight on this – for the area--recent system including the ecological but also the sociological and economical aspects in order to develop this extensive traditional fishery to a more productive semi-intensive aquaculture system. With the retreat of the flood ‘Whedos’ usually become infested with numerous hydato-and tenagophytes, while the presence and density of the free-floating macrophytes were positively related to the nutrient content of the ‘Whedo’. Extensive plant infestation also affects water quality through the decomposition of organic material and its accumulation in thick mud layers on the pond bottom as well as through the nocturnal oxygen consumption. Unfavourable water quality, especially low dissolved oxygen as well as high conductivity and nitrite levels, was identified to be the main factor determining which fish species were able to survive the harsh conditions prevailing in the ‘Whedos’ during the dry season. With the deteriorating water quality with advancing dry season, fish diversity decreased significantly leaving only species that are highly adapted to such unfavourable conditions. The most abundant species were Clarias gariepinus, Heterotis niloticus, Oreochromis niloticus L., Hemichromis c.f. letourneauxi, Polypterus senegalus and Epiplatys spilargyreius. Besides, the investigations also concentrated on the fish diversity of the rivers Niger and Sota with the results that for three species distribution gaps could be closed and for further three species their already known distribution could be expanded. But otherwise it could also be detected that some economically important species that were abundant in the past. In regard to the ‘Whedo’-management, the investigations showed that the owners lack most of the knowledge on appropriate management strategies, e.g. the feeding and stocking regime. The exploitation period depends on the extent of the previous annual flood and the location of the ‘Whedo’ within the floodplain, but the main season is from February to April. The biomass harvested on a hectare basis separated for each of the ‘Whedos’ averaged 17 tons/ha in 2008 and 8.6 tons/ha in 2009. However, 72 percent of the total biomass of Clarias only had an average weight of 40 grams. Therefore, two separated feeding trials were conducted and in total 6 supplementary feeds were tested on Clarias gariepinus. Groundnut cake, fish trash, rice bran, blood meal and azolla meal were used in different combination and rations to formulate the experimental diets. Diet containing 19 percent blood meal resulted in the best economical benefits showing that the use of high quality feed ingredients such as groundnut cake is not recommendable because local fish prices are too low to compensate the additional feeding costs. Instead of high quality feed farmers should focus on ingredients that are free of charge and easy to process. The supplementation based on 19 percent blood meal resulted in the doubling of the net profit compared to the income based on feeding only rice bran, thus provided higher additional income, enhancing the livelihood of the fish farmers. Concluding, the ‘Whedo’-aquaculture system is still in its infancy but nevertheless is an attractive system for the rural population because of existing knowledge of post-flood wetland fisheries as well as the low investment needed for its installation. Additionally, the local fish supply will increase and hence not only contribute to a better provision of protein-rich food and reduced pressure on the wild fish stocks but might also prevent fish prices to increase in a way that the poor won’t be able anymore to afford their most important source of animal protein. But fish farmers need more knowledge on appropriate management strategies and thus should be provided with technical support to guarantee a successful development and not to discourage the owners as a consequence of avoidable failures. Furthermore, the use of supplementary feed offers a cheap and effective means to increase the biomass production and thus enhance the extensive fishery to a semi-intensive aquaculture system.
Spir proteins are the founding members of the novel class of WH2-actin nucleators. A C-terminal modified FYVE zinc finger motif is necessary to target Spir proteins towards intracellular membranes. The function and regulation of the Spir actin organizers at vesicular membranes is almost unknown. Live cell imaging analyses performed in this study show that Spir-2 is localized at tubular vesicles. Cytoplasmic Spir-2-associated vesicles branch and form protrusions, which can make contacts to the microtubule network, where the Spir-2 vesicles stretch and slide along the microtubule filaments. The analysis of living HeLa cells expressing eGFP-tagged Spir-2, Spir-2-ΔKIND and Spir-2-ΔKW (lacking the 4 WH2 domains and the KIND domain) showed Spir-2-associated tubular structures which differ in their length and motility. Throughout the course of that study it could be shown that the tail domain of the actin motor protein myosin Vb, as a force-generating molecule, is colocalizing and co-immunoprecipitating with Spir-2-ΔKW. By using the tail domain of myosin Vb as a dominant negative mutant for myosin Vb-dependent vesicle transport processes it could be shown that Spir-2-ΔKW/MyoVb-cc-tail- associated vesicles exhibit an increased elongation. Moreover, using the microtubule depolymerizing drug nocodazole it could be shown that the elongation and the motility of Spir-2-ΔKW-associated vesicles depends on an intact microtubule cytoskeleton. Motility and morphological dynamics of Spir-2-associated vesicles is therefore dependent on actin, actin motorproteins and microtubule filaments. These results propose a model in which myosin/F-actin forces mediate vesicle branching, allowing the vesicles to move to and in between the microtubule filaments and thereby providing a new degree of freedom in vesicular motility. To determine the exact subcellular localization of Spir-2, colocalization studies were performed. It could be shown that Spir-2 shows a partial colocalization to Rab11a-positive compartments. Furthermore, Spir-2 exhibits an almost identical localization to Arf1 and the Arf1 small G protein but not Rab11a could be immunoprecipitated with Spir-2-ΔKW. This suggests, that Arf1 recruits Spir-2 to Arf1/Rab11a-positive membranes. Another important function of the Spir-2 C-terminus is the membrane targeting by the FYVE domain. By performing a protein-lipid overlay assay, it has been shown that purified GST- and 6xHis-tagged Spir-2-ΔKW bind phosphatidic acid suggesting a mechanism in which Spir-2 is recruited to phosphatidic acid-enriched membranes. To further elucidate the mechanism in which Spir-2 membrane-targeting could be regulated, interaction studies of C-terminal parts of Spir-2 revealed that the Spir-2 proteins interact directly.
The Ecology and Population structure of the invasive Yelllow Crazy Ant Anoplolepis gracilipes
(2011)
The invasive Yellow Crazy Ant Anoplolepis gracilipes is a widespread tropical ant species which is particularly common in anthropogenically disturbed habitats in South-East Asia and the Indopacific region. Its native range is unknown, and there is little information concerning its social structure as a potential mechanism facilitating invasion as well as its ecology in one of the putative native ranges, South-East Asia. Using mitochondrial DNA sequences, I demonstrated that the majority of the current Indopacific colonies were likely introduced from South-East Asian populations, which in turn may have been introduced much earlier from a yet unidentified native range. By conducting behavioral, genetic and chemical analyses, I found that A. gracilipes supercolonies contain closely related individuals, thus resembling enlarged versions of monogynous, polydomous colonies of other ant species. Furthermore, mutually aggressive A. gracilipes supercolonies were highly differentiated both genetically and chemically, suggesting limited or even absent gene flow between supercolonies. Intranidal mating and colony-budding are most likely the predominant, if not the exclusive mode of reproduction and dispersal strategy of A. gracilipes. Consequently, a positive feedback between genetic, chemical and behavioral traits may further enhance supercolony differentiation though genetic drift and neutral evolution. This potential scenario led to the hypothesis that absent gene flow between different A. gracilipes supercolonies may drive them towards different evolutionary pathways, possibly including speciation. Thus, I examined one potential way by which gene flow between supercolonies of an ant species without nuptial flights may be maintained, i.e. the immigration of sexuals into foreign supercolonies. The results suggest that this option of maintaining gene flow between different supercolonies is likely impaired by severe aggression of workers towards allocolonial sexuals. Moreover, breeding experiments involving males and queens from different supercolonies suggest that A. gracilipes supercolonies may already be on the verge of reproductive isolation, which might lead to the diversification of A. gracilipes into different species. Regarding the ecological consequences of its potential introduction to NE-Borneo, I could show that A. gracilipes supercolonies may affect the local ant fauna. The ant community within supercolonies was less diverse and differed in species composition from areas outside supercolonies. My data suggest that the ecological dominance of A. gracilipes within local ant communities was facilitated by monopolization of food sources within its supercolony territory, achieved by a combination of rapid recruitment, numerical dominance and pronounced interspecific aggression. A. gracilipes’ distribution is almost exclusively limited to anthropogenically altered habitat, such as residential and agricultural areas. The rate at which habitat conversion takes place in NE-Borneo will provide A. gracilipes with a rapidly increasing abundance of suitable habitats, thus potentially entailing significant population growth. An potentially increasing population size and ecological dominance, however, are not features that are limited to invasive alien species, but may also occur in native species that become ‘pests’ in an increasing abundance of anthropogenically altered habitat. Lastly, I detected several ant guests in supercolonies of A. gracilipes. I subsequently describe the relationship between one of them (the cricket Myrmecophilus pallidithorax) and its ant host. By conducting behavioral bioassays and analyses of cuticular hydrocarbon (CHC) profiles, I revealed that although M. pallidithorax is attacked and consumed by A. gracilipes whenever possible, it may evade aggression from its host by a combination of supreme agility and, possibly, chemical deception. This thesis adds to our general understanding of biological invasions by contributing species-specific data on a previously understudied invasive organism, the Yellow Crazy Ant Anoplolepis gracilipes. Introductions which may have occurred a long time ago may make it difficult to determine whether a given species is an introduced invader or a native pest species, as both may have pronounced ecological effects in native species communities. Furthermore, this thesis suggests that supercolonialism in invasive ants may not be an evolutionary dead end, but that it may possibly give rise to new species due to reproductive boundaries between supercolonies evoked by peculiar mating and dispersal strategies.
Stroke, after myocardial infarction and cancer is the third most common cause of death worldwide and 1/6th of all human beings will suffer at least one stroke in their lives. Furthermore, it is the leading cause for adult disability with approximately one third of patients who survive for the next 6 months are dependent on others. Because of its huge socioeconomic burden absorbing 6% of all health care budgets and with the fact that life expectancy increases globally, one can assume that stroke is already, and will continue to be, the most challenging disease. Ischemic stroke accounts for approximately 80% of all strokes and results from a thrombotic or embolic occlusion of a major cerebral artery (most often the middle cerebral artery, MCA) or its branches Following acute ischemic stroke, the most worrisome outcome is the rapidly increasing intra-cranial pressure due to the formation of space-occupying vasogenic oedema which can have lethal consequences. Permeability changes at the Blood-Brain Barrier (BBB) usually accompanies the oedematous development and their time course can provide invaluable insight into the nature of the insult, activation of compensatory mechanisms followed by long term repair. Rodent models of focal cerebral ischemia have been developed and optimized to mimic human stroke conditions and serve as indispensable tools in the field of stroke research. The presented work constituting of three separate but complete works by themselves are sequential, where, the first part was dedicated to the establishment of non-invasive small animal imaging strategies on a 3 tesla clinical magnetic resonance scanner. This facilitated the longitudinal monitoring of pathological outcomes following stroke where identical animals can serve as its own control. Tissue relaxometric estimations were carried out initially to derive the transverse (T2), longitudinal (T1) and the transverse relaxation time due to magnetic susceptibility effects (T2*) at the cortical and striatal regions of the rodent brain. Statistically significant differences in T2*-values could be found between the cortex and striatal regions of the rodent brain. The derived tissue relaxation values were considered to modify the existing imaging protocols to facilitate the study of the rodent model of ischemic stroke. The modified sequence protocols adequately characterized all the clinically relevant sequels following acute ischemic stroke, like, the altered perfusion and diffusion characteristics. Subsequent to this, serial magnetic resonance imaging was performed to investigate the temporal and spatial relationship between the biphasic nature of BBB opening and, in parallel, the oedema formation after I/R injury in rats. T2-relaxometry for oedema assessment was performed at 1 h after ischemia, immediately following reperfusion, and at 4, 24 and 48 hours post reperfusion. Post-contrast T1-weighted imaging was performed at the last three time points to assess BBB integrity. The biphasic course of BBB opening with significant reduction in BBB permeability at 24 hours after reperfusion was associated with a progressive expansion of leaky BBB volume, accompanied by a peak ipsilateral oedema formation. At 48 hours, the reduction in T2-value indicated oedema resorption accompanied by a second phase of BBB opening. In addition, at 4 hours after reperfusion, oedema formation could also be detected at the contralateral striatum which persisted to varying degrees throughout the study, indicative of widespread effects of I/R injury. The observations of this study may indicate a dynamic temporal shift in the mechanisms responsible for biphasic BBB permeability changes, with non-linear relations to oedema formation. Two growth factor peptides namely pigment epithelium derived factor (PEDF) and epidermal growth factor (EGF) with widely different trophic properties were considered for their beneficial effects, if any, in the established rodent model of I/R injury and studied up to one week employing magnetic resonance imaging. Both the selected, trophic factors demonstrated significant neuroprotection as demonstrated by a reduction in infarct volume, even though PEDF was found to be the most potent one. PEDF also demonstrated significant attenuation of oedema formation in comparison to both the control and EGF groups, even though EGF could also demonstrate oedema suppression. In the present work, we noticed that interventions with macromolecule protein/peptides by itself could mediate remote oedema at distant sites even though the significance of such an observation is not clear at present. Susceptibility (T2*) weighted tissue relaxometric estimations were considered at the infarct region to detect any metabolic changes arising out of any neuroprotection and/or cellular proliferation / neurogenesis. PEDF group demonstrated a striking reduction of the T2*-values, which is indicative of an increased metabolic activity. Moreover, all the groups (Control, EGF and PEDF) demonstrated significantly elevated T2*-values at the contralateral striatum, which is indicative of widespread metabolic suppression usually associated with a variety of traumatic brain conditions. Moreover, as expected from the properties of PEDF, it demonstrated an extended BBB permeability suppression throughout the duration of the study. This study underlines the merits of considering non-invasive imaging strategies without which it was not possible to study the required parameters in a longitudinal fashion. All the observations are adequately supported by reasonably well defined mechanisms and needs to be further verified and confirmed by an immunohistochemical study. These results also need to be complemented by a functional study to evaluate the behavioural outcome of animals following these treatments. These studies are progressing at our laboratory and the results will be duly published afterwards.
Bordetellen sind Gram-negative Kokkobazillen, die phylogenetisch zu den β-Proteobakterien zählen und in der Familie der Alcaligenaceae eingeordnet sind. Der bedeutendste Vertreter der Gattung, die nach heutigem Kenntnisstand neun Arten umfasst, ist Bordetella pertussis, der Erreger des Keuchhustens. Der Keim ist obligat humanpathogen und besitzt zahlreiche Virulenzfaktoren, um die Epithelzellen des Respirationstraktes zu besiedeln und zu zerstören, wodurch es zu dem charakteristischen Krankheitsverlauf kommt. Neben B. pertussis werden noch B. bronchiseptica und B. parapertussis dem sogenannten B. bronchiseptica-Cluster zugeteilt. Alle Vertreter des B. bronchiseptica-Clusters sind in der Lage, bei verschiedenen Wirtsspezies respiratorische Erkrankungen mit unterschiedlichem Schweregrad auszulösen. Dabei weist B. bronchiseptica ein breiteres Wirtsspektrum auf und kann Atemwegserkrankungen in einer Vielzahl von Säugetieren auslösen, wohingegen B. parapertussis vornehmlich Schafe und Menschen infiziert und bei letzteren eine schwächere Form des Keuchhustens bewirkt. Das Hfq-Protein wurde ursprünglich als Wirtsfaktor identifiziert, welcher für die Replikation des RNA-Phagen Qβ in Escherichia coli benötigt wird (host factor for Qβ oder HF-1). Es ist in Struktur und Funktion homolog zu den Sm-Proteinen aus Eukaryoten, die am Splicing von mRNAs involviert sind. Die Beteiligung des Hfq-Proteins an regulatorischen Vorgängen, die durch kleine nicht-kodierende RNAs (sRNAs) vermittelt werden, wurde erstmals in einer Studie zum Mechanismus der rpoS-Regulation durch die kleine regulatorische RNA OxyS ersichtlich. Seitdem konnte für eine Vielzahl an sRNAs gezeigt werden, dass sie an Hfq gebunden vorliegen und die Hilfe des Proteins bei der post-transkriptionellen Kontrolle ihrer Ziel-mRNAs benötigen. In dieser Hinsicht übernimmt Hfq die Rolle eines RNA-Chaperons, indem es trans-kodierte sRNAs stabilisiert und die Basenpaarung mit ihren Ziel-mRNAs fördert. Dabei beeinflusst die Bindung der sRNA-Regulatoren an ihre Ziel-mRNAs deren Translation, sowohl aktivierend als auch inhibierend. Bislang wurden Hfq-Homologe in der Hälfte aller sequenzierten Gram-positiven und Gram-negativen Bakterienarten gefunden. Eine BLAST-Analyse ergab, dass B. pertussis und B. bronchiseptica Homologe zum Hfq-Protein aufweisen und diese in der veröffentlichten Genomsequenz bereits als Hfq-Protein annotiert sind. Fokus dieser Arbeit war weitestgehend, die Funktion des Hfq-Proteins in B. pertussis und vergleichend in B. bronchiseptica zu charakterisieren. Mittels Primer Extension-Analyse konnte zunächst der Startpunkt des hfq-Transkripts in B. pertussis und B. bronchiseptica unter logarithmischen Wachstumsbedingungen bestimmt werden. Dieser Startpunkt war zudem unter stationären Wachstumsbedingungen und nach Hitzestress aktiv, was in Diskrepanz zur Beobachtung in E. coli steht. Ferner konnte festgestellt werden, dass die hfq-Transkription nach Induktion verschiedener Stressformen in beiden Organismen erhöht war. Nach Generierung der jeweiligen Δhfq-Mutanten in beiden Organismen wurden diese charakterisiert. Die B. pertussis Δhfq-Mutante zeigte ein deutliches Wachstumsdefizit gegenüber dem Wildtyp, im Gegensatz zu B. bronchiseptica Δhfq, die sich im Wachstum wie der Wildtyp verhielt. Beide Mutanten zeigten sich sensitiver gegenüber H2O2-Stress als der Wildtyp, nicht jedoch gegenüber weiteren oxidativen Stressbedingungen oder Membranstress induzierenden Substanzen. Die Δhfq-Mutante in B. pertussis war zudem in ihrer Fähigkeit zur Biofilmbildung beeinträchtigt, was jedoch nicht für B. bronchiseptica Δhfq galt. Da Hfq an sRNA-mRNA-Interaktionen, welche die Translation der mRNAs beeinflussen, beteiligt ist, sollte über 2D-Gelelektrophorese das Hfq-regulierte Proteom in B. pertussis und B. bronchiseptica bestimmt werden. Auffällig war, dass viele periplasmatische Transport-bindeproteine von der Δhfq-Mutation betroffen waren. Es zeigten sich aber auch Stoffwechselenzyme und wichtige Housekeeping-Faktoren, wie z. B. der Elongationsfaktor EF-Tu und das Chaperon GroEL, in der Δhfq-Mutante dereguliert. Generell scheint das Hfq-regulierte Proteom in B. pertussis und B. bronchiseptica nur einen kleinen Teil des gesamten Proteoms auszumachen. Zudem ist das Hfq-regulierte Proteom variabel zwischen verschiedenen Wachstumsbedingungen, aber auch zwischen den beiden Organismen trotz der engen Verwandtschaft. Die Expression ausgewählter Virulenzfaktoren zeigte keinen Unterschied zwischen Δhfq-Mutante und B. pertussis-Wildtyp.
An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade.