@phdthesis{Hartmann2010,
  author    = {Hartmann, Thomas},
  title     = {Nitrogen metabolism in Aspergillus fumigatus with emphasis on the oligopeptide transporter (OPT) gene family},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54027},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {The saprophytic filamentous fungus Aspergillus fumigatus has been gaining importance as an opportunistic human pathogen over the past decades. Advances in modern medicine have created a growing group of patients susceptible to infection with A. fumigatus, often contracting potentially deadly invasive aspergillosis. The virulence of this pathogen appears to be a multifactorial trait, a combination of physiological characteristics that enables the fungus to infect immunocompromised humans. This work concentrates on the nitrogen metabolism of A. fumigatus, which is essential for meeting the nutritional needs inside the human host. Using DNA microarrays, the transcriptional response during growth on three different secondary nitrogen sources was examined, which revealed the metabolic versatility of A. fumigatus, especially when challenged with proteins as the sole source of nitrogen. In-depth transcriptional profiling of the eight-member oligopeptide transporter (OPT) gene family underlined the importance of oligopeptide transport for growth on complex nitrogen sources like BSA or collagen. Heterologous expression of the opt genes in Saccharomyces cerevisiae showed their functionality as oligopeptide transporters, and characterized their substrate specificity. Using a Cre/loxP based genetic tool, a complete deletion of all opt genes in A. fumigatus was achieved. The resultant strain exhibited diminished growth on medium where the oligopeptide GPGG was the sole nitrogen source, but did not show any other in vitro phenotype. The opt deletion strain was not attenuated in virulence in a murine model of pulmonary aspergillosis, suggesting that the OPT gene family is not necessary for successful infection. The connection of oligopeptide transport and extracellular proteolytic activity was investigated by deleting the genes encoding Dpp4 and Dpp5, two dipeptidyl peptidases, or PrtT, the transcriptional regulator of major secreted proteases, in the complete opt deletion background. In contrast to the deletion of dpp4 and dpp5, which did not result in any additional phenotype, the absence of prtT led to a drastic growth defect on porcine lung agar. This suggests a synergistic action of extracellular proteolytic digest of proteins and transport of oligopeptide degradation products into the cell. Finally, this work established the bacterial β-Rec/six site-specific recombination system as a novel genetic tool for targeted gene deletion in A. fumigatus.},
  subject      = {Aspergillus fumigatus},
  language  = {en}
}
@phdthesis{Rupp2009,
  author    = {Rupp, Ingrid},
  title     = {Die Gametogenese des humanpathogenen Malariaerregers Plasmodium falciparum - eine Charakterisierung von daran beteiligten Proteasen sowie die Beschreibung und Funktionsanalyse von dabei auftretenden interzellul{\"a}ren Gametenfilamenten},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-47830},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {Malaria stellt mit einer Mortalit{\"a}t von {\"u}ber einer Million Menschen pro Jahr die bedeutsamste Tropenkrankheit f{\"u}r den Menschen dar. Wachsende Resistenzen der Malariaerreger gegen{\"u}ber den verf{\"u}gbaren Medikamenten erh{\"o}hen mehr denn je den Druck, neue Therapiem{\"o}glichkeiten sowie einen Impfstoff gegen diese Krankheit zu entwickeln. Eine Unterbrechung des sexuellen Fortpflanzungszyklus im Laufe der Transmission von Mensch zu Stechm{\"u}cke w{\"u}rde zu einem Verbreitungsstopp des Erregers f{\"u}hren. Sowohl die Identifizierung von molekularen Wechselwirkungen als auch die Erforschung von an Fertilisationsereignissen beteiligten Prozessen sind wichtige Schritte, um die Sexualphase des Erregers aufzukl{\"a}ren und neue Angriffspunkte f{\"u}r Medikamente oder Vakzine zu entwickeln. Dem Genom von P. falciparum konnten 92 putative Proteasen zugeordnet werden, von denen nur ein geringer Bruchteil charakterisiert worden ist. Unter Anwendung von Protease-Inhibitoren konnte in dieser Arbeit gezeigt werden, dass die Exflagellation der m{\"a}nnlichen Gameten die Beteiligung von Proteasen verschiedener Kategorien ben{\"o}tigt. Die Ergebnisse belegten, dass die Aktivit{\"a}t von zwei oder mehr Serinproteasen, von Falcipain-{\"a}hnlichen Cysteinproteasen, von nicht-Thermolysin-{\"a}hnlichen Zink-Metalloproteasen und von Aspartatproteasen f{\"u}r den erfolgreichen Abschluss der m{\"a}nnlichen Gametogenese eine wichtige Voraussetzung ist. Die Lokalisation des Cysteinproteasen- und Falcipain-hemmenden Inhibitors bADA konnte erstmals im Zytosol von Sexualstadien nachgewiesen werden. In dieser Arbeit wurden zus{\"a}tzlich die Proteasen Calpain, DPAP2, GPI8, Metacaspase 2, Plasmepsin 6 und PfSub3 n{\"a}her untersucht. RT-PCR-Analysen konnten die Transkription der sechs ausgesuchten Proteasen in gemischten asexuellen Parasiten sowie zum Großteil in Gametozyten, Gameten und Zygoten belegen. Die Transformation von asexuellen Parasiten mit entsprechenden knockout-Konstrukten deckte f{\"u}r Metacaspase 2 und PfSub3 auf, dass sie im asexuellen Vermehrungszyklus nicht essentiell und die entsprechenden Genloci f{\"u}r Rekombinationsereignisse zug{\"a}nglich sind. Die Ergebnisse der {\"u}brigen Transformationen deuteten darauf hin, dass Calpain essentiell im asexuellen Vermehrungszyklus und dass der Genlocus von Plasmepsin 6 f{\"u}r Rekombinationsereignisse unzug{\"a}nglich ist. Proteinexpressionsstudien anhand von Western-Blot-Analysen und Immunfluoreszenzstudien f{\"u}r PfSub3 konnten Hinweise darauf liefern, dass diese Serinprotease in asexuellen Parasiten, nicht-aktivierten sowie aktivierten Sexualstadien exprimiert wird. Aufgrund der in dieser Arbeit generierten Ergebnisse konnten im Laufe der Gametogenese auftretende Gametenfilamente morphologisch beschrieben sowie Hinweise auf ihre m{\"o}gliche Funktion erlangt werden. Durch die Anwendung von Immunfluoreszenzstudien, rasterelektronenmikroskopischen Aufnahmen sowie die Analyse lebender Gameten konnte gezeigt werden, dass die bis zu 180 µm langen Filamente am Ende geschlossen sind und einen Durchmesser von ca. 200 nm aufweisen. Die tubul{\"a}ren Zellausl{\"a}ufer konnten weiterhin als verzweigte sowie nicht-verzweigte Ausl{\"a}ufer der parasit{\"a}ren Plasmamembran dargestellt werden, die mit Zytoplasma gef{\"u}llt sind. Es konnte belegt werden, dass die Aktin-assoziierten Filamente in periodischen Abst{\"a}nden von beulenartigen Ausw{\"o}lbungen unterbrochen werden und dass sie in rasterelektronenmikroskopischen Analysen ein perlschnurartiges Erscheinungsbild aufweisen. Weiterhin wurde dokumentiert, dass die Zellausl{\"a}ufer mit typischen sexualstadienspezifischen Proteinen wie Pfs25, Pfs230, Pfs48/45 und PfCCp4 assoziiert vorliegen, wobei das Fehlen einzelner dieser Proteine jedoch nicht das Ausbilden der Gametenfilamente verhinderte. Als typisches Charakteristikum der Filamente konnte ihre Eigenschaft beschrieben werden, mehrere Makrogameten und zum Teil Gametozyten in einem Zellkluster miteinander netzartig zu verbinden, wobei bis zu neun Filamente von einem Makrogameten ausgehend beobachtet werden konnten. Die Gametenfilamente zeigten ebenfalls die F{\"a}higkeit, an umliegende nicht-infizierte Erythrozyten sowie mit asexuellen Parasiten infizierte Erythrozyten zu adh{\"a}rieren. Die Filamente waren bereits f{\"u}nf Minuten nach der Aktivierung der Gametozyten und im Laufe der Gametogenese bei 33 bis 73 \% der Zellen nachweisbar. Die Gametenfilamente blieben bis zu 12 Stunden nach Aktivierung der Gametozyten mit der Zelloberfl{\"a}che verbunden. Der aktive Einzug eines Zellfilaments sowie die Bildung der Gametenfilamente im Mitteldarm der Stechm{\"u}cke konnte ebenfalls demonstriert werden. Die in dieser Arbeit dargestellten Ergebnisse lieferten unter anderem den Grundbaustein einer formulierten Funktionshypothese f{\"u}r diese Gametenfilamente. Es wird angenommen, dass die Filamente aufgrund ihrer adh{\"a}siven Eigenschaften im Laufe der Befruchtung von Plasmodium im Mitteldarm der Stechm{\"u}cke auftreten. M{\"o}glicherweise bedienen sich vitale Gameten dieser Strukturen, um andere Sexualstadien zu finden und sie zu verbinden.},
  subject      = {Plasmodium falciparum},
  language  = {de}
}
@phdthesis{Friedrich2009,
  author    = {Friedrich, Torben},
  title     = {New statistical Methods of Genome-Scale Data Analysis in Life Science - Applications to enterobacterial Diagnostics, Meta-Analysis of Arabidopsis thaliana Gene Expression and functional Sequence Annotation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39858},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.},
  subject      = {Genomik},
  language  = {en}
}