On the structure differences of short fragments and amino acids in proteins with and without disulfide bonds

Of the 20 standard amino acids, cysteines are the only amino acids that have a reactive sulphur atom, thus enabling two cysteines to form strong covalent bonds known as disulfide bonds. Even though almost all proteins have cysteines, not all of them have disulfide bonds. Disulfide bonds provide structural stability to proteins and hence are an important constraint in determining the structure of a protein. As a result, disulfide bonds are used to study various protein properties, one of them being protein folding. Protein structure prediction is the problem of predicting the three-dimensional structure of a protein from its one-dimensional amino acid sequence. Ab initio methods are a group of methods that attempt to solve this problem from first principles, using only basic physico-chemical properties of proteins. These methods use structure libraries of short amino acid fragments in the process of predicting the structure of a protein. The protein structures from which these structure libraries are created are not classified in any other way apart from being non-redundant. In this thesis, we investigate the structural dissimilarities of short amino acid fragments when occurring in proteins with disulfide bonds and when occurring in those proteins without disulfide bonds. We are interested in this because, as mentioned earlier, the protein structures from which the structure libraries of ab initio methods are created, are not classified in any form. This means that any significant structural difference in amino acids and short fragments when occurring in proteins with and without disulfide bonds would remain unnoticed as these structure libraries have both fragments from proteins with disulfide bonds and without disulfide bonds together. Our investigation of structural dissimilarities of amino acids and short fragments is done in four phases. In phase one, by statistically analysing the phi and psi backbone dihedral angle distributions we show that these fragments have significantly different structures in terms of dihedral angles when occurring in proteins with and without disulfide bonds. In phase two, using directional statistics we investigate how structurally different are the 20 different amino acids and the short fragments when occurring in proteins with and without disulfide bonds. In phase three of our work, we investigate the differences in secondary structure preference of the 20 amino acids in proteins with and without disulfide bonds. In phase four, we further investigate and show that there are significant differences within the same secondary structure region of amino acids when they occur in proteins with and without disulfide bonds. Finally, we present the design and implementation details of a dihedral angle and secondary structure database of short amino acid fragments (DASSD) that is publicly available. Thus, in this thesis we show previously unknown significant structure differences in terms of backbone dihedral angles and secondary structures in amino acids and short fragments when they occur in proteins with and without disulfide bonds

Dog Population Management and Dog Bite Prevention in Rural and Remote Northern Saskatchewan Aboriginal Communities

Communities employ a wide variety of methods to reduce critical encounters and dog population numbers. However, systematic studies evaluating the success of approaches and techniques are currently lacking. Nor has significant consideration of community decision-making processes and policy development, or of the long-term sustainability of these programs been completed. Therefore, to assess the perception of dog-related issues, methods of policy creation and implementation, and true within-community dog demographic characteristics and rate of aggressive encounters a community-based research project was developed. A multiphase, convergent mixed methods study design in four separate northern Saskatchewan communities was implemented to evaluate these concerns. Methods of community-driven policy creation and implementation were recorded, management plans and strategies were monitored, and options were evaluated for successful reduction in dog bites and violent encounters. Community-based participatory methods created exchange and discussion with all levels of society, providing in-depth two-way channels for knowledge translation for researchers and community members. Policy creation and implementation was found to vary significantly between communities. Policies surrounding dog ownership and bite prevention are often dependent upon perceived risks for imminent human-canine aggressive encounters. Regrettably, sustainable interventions require sustained key community partner support and resource access. Community engagement and knowledge translation creates long-term, trusting relationships permitting more in-depth understanding of group choices. In addition, involving community members in research and data collection provides public appreciation of the scope and breadth of community issues and opinions. Enabling and empowering communities entails constant communication and education of all parties. No single model can be effective in all situations. Although enforceable legislation and widespread canine sterilisation are key aspects for community dog issues, comprehensive all-inclusive community education is indispensable. Wide-spread education and communication have the potential to dramatically decrease the number of aggressive dog:human encounters and fulfil goals for dog-human relationships that occur in indigenous communities in Canada

Molekulare Struktur- und Funktionsanalyse der Transkriptionskontrolle der GeneDDX3X und DDX3Y in der männlichen Keimbahn

Das Y-chromosomale Gen DDX3Y und das X-homologe Gen DDX3X kodieren zwei RNA-Helikasen der DEAD-Box Familie, die beide funktionell in verschiedenen Phasen der Human Spermatogenese aktiv sind (Ditton et al., 2004). Deletionen der „Azoospermia Factor a“ (AZFa) Region in Yq11, in der DDX3Y lokalisiert ist, führen zu einem Totalverlust der männlichen Keimzellen, dem „Sertoli Cell-only“ (SCO)-Syndrom. Beide Gene weisen in den kodierenden Sequenzen eine hohe Konserviertheit (92,4%) ihrer Aminosäure-Sequenzen auf, was auf eine funktionelle Selektion beider Genkopien hindeutet. Die Promoter- und 5´UTR-Sequenzen haben dagegen, seit Fehlen der Rekombination der Säuger Gonosomen, deutliche Chromosomen-spezifische Sequenzveränderungen durchlaufen. Diese Veränderungen haben zur Entstehung einer komplexen hodenspezifischen Transkriptions- und Translationskontrolle beider Gene geführt. Auf Grund der Allel-spezifischen Sequenzevolution sind unterschiedliche Core-Promotermodule zur Keimbahn-spezifischen Expressionskontrolle etabliert worden. Einige Sequenzmotive geben auch erste Hinweise auf unterschiedliche Chromatinstrukturen der beiden Promoterdomänen. Durch die Kombination von vergleichender Genomik in sechs Säugerspezies (Mensch, Schimpanse, Rhesusaffe, Weißbüschelaffe, Rind, Maus) für beide Gene und gezielten Experimenten, konnten sowohl Allel-spezifische, als auch Spezies-spezifische cis-regulative Module identifiziert werden. So konnten in den Human DDX3(X/Y) Promoterregionen neun konservierte Sequenzblöcke kartiert werden. Ein solcher Sequenzblock ist der Y-spezifische MSY2 Minisatellit (Bao et al., 2000). Eine MSY2, bzw. homologe MSY2-X Basissequenz konnte in allen Spezies für DDX3Y und DDX3X stromaufwärts zu den Transkriptionseinheiten identifiziert werden. Eine Vervielfältigung der MSY2 Sequenz erfolgte allerdings nur in Primaten und nur in der Keimzell-spezifischen Promoterdomäne von DDX3Y. In den neun Human X-Y Sequenzblöcken wurden 24 X-Y konservierte Transkriptionsfaktorbindestellen (TFBS) identifiziert. Besonders auffällig ist eine in allen untersuchten Spezies X-Y konservierte SOX5-TFBS, die in den MSY2 und MSY2-X Sequenzen lokalisiert ist. Insgesamt konnten 30 X-Y konservierte TFBS in den Human Promotersequenzen kartiert werden. Die Mehrzahl der dazugehörigen TFs weist eine Expression in Hodengewebe auf. Sechs gemeinsame TF-Module konnten identifiziert werden, wovon eines positionshomolog lokalisiert ist. Die konservierten TFBS und gemeinsamen TF-Module deuten somit auf einen gewissen Grad von Co-Regulation der beiden Gene in der männlichen Keimbahn hin. Daneben konnten zusätzlich mehrere Allel-spezifisch repetitive TFBS und TFBS-Cluster kartiert werden, die offensichtlich eine genspezifische Transkriptionskontrolle vermitteln. Die Kombination aus in-silico Analysen und gezielten Luziferase-Reportergenanalysen demonstriert somit eine erfolgreiche Strategie zur Identifizierung wesentlicher cis-Kontrollelemente für die DDX3(X/Y) Keimbahnexpression. Diese cis-Module sind daher gute Sequenzmotive für die molekulargenetische Mutationsanalyse bei infertilen Männern mit Verdacht auf Fehlfunktion der DDX3(X/Y) Expression