Gene trees enable the detailed study of the evolution of genes. These trees can be additive, based purely on mutational counts, or ultrametric, where the branches represent the time elapsed. The purpose of this thesis will be to relate evolutionary rate changes to gene function by examining the effects of a tree transformation on gene trees. The study will focus on the coffee genomes C. arabica, C. eugenioides and C. canephora. Three gene family trees will be studied: N-methyltransferases, FAD2 enzymes and terpenes. An additive tree can be transformed into an ultrametric tree using a transformation method. By taking each additive tree branch length and dividing the branch by the ultrametric branch length we can get a branch mutation rate. To quantify functional divergence, we compared aligned sequences of genes from groups of interest to calculate the ratio of non-synonymous mutations to synonymous mutations (Kn/Ks). Kn/Ks was found to correlate with the branch mutation rate in some but not all groups of interest. In groups in which correlation was present, there was also a correlation between Kn/Ks and branch length. The branch mutation rate can be used in some cases to further prove that functional divergence is present.

Legionella pneumophila est la bactérie responsable de la légionellose, une pneumonie atypique dans les pays industrialisés. Les souches pathogènes sont issues de notre environnement après multiplication à l’intérieur d’amibes, sont disséminées par la technologie humaine, puis peuvent infecter les macrophages alvéolaires humains. Ce travail vise à caractériser une famille d’effecteurs du système de sécrétion de type IV Dot/Icm transloqués dans le cytoplasme de la cellule hôte, des protéine kinases, et en particulier à établir le rôle de la protéine kinase LegK2 dans la virulence. L’analyse in silico et des tests de phosphorylation in vitro ont permis d’identifier 5 protéine kinases fonctionnelles, LegK1-LegK5, codées par la souche épidémique L. pneumophila Lens. Des tests de translocation ont montré qu’à l’exception de LegK5, les protéine kinases de Legionella sont transloquées dans la cellule hôte de façon Icm/Dot dépendante. LegK2 joue un rôle clé dans la virulence, comme démontré par inactivation de gène. Les vacuoles contenant le mutant legK2 présentent un recrutement moins efficace de reticulum endoplasmique, ce qui entraine une réplication intracellulaire retardée. Un mutant de substitution déficient pour l’activité kinase présente les mêmes défauts de virulence, ce qui démontre le rôle central de la phosphorylation dans le contrôle de ce processus. Les mécanismes moléculaires contrôlés par LegK2 sont actuellement recherchés par identification de partenaires et/ou substrats protéiques / Legionella pneumophila is the most common causative agent of the severe pneumony legionellosis. Legionella pathogenic strains are emerging from the environment after intracellular multiplication in amoeba, are dissiminated by water aerosols technologies, and are able to infect alveolar macrophages of human lungs. This work aims to characterize one family of effectors translocated into the host cytoplasm, namely the protein kinase family, and particularly the role of LegK2 protein kinase in virulence. In silico analysis and in vitro phosphorylation assays allowed the identification of 5 functional protein kinases LegK1-LegK5 encoded by the epidemic L. pneumophila Lens strain. Translocation assays showed that except LegK5, the Legionella protein kinases are translocated. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles display less efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. A kinase-dead substitution mutant of legK2 exhibits the same virulence defects. Molecular mechanisms controled by LegK2 have been investigated by searching LegK2 partner and substrate proteins

Genome-wide association studies (GWAS)s provide an unbiased means of exploring the landscape of complex genetic disease. As such, these studies have identified genetic variants that are robustly associated with a multitude of conditions. I hypothesize that these genetic variants serve as excellent tools for evaluation of the genetic interface between epidemiologically related conditions. Herein, I test the association between SNPs associated with either (i) plasma lipids, (ii) rheumatoid arthritis (RA) or (iii) diabetes mellitus (DM) and late-onset Alzheimer’s disease (AD) to identify shared genetic variants. Regarding the most significantly AD-associated variants, I have also attempted to elucidate their molecular function. Only cholesterol-associated SNPs, as a group, are significantly associated with AD. This association remains after excluding APOE SNPs and suggests that peripheral and or central cholesterol metabolism contribute to AD risk. The general lack of association between RA-associated SNPs and AD is also significant in that these data challenge the hypothesis that genetic variants that increase risk of RA confer protection against AD. Functional studies of variants exhibiting novel associations with AD reveal that the lipid-associated SNP rs3846662 modulates HMGCR exon 13 splicing differentially in different cell types. Although less clear, trends were also observed between the RA-associated rs2837960 and the expression of several BACE2 isoforms, and between the DM-associated rs7804356 and expression of a rare SKAP2 isoform, respectively. In conclusion, the overlap of lipid-, RA- or DM-associated SNPs with AD is modest but in several instances significant. Continued analysis of the interface between GWAS of separate conditions will likely facilitate novel associations missed by conventional GWAS. Furthermore, the identification of functional variants associated with multiple conditions should provide insight into novel mechanisms of disease and may lead to the identification of new therapeutic targets in an era of personalized genomic medicine.

Infektionen mit dem intestinalen Parasiten Giardia duodenalis, verursachen weltweit eine der häufigsten humanen Parasitosen. Bislang konnten keine eindeutigen Virulenz- oder Pathogenitätsmarker des Erregers beschrieben werden. Es wird allerdings vermutet, dass potentielle G. duodenalis Virulenzfaktoren Enzyme sind, die während des Kontaktes des Erregers mit den Dünndarmepithelzellen sezerniert werden. Eines dieser Enzyme ist die Arginin Deiminase (ADI), die Arginin zu Citrullin umwandelt. Ziel dieser Arbeit war es Merkmale zu identifizieren, die für die ADI als Virulenzfaktor sprechen. Dazu wurde das Enzym zunächst hinsichtlich seiner Bedeutung für die Wirt-Pathogen-Interaktion untersucht. Die mit rekombinanter, katalytisch aktiver ADI (Assemblage A) behandelten LPS-stimulierten humanen moDC zeigten eine Veränderung in ihrem Phänotyp als auch in ihrer Cytokinsekretion. Diese ließ sich auf die durch das Enzym hervorgerufene Arginindepletion und/oder auf die Bildung der Metabolite, Citrullin und NH4+, zurückführen. Weiterhin konnte gezeigt werden, dass Parasitenisolate verschiedener G. duodenalis Assemblage A-Subtypen, vermutlich durch die katalytische Aktivität der ADI, die Stickstoffmonoxid-Bildung einer intestinalen Epithelzelllinie inhibiert. Neben dem Einfluss auf die Wirtsimmunantwort wurde auch die Variabilität in der kodierenden Sequenz des Enzyms in verschiedenen Parasitenisolaten analysiert. Anschließend erfolgte die funktionelle Charakterisierung des nativen (verschiedene Assemblage A-Subtypen) als auch des rekombinant aufgereinigten Enzyms (Assemblage A, B und E). Dabei zeigten sich Unterschiede in der Substrataffinität der ADI für Arginin, sowohl zwischen unterschiedlichen Assemblage A-Subtypen als auch unterschiedlichen Assemblage-Klassen. Zusammenfassend wurde gezeigt, dass die G. duodenalis ADI immunmodulatorische Effekte hat und das vermutlich eine Korrelation zwischen der Variation in der Primärstruktur und der Funktion des Enzyms besteht. / Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite that causes giardiasis, one of the most prevalent parasitic diseases worldwide. So far, little is known concerning host-parasite interaction, in particular what determines the parasite’s pathogenicity. Several potential virulence factors, among them the arginine deiminase (ADI) that hydrolyzes arginine into citrulline and NH4+, are discussed. The ADI was identified to be released upon contact with intestinal epithelium by Giardia trophozoites and was recognized as an immunoreactive protein during acute human giardiasis. Aim of the study was to identify hints for G. duodenalis ADI to be a virulence factor. First, to analyze the enzyme’s impact on host-parasite interplay, its influence on human monocyte-derived dendritic cells (moDC) was investigated. Treatment of LPS-stimulated cells with recombinant ADI of assemblage A changed DC phenotype (CD83, CD86) and cytokine secretion (TNF-α, IL-10, IL-12p40). These immunomodulatory changes in DC response were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Furthermore, trophozoites of different assemblage subtypes were shown, probably due to consumption of arginine by ADI, to reduce nitric oxide formation by intestinal epithelial cells in vitro. Second, variation in the ADI coding sequence of different G. duodenalis isolates being collected in a Giardia biobank was analyzed by sequencing. Subsequently, functional genetics were performed with native ADI of different assemblage A subtypes expressed by these strains as well as with purified, recombinant ADI of assemblage A, B and E. It was recognized that enzymes of the same subtype as well as of different assemblages types had different substrate affinities for arginine. To sum up, this report identified G. duodenalis ADI to be immunomodulatory and gives first indications of a correlation between enzyme function and variation of the protein primary structure.