Mannheimia haemolytica (M. haemolytica)-induced bovine respiratory disease causes millions of dollars in economic losses to Canadian cattle industry. Contemporary management strategies built around the use of antimicrobials are proving to be increasingly unavailing and lead to drug residues in meat which may contribute to the development of multi drug resistant bacteria. Many M. haemolytica vaccines are effective in stimulating antibody responses but studies of vaccina-tion in young calves and the cattle exposed to M. haemolytica (high-risk cattle) have shown poor vaccine efficacy. Antimicrobial peptides (AMPs) may help in the management of respiratory disease caused by M. haemolytica while minimizing the risk of drug residues in animal-derived food products. AMPs are positively charged molecules that can kill bacteria primarily through the electrostatic interactions with the anionic bacterial lipid bilayer. Since the primary target of AMPs is the bac-terial surface charge, which is evolutionarily conserved, the development of resistance towards AMPs seems less likely. These peptides hold potential to replace or reduce the use of antibiotics. Human β-Defensin 3 (HBD3) and Microcin J25 (MccJ25) are cationic peptides that have shown good activity against many Gram-negative bacteria. Five peptides, namely native HBD3, three synthetic HBD3 analogues (28 amino acid, 20AA, and 10AA), and MccJ25 were selected for microbicidal activity against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis (SPPS). In all the three analogue, replacement of cysteine with valine rendered them linear and increased their antibacterial activity. Minimum Bactericidal concentration (MBC) assays were performed with the final inoculum size of 1-5x105 cells/ml, with the exception of the 10AA analogue which was incubated with 104 cells/ml final inoculum size. The antimicrobial assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogue with an MBC of 50 µg/ml. MccJ25 had limited effect with an MBC greater than 100 µg/ml. The MBC value of 6.3 µg/ml achieved with the 10AA analogue is likely a result of lower final inoculum size. AMPs have several immunomodulatory functions, and these peptides can act as chemoattractant, induce cytokine release that in turn leads to chemotaxis of monocytes and neutrophils. Since neutrophils play an important role in the pathogenesis of BRD, the chemotactic effect of HBD3, 20AA and 28AA peptides on bovine neutrophils was studied using Boyden chamber. Peripheral blood neutrophils isolated from normal healthy cattle showed chemotaxis towards HBD3 and 20AA peptides (P<0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). Based on these data, it can be concluded that HBD3 and its analogues showed antimicrobial ef-fects against M. haemolytica but MccJ25 had limited microbicidal activity against M. haemolytica. While HBD3 and 20AA analogue were chemotactic for bovine peripheral blood neutrophils, none of the peptides inhibited fMLP-induced migration of neutrophils. These peptides hold potential for further in vivo testing to develop them for use to manage M. haemolytica-induced respiratory disease in cattle.

In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

Mon travail de thèse porte sur la protéine AhPDF1 de la plante Arabidopsis halleri. AhPDF1 est une défensine de 51 résidus, riche en cystéines qui participe à la défense de la plante en jouant un rôle antifongique. La défensine AhPDF1 possède 8 cystéines impliquées dans ses 4 ponts disulfure, elle présente un repliement en CSαβ. Des travaux récents sur AhPDF1 ont permis d’identifier une nouvelle fonction : la tolérance aux métaux lourds, en particulier la tolérance au zinc. L’objectif général du projet dans lequel s’intègre ma thèse est donc de comprendre, au niveau atomique et en lien avec l’état d’oxydation des cystéines, le mécanisme par lequel les défensines de plantes confèrent la tolérance au zinc. Dans une majeure partie de ma thèse j’ai travaillé à la production de la défensine AhPDF1 d’abord dans Escherichia coli puis dans Pichia pastoris. J’ai ensuite mis au point la synthèse chimique de la protéine AhPDF1 et optimisé l’étape la plus délicate, celle du repliement oxydatif. Après avoir produit la défensine AhPDF1 en quantité et qualité suffisante, j’ai réalisé son étude structurale par RMN. De plus cette structure m’a servi de base pour modéliser, par homologie, toutes les autres défensines actuellement identifiées d’Arabidopsis halleri et Arabidopsis thaliana. Enfin, j’ai appris à maîtriser les conditions qui permettent de conserver la protéine dans un état partiellement réduit et j’ai réalisé les premiers essais de chélation de la défensine avec le zinc. / My thesis focuses on the AhPDF1 plant defensin from Arabidopsis halleri. AhPDF1 is a 51-residue, cysteine-rich protein involved in the plant defense, and playing an antifungal role. AhPDF1 defensin has eight cysteins involved in its four disulfide bridges, and presents a folding in CSαβ. Recent work on AhPDF1 allowed to identify a new function : the tolerance to heavy metals, especially zinc tolerance. The overall objective of the project that fits my thesis is to understand, at the atomic level and in relation to the oxidation state of cysteins, the mechanism by which the plant defensins confer zinc tolerance. In a major part of my thesis I worked on the production of AhPDF1 defensin first in Escherichia coli and in Pichia pastoris. Then, I developed the chemical synthesis of AhPDF1 and optimized the most delicate step of the oxidative folding. After producing AhPDF1 defensin in sufficient quantity and quality, I realized its structural study by NMR. Furthermore, this structure was used as starting structure, for modeling by homology all other defensins currently identified from Arabidopsis halleri and Arabidopsis thaliana. Finally, I learnt how to master the conditions that maintain the protein in a partially reduced state in order to achieve the first assay of zinc chelation with defensin.

Total RNA was extracted from chicken oviduct epithelial cells. Avian Beta-defensin (AvBD)-2, -6, and -12 cDNAs were amplified by reverse transcription-PCR and cloned into pRSET A style='msoareast-language:ZH-CN'>, a protein expression vector. The class=SpellE>hexa-histidine-tagged class=SpellE>AvBD peptides were expressed in Escherichia coli (E. coli) BL21(DE3) class=SpellE>plysS and affinity-purified. The antimicrobial activities of the recombinant AvBDs against E. coli style='msoareast-language:ZH-CN;mso-bidiont-style:italic'>, Salmonella class=SpellE>enterica style='mso-bookmark:OLE_LINK9'> serovar Typhimurium (S. class=SpellE>typhimurium), and Staphylococcus aureus style='msoareast-language:ZH-CN'> (S. aureus) were determined. style='msoareast-language:ZH-CN;mso-bidiont-style:italic'> At 8, 16 and 32 µg/ml, all three rAvBDs killed and inhibited the growth style='msoareast-language:ZH-CN'> of E. coli style='msoareast-language:ZH-CN;mso-bidiont-style:italic'>, S. typhimurium, and S. aureus. The killing of rAvBD-2, -6, and -12 against stationary phase E. coli and S. class=SpellE>aureus was pH dependent in the range investigated. style='msoareast-language:ZH-CN'> In addition, the killing-curves showed that rAvBDs exerted their antimicrobial function within 30 minutes of treatment, suggesting the fast killing mechanisms of rAvBDs.

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In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

L'ulcère de Buruli (UB), infection à Mycobacterium ulcerans, troisième mycobactériose mondiale, connait une émergence rapide depuis 1980, essentiellement dans les pays d'Afrique subsaharienne. Jusqu’ici, les connaissances épidémiologiques sur l’UB étaient fondées sur des séries de cas cliniques non confirmés par laboratoire. Nous avons constitué la plus grande cohorte de cas confirmés à ce jour rassemblant plus de 1200 patients traités au CDTUB de Pobè au Bénin entre 2005 et 2011, afin de décrire l'épidémiologie clinique de la maladie et d'explorer l’architecture génétique de la susceptibilité à cette maladie. Les patients atteints d’UB sont des enfants (âge médian au diagnostic de 12 ans), présentant une lésion unique (96%), large (plus de 15 cm, 36%), ulcérative (66%) du membre inférieur (60%). Nous rapportons une présentation clinique atypique de l’UB, dans laquelle les patients présentent exclusivement une ostéomyélite à M. ulcerans. Le sex-ratio varie avec l’âge : les garçons sont majoritaires parmi les enfants (57% de patients masculins chez les moins de 15 ans), et les femmes parmi les adultes (33% de patients masculins). La présentation clinique dépend de l’âge et du sexe. 9% des patients masculins ont présenté une ostéomyélite contre 4% des patients féminins. Un an après la fin du traitement, 22% des patients présentent des séquelles fonctionnelles fixées. Une présentation clinique comportant une lésion oedémateuse, osseuse, de grande taille ou plusieurs lésions est significativement associée avec le développement de séquelles fonctionnelles (OR 7.64, IC95% [5.29-11.31]). Les patients coinfectés par le VIH ont un risque significativement plus élevé de développer un UB sévère (OR 2.77, IC95% [1.32-6.33]). Nous avons exploré l’architecture génétique de la susceptibilité à l’UB dans une perspective mendélienne et une perspective complexe. Le cas le plus sévère de la maladie observé dans ce centre appartient à une famille consanguine dans laquelle la ségrégation du phénotype suggère un défaut génétique mendélien récessif. Une analyse de liaison génétique par cartographie d'homozygotie suggère l’implication du locus des béta-défensines sur le chromosome 8 dans la pathogénèse de l'UB, et mène à l’identification d’une délétion homozygote ségrégeant parfaitement avec la maladie. Dans une perspective complexe, une étude d’association pangénomique a été réalisée après génotypage d’une cohorte de 400 cas et 400 témoins exposés sur plus de 2 millions de SNPs par la puce Illumina Omni2.5 et a permis l’identification de nombreux signaux d’intérêt. L’étude de réplication est en cours. La compréhension de la physiopathologie de l'infection à M. ulcerans est cruciale pour générer de nouvelles pistes thérapeutiques et vaccinales. La dissection du contrôle génétique de l'infection par l'hôte est en ce sens indispensable. / Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most frequent mycobacteriosis worldwide. It has been rapidly emerging in sub-Saharan African countries since 1980. Until now, knowledge of BU epidemiology relied on series of non laboratory-confirmed clinical cases. From 2005-2011, we recruited the current largest cohort of laboratory-confirmed cases (more than 1,200 patients) at the Pobe CDTUB, Benin, to describe the clinical epidemiology of the disease and to explore the genetic architecture of human susceptibility to BU. Typically, patients with BU were children (median age at diagnosis 12 years) presenting with a unique (96%) large (≥15 cm, 36%) ulcerative (66%) lesion of the lower limb (60%). Atypical clinical presentation of BU included osteomyelitis with no identifiable present or past BU skin lesions. The sex ratio of BU widely varied with age, with male patients accounting for 57% of patients aged 15 years and younger, but only 33% of those older than 15 years. Clinical presentation of BU was significantly dependent on age and sex. 9% male patients had BU osteomyelitis, whereas only 4% of female patients did. 1 year after treatment, 22% of patients with follow-up information presented with permanent functional sequelae. Presentation with oedema, osteomyelitis, or large (≥15 cm in diameter), or multifocal lesions was significantly associated with occurrence of permanent functional sequelae (OR 7•64, 95% CI 5•29–11•31) and operationally defines severe BU. When coinfected with HIV, patients had a significantly higher risk to develop severe BU (OR 2.77, IC95% [1.32-6.33]). We explored the genetic architecture of susceptibility to BU in both mendelian and complex genetic frameworks. The most severe case of the disease to have been treated at the Pobe CDTUB belonged to a consanguineous family in which the segregation of the phenotype was indicative of a recessive mendelian genetic defect. Genetic linkage analysis by homozygosity mapping suggested the implication of the beta-defensin locus on chromosome 8 in BU pathogenesis and lead to the identification of a homozygous deletion, which co-segregated perfectly with the disease in the family. In a complex genetics approach, we undertook a genome-wide association study, which involved the genotyping of more than 2 million SNPs (Illumina Omni2.5) in a cohort of 400 cases and 400 exposed controls. We identified many signals of interest. The replication study is ongoing. Understanding BU physiopathology is crucial to the development of efficient vaccines and drugs. Dissection of the genetic control of the infection by M. ulcerans by its human host therefore constitutes an indispensable step.

Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with Major Depressive Disorder (MDD), resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria are also able to produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which although found in trace concentrations in the mammalian brain, can affect CNS function. Specifically, trace amines can affect catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. We investigated whether microbiota from patients with GAD with no signs of immune activation can alter behaviour in gnotobiotic mice and whether this is accompanied by changes in metabolites within the gastrointestinal tract. Germ-free NIH Swiss mice (n=35) were colonized with microbiota from either a GAD patient (n=18) with severe anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Three weeks post- colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Microbiota profiles were assessed by 16S rRNA based Illumina, fecal β-defensin-3 level was measured by ELISA. After sacrifice, mouse brain BDNF and GDNF expression was assessed by immunofluorescence, and gene expression in the hippocampus, amygdala, and olfactory bulbs was assessed by Nanostring. Stool and cecum metabolites were measured in all colonized mice by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). There were no differences in fecal β-defensin levels between mice colonized with GAD microbiota as compared to mice colonized with HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice. Mice colonized with microbiota from a GAD patient had distinct bacterial profiles from mice colonized with HC microbiota. Compared to HC mice, GAD mice had lower levels of dopamine, octopamine and acetylcholine in cecum contents. Furthermore, GAD mice had higher expression of BDNF in the amygdala, lower expression of BDNF in the hippocampus, and lower expression of GDNF in the midbrain. GAD mice also had lower expression of CCR2 in the hippocampus, higher Cnlp/CAMP in the amygdala and olfactory bulb, and higher Nfkb1 in the olfactory bulb compared to HC mice. Our results suggest that microbiota from a selected patient with GAD has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of immune system, likely by altered production of biogenic amines and neurotransmitters. / Thesis / Master of Science (MSc)