Le domaine TIR (Toll/interleukin (IL)-1 receptor) est une composante essentielle du système immunitaire inné, celui-ci est présent dans les récepteurs TLR (Toll-like receptor) et les protéines adaptatrices associées comme MyD88 et TIRAP. La détection de pathogènes déclenche l'interaction entre les domaines TIR permettant ainsi l'initiation et la propagation de la signalisation par les TLRs. Aussi, de nombreux pathogènes produisent des effecteurs contenant un domaine TIR tels que BtpA et BtpB chez Brucella abortus, TirS chez Staphylococcus aureus ou TcpC chez l'uropathogènique Escherichia coli. Tous ces effecteurs bloquent la signalisation des TLRs et sont capables de perturber les voies de signalisation de l'immunité innée pendant l'infection. Cependant les mécanismes moléculaires impliqués restent la plupart du temps non caractérisés et dans certains cas controversés. Dans le but de mieux comprendre le fonctionnement de ce type d'effecteurs bactériens, j'ai caractérisé chez Pseudomonas aeruginosa PA7 un nouvel effecteur contenant un domaine TIR que nous avons renommé PumA pour Pseudomonas UBAP1 Modulator A. En parallele, j'ai aussi participé à des projets de caractérisation de deux autres effecteurs avec un TIR domain : BtpB et TirS. Ainsi, PumA est un facteur essentiel pour la virulence de P. aeruginosa PA7 et son domaine TIR est essentiel pour interaction avec deux protéines adaptatrice, TIRAP et MyD88. Durant l'infection de cellules épithéliales pulmonaires par P. aeruginosa PA7, PumA est responsable du contrôle de la translocation du facteur de transcription NF-κB dans le noyau. De plus, la production de PumA dans une souche de P. aeruginosa non-TIR confère à cette bactérie de nouvelles propriétés d'immuno-modulation. PumA cible aussi UBAP1, une protéine du complexe de tri endosomal requis pour le transport, ESCRT-I (endosomal sorting complex require for transport I) qui a été récemment montré pour moduler l'activation de récepteur de cytokine. Nos résultats montrent que UBAP1 peut s'associer avec TIRAP et MyD88, provoquant le mouvement de MyD88 à la membrane cytoplasmique, suggérant une nouvelle voie cellulaire commune entre UBAP1 et les TLRs, et révélant UBAP1 comme nouvelle cible pour des effecteurs bactériens dans le cadre du contrôle des réponses immunitaires de l'hôte / In higher eukaryotes, the innate immune system provides the first line of defense against invading pathogens. The Toll/interleukin-1 receptor (TIR) domain is an essential component of the innate immune system. This domain is present in Toll-like receptors (TLRs) and associated adaptor proteins such as MyD88 and TIRAP. Pathogen detection requires interaction between the TIR domains, which initiates and triggers propagation of TLR signaling. However, many pathogens produce a TIR domain-containing protein such as BtpA and BtpB in Brucella abortus, TirS in Staphylococcus aureus or TcpC in the uropathogenic strain Escherichia coli. These effectors block TLR signaling and are able to disrupt innate immune response during infection. However, the molecular mechanisms involved remain mostly uncharacterized and in some cases controversial. The objective of this thesis was to study bacterial effectors containing a TIR domain particularly at the molecular level. For this, we focused on Pseudomonas aeruginosa PA7, an atypical multi-drug resistant strain that contains an effector with a TIR domain that we named PumA, for Pseudomonas UBAP1 Modulator A. In addition, during these four years of thesis work I also participated in the characterization of two other effectors with a TIR domain: BtpB in B. abortus and TirS in S. aureus.We found that PumA is essential for virulence of P. aeruginosa PA7 and its TIR domain is the key element for interaction with two adaptor proteins MyD88 and TIRAP. During infection of lung epithelial cells by P. aeruginosa PA7, PumA is responsible for controlling the translocation of NF-?B into the nucleus indicative of activation of this transcription factor. In addition, production of PumA by a TIR-deficient strain of P. aeruginosa confers to this bacterium a new immuno-modulation property. Furthermore, PumA targets ubiquitin-associated protein 1 (UBAP1), a protein of the endosomal sorting complex required for transport I (ESCRT-I) which has recently been shown to modulate cytokine receptor activation. Our results also show that UBAP1 can associated with TIRAP and MyD88, causing movement of MyD88 to the cytoplasmic membrane and suggesting a new cellular pathway between UBAP1 and TLRs. In summary, our data reveal UBAP1 as a novel target for bacterial effectors implicated in control of host immune responses

The grey squirrel (Sciurus carolinensis) has wildtype and melanic (dark) colour morphs. Melanism is associated with variations in the melanocortin-1 receptor (MC1R) gene in a number of species. The MC1R protein is a G-protein coupled receptor, predominantly expressed in melanocytes, where it is a key regulator of pigment production. To investigate the genetic and molecular basis of melanism, the MC1R genes of the wildtype and melanic grey squirrel were sequenced. The wildtype (MC1R-wt) and melanic (MC1RΔ24) variants of the MC1R were then functionally characterised in a cell-based assay. The MC1R gene of the grey squirrel was found to have a 24 base pair (bp) deletion associated with melanism. The MC1R is typically activated by its agonist, the alpha-melanocyte stimulating hormone (α-MSH), which stimulates dark pigment production by raising intracellular cAMP levels. Conversely, the MC1R is inactivated by its inverse agonist, the agouti signalling protein (ASIP), which stops dark pigment production by lowering intracellular cAMP levels. To investigate the effects that the 24 bp deletion have on receptor function, MC1R-wt and MC1RΔ24 genes were transfected into HEK293 cells. Cells expressing either MC1R-wt or MC1RΔ24 were stimulated with α-MSH or ASIP and intracellular cAMP levels were measured. Unstimulated MC1RΔ24 cells showed higher basal activity than the MC1R-wt cells. Both MC1R-wt and MC1RΔ24 cells responded to α-MSH with a concentration-dependent increase in intracellular cAMP. However, while the MC1Rwt cells responded to ASIP with a concentration-dependent decrease in intracellular cAMP, MC1RΔ24 cells responded with an increase in cAMP. Melanism in the grey squirrel is associated with a 24 bp deletion in the MC1R. Cells expressing MC1RΔ24 have higher basal levels of cAMP than MC1R-wt cells. ASIP acts as an inverse agonist to the MC1R-wt but as an agonist to the MC1RΔ24. As MC1RΔ24 cells have higher levels of cAMP, and higher levels of cAMP lead to dark pigment production, the 24 bp deletion is the likely molecular cause of melanism in the grey squirrel.

The sigma-1 receptor, although originally classified as an opioid receptor is now thought of as distinct receptor class, sharing no homology with any other known mammalian protein. The receptor has been implicated with a number of diseases including cancer and depression. Modulation of the receptors activity with agonists has potential antidepressant activity whereas antagonists lead to death of cancer cells. Using radioligand binding assays, utilizing the cancer cell line MDA-MB-468, which highly expresses the sigma-1 receptor, a series of novel specific, high affinity, sigma-1 receptor ligands have been characterised. These ligands differed from any previous sigma- 1 receptor ligand in that they are very simple ammonium salts, containing a single nitrogen atom and either straight or branched carbon chains. The binding studies revealed that the straight-chain ammonium salts gave nH values of 1 whereas the branched-chain ammonium salts had statistically significant lower nH values. The ammonium salts were tested for sigma-1 receptor activity in vitro using ratiometric Fura-2 calcium assays and the MTS cell proliferation assay. Branched-chain ammonium salts appeared to have sigma-1 receptor antagonist like effects on cytoplasmic calcium and cell proliferation, whereas the straight-chain ammonium salts behaved as sigma-1 receptor agonists. Three ammonium salts stood out as potential effective sigma-1 receptor drugs, the straight-chain ammonium salt dipentylammonium, and two branched-chain ammonium salts, bis(2-ethylhexyl)ammonium and triisopentylammonium. The ammonium salts were then tested in vivo. Dipentylammonium showed significant antidepressant properties when tested in behavioural models for depression and bis(2-ethylhexyl)ammonium and triisopentylammonium were able to significantly inhibit the growth of tumours implanted in mice. Finally I looked at the coupling of the sigma-1 receptor with G-proteins and show that sigma-1 receptor antagonists dose dependently reduce G-protein activity and inhibition of G-proteins enhanced the sigma-1 antagonists' effects of calcium signalling.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

<p><strong><p>Abstract</p><em><p>Background and aim:</p>Diabetes mellitus (DM) is a chronic and progressive illness that affects all type of populations and ages. According to World health organization (WHO) by 2030 it will be 366 million people effected world wild. Many new drugs are Glucagon-like peptide-1 (GLP-1) based therapy for treatment of type 2diabetes. GLP-1 is released from the intestinal L-cells, and is a potent stimulator of glucose-dependent insulin secretion. The aim of this study was to investigate the effect of GLP-1 and its stable analogs on cell proliferation of GLP-1 secreting GLUTag cells. <em><p>Material and methods:</p>GluTag cells were incubated for 48h in DMEM medium containing (0.5% fetal bovine serum and 100 IU/ml penicillin and 100 μg/ml streptomycin and 3mM glucose concentration) in the present or absence of the agents. DNA synthesis was measured using 3H- thymidine incorporation and Ki67 antigen staining. Western blot were performed to investigate the present of GLP-1 receptor in GLUTag cells. <em><p>Results/conclusions:</p><p>These results suggest that GLP-1 regulates proliferation of the GLP-1-secreting cell through a feedback mechanism via its receptor. Since serum GLP-1 levels are decreased in type 2 diabetic patients, the effect of GLP-1 on the GLP-1-secreting cell proliferation suggested here provides a novel beneficial long-term effect of the incretin-based drugs in clinical practice i.e. through increase of the GLP-1-secreting cell mass, augmenting the incretin effect. In addition, the feedback mechanism action of GLP-1 reveals a new insight in regulation manner of the L-cell proliferation.</p>GLP-1(7-36) increased cell proliferation in GLUTag cells, an effect which was blocked by the GLP-1 receptor antagonist exendin(9-39). The GLP-1 receptor was expressed in GluTag cells. <em><p>Keywords:</p>Incretin hormone<em>, GLP-1, GLP-1 receptor, Exendin-4, Diabetes </em></em></em></em></em></strong></p>

Abstract Background and aim: Diabetes mellitus (DM) is a chronic and progressive illness that affects all type of populations and ages. According to World health organization (WHO) by 2030 it will be 366 million people effected world wild. Many new drugs are Glucagon-like peptide-1 (GLP-1) based therapy for treatment of type 2diabetes. GLP-1 is released from the intestinal L-cells, and is a potent stimulator of glucose-dependent insulin secretion. The aim of this study was to investigate the effect of GLP-1 and its stable analogs on cell proliferation of GLP-1 secreting GLUTag cells. Material and methods: GluTag cells were incubated for 48h in DMEM medium containing (0.5% fetal bovine serum and 100 IU/ml penicillin and 100 μg/ml streptomycin and 3mM glucose concentration) in the present or absence of the agents. DNA synthesis was measured using 3H- thymidine incorporation and Ki67 antigen staining. Western blot were performed to investigate the present of GLP-1 receptor in GLUTag cells. Results/conclusions: These results suggest that GLP-1 regulates proliferation of the GLP-1-secreting cell through a feedback mechanism via its receptor. Since serum GLP-1 levels are decreased in type 2 diabetic patients, the effect of GLP-1 on the GLP-1-secreting cell proliferation suggested here provides a novel beneficial long-term effect of the incretin-based drugs in clinical practice i.e. through increase of the GLP-1-secreting cell mass, augmenting the incretin effect. In addition, the feedback mechanism action of GLP-1 reveals a new insight in regulation manner of the L-cell proliferation. GLP-1(7-36) increased cell proliferation in GLUTag cells, an effect which was blocked by the GLP-1 receptor antagonist exendin(9-39). The GLP-1 receptor was expressed in GluTag cells. Keywords: Incretin hormone, GLP-1, GLP-1 receptor, Exendin-4, Diabetes

Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.

Interleukin-1 (IL-1) is a prototypic multifunctional cytokine. IL-1 family include interleukin-1 a (IL-1 a), interleukin-1b (IL-1 b) and interleukin-1 receptor antagonist (IL-1 Ra). IL-1 b is the archetypeal pleiotropic cytokine which have been produced by many cells and exerting its biological effects on almost all cell types. IL-1 b is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory and an inhibitor of gastric emptying. IL-1 Ra competes with IL-1 b for cell surface receptor occupancy. Host genetic factors that affect interleukin-1 (IL-1) have been reported to influence the susceptibility of Caucasians to gastric cancer. Whether Asians have the same genetic susceptibility remains unclear. In this study, the genetic associations of IL-1B and IL-1RN polymorphisms with gastric cancer and duodenal ulcer in Taiwan were evaluated. Genomic DNA from 140 unrelated Taiwanese patients with gastric adenocarcinoma, 94 with duodenal ulcer and 165 ethically matched healthy controls was typed for polymorphisms at positions ¡V31, -511, and +3954 in the IL-1B gene, and the variable number of tandem repeats polymorphisms in intron 2 of the IL-1RN gene. The allele frequencies of IL-1RN 2R in gastric cancer cases were much higher than those in healthy controls (9% vs. 3%, p = 0.781). The allele frequencies of IL-1B ¡V31, IL-1B ¡V511 and IL-1B +3954 did not differ. An increased risk of the development of intestinal type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 4.06 (95% confidence interval [CI]: 1.68 ¡V 9.79, p-value=0.085). And another increased risk of the development of diffuse type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 3.15 (95% confidence interval [CI]: 1.16 ¡V 8.56, p-value=0.061). A significant association was found in IL-1RN 2R/4R genotype and the risk of the development of duodenal ulcer, with an odds ratio (OR) of 2.57 (95% CI: 1.03 ¡V 6.38, p = 0.292). No significant relationship was noted in duodenal ulcer patients with IL-1B genotype examed in this study. Additionally, a synergistic interaction between blood type A and IL-1 RN 2R carriers existed in gastric cancer patients (OR= 4.51; 95% CI: 1.20 ¡V 16.88, p-value=0.516). The synergistic interaction was even stronger between blood type O and IL-1 RN 2R carriers of duodenal ulcer patients (OR= 10.3; 95% CI: 2.10 ¡V 50.61, p-value=0.160). In conclusion, the genetic polymorphisms of IL-1RN 2R and blood type A are associated with the development of gastric cancer. The genetic polymorphisms of IL-1RN 2R and blood type O are associated with the development of duodenal ulcer.

A scourge of Type I and Type II diabetes impacts the health of hundreds of millions worldwide. The number and prevalence of diabetics are expected to rise dramatically in the next two decades. Diabetes is defined by chronic hyperglycemia which can result in a number of detrimental and costly metabolic, renal, cardiovascular and neurological disorders. Identification of at risk individuals and effective blood glucose management are critical to improving diabetic outcomes and preventing hyperglycemic complications. Diabetes prevention and treatment is limited by the understanding of islet function and mass in the diabetogenic and diabetic state. The islets of Langerhans are dispersed throughout the pancreas and comprise <2% of the pancreatic mass. The reclusive nature of islet cells presents unique challenges understanding disease development. No agent capable of exclusively targeting pancreatic β-cells within the islet has been discovered and the lack of targeting agent specificity impedes efforts to: quantify β-cell mass and develop novel therapeutics. We propose β-cell targeting can be improved by targeting unique combinations of receptors simultaneously with multivalent ligands. A synthetic multivalent agent composed of two β-cell specific diabetic therapeutics, glucagon-like peptide-1 (GLP-1) and glibenclamide (Glb), targeted against the GLP-1R and the sulfonylurea-1 receptor (SUR1) is a lead compound for the development of specific bi-functional islet cell targeting agents for use in the in vivo detection and treatment of β -cells. Herein, we describe the synthesis and initial characterization of a heterobivalent ligand composed of GLP-1 coupled to Glb. The heterobivalent ligand binds to an unaltered β-cell line with increased specificity relative to a human pancreatic exocrine cell line. Additionally, receptor cross-linking modifies β-cell signaling. Exposure of β-cells to the heterobivalent ligand results in antagonism of SUR1-Ca²⁺ signaling and equipotent agonism of GLP-1R-cAMP signaling, in comparison to the cognate monomeric ligands (Glb and GLP-1). Perturbations in intracellular signaling modifies β-cell insulin secretion resulting in decreased basal insulin secretion and with maintained yet reduced ability to potentiate β-cell glucose stimulated insulin secretion. GLP-1/Glb β-cell specificity and functional modulation suggests combinatorial receptor targeting is an effective strategy for the development of bi-functional cell-specific targeting agents, warranting further investigation and optimization.

The melanocortin system regulates various physiological processes including feeding behavior, sexual function, skin pigmentation and photoprotection via five G-protein coupled receptors and several endogenous ligands. There is a need for selective and potent ligands to the human melanocortin receptors (hMCRs) that can chemically resolve these various functions. This thesis presents three studies aimed at refining the understanding of the structural differences between binding pockets of the hMCR subtypes. In the first study α-N-methylated analogues of the non-selective agonist, MT-II, are evaluated for their in vitro function. This study produced the most potent hMC1R selective agonist to date. The following two studies examine the effects of mutations on the biological activity of melanocortin receptor subtypes 1 and 4. Much of the mutation study data is preliminary and requires a demonstration of reproducibility.