La fibrose kystique (FK) est une maladie génétique causée par la mutation du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Celle-ci présente un défaut sur le canal à ions chlorure affectant ainsi entre autres la viscosité des muqueuses au niveau du système respiratoire. Cet environnement est alors propice aux colonisations bactériennes opportunistes sous la forme de biofilm. Burkholderia cepacia, bacille Gram-négatif mobile, multi-résistant aux antibiotiques et hautement transmissible, s'avère d'une extrême virulence pour les patients atteints de FK. Cette bactérie pathogène désigne en fait un ensemble de neuf souches rassemblées sous le nom de « complexe B. cepacia » (CBC). Au moins huit de ces neuf souches produisent un exopolysaccharide nommé Cepacian. Ceci est constitué d'un motif de répétition heptasaccharidique composé notamment de l'enchaînement α-D-Rhap-(1→4)-α-D-GlcpA. Le D-rhamnose (ou 6-deoxy-D-mannose) est un sucre rare et un composant de glycoconjugués des parois de bactéries pathogènes mais est absent chez l'homme. Ce dernier est donc un excellent candidat antigénique dans le cadre de la préparation d'un vaccin entièrement synthétique et spécifique. L'élaboration de celui-ci consiste en un activateur universel immunogénique peptidique (Tc-épitope) et/ou protéique (semi-synthétique) fonctionnalisé par une unité antigénique spécifique. Dans ce but, un tri-O-saccharide de constituant de LPS de B. cepacia et composé majoritairement de D-rhamnose, ainsi que des fragments du motif répétition de l'exopolysaccharide du CBC ont été synthétisés. Des méthodes de synthèses orthogonales ont été optimisées avec de hauts rendements sur les cinq types de sucres du CBC, spécifiquement le D-rhamnose, et l'acide glucuronique ainsi qu'une série de glycosylations. La synthèse linéaire en 18 étapes conduit à l'α-D-Rhap-(1→4 )-α-D-Galp-(1→3)-α-D-Rhap avec 11.3% de rendement global. La dernière étape sera la conjugaison de Tc-épitope par ce dernier afin d'obtenir un vaccin entièrement synthétique. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Vaccin synthétique, T-cell épitope, Burkholderia cepacia, Fibrose kystique, Oligosaccharide, D-rhamnose, Complexe B. cepacia.

Le facteur d'inhibition de la migration des macrophages (MIF) joue un rôle important dans la régulation du système immunitaire inné et adaptatif. Le MIF est impliqué dans plusieurs maladies inflammatoires chroniques dû à sa capacité à induire la sécrétion du facteur nécrosant tumoral (TNF-a) et de l'interleukine-12 (IL-12), en plus de favoriser l'expression des récepteurs de type Toll (TLR) 4 chez les macrophages. Paradoxalement, la neutralisation du MIF augmente l'activation des lymphocytes T cytotoxiques et diminue la réponse humorale in vivo qui sont des résultats inattendus considérant le rôle du MIF comme pro-inflammatoire et inducteur d'IL-12. Étant donné que la sécrétion de MIF est induite chez les souris BALB/c infectées avec Plasmodium chabaudi adami (DK) et que la résolution de cette infection nécessite une réponse Th1 couplée à une production d'IFN-y, nous avons évalué l'effet de la déficience en MIF sur la cinétique d'infection ainsi que sur l'activation des lymphocytes CD4+. Nos résultats montrent un meilleur contrôle de l'infection avec Plasmodium chabaudi adami (DK) chez des souris déficientes en MIF (MIF/KO), caractérisé par un pic et une parasitémie cumulative significativement plus faible. D'autre part, une anémie plus modérée, déterminée par une plus faible expression des précurseurs érythroïdes et des réticulocytes a aussi été mesurée chez des souris MIF/KO lors de l'infection. Au jour 4 post-infection, le pourcentage et le nombre absolu de cellules CD4+ spléniques activées était plus important chez les souris MIF/KO, de plus, ces cellules sécrétaient plus d'IFN-y, moins d'IL-10 et d'IL-4. De manière intéressante, la production précoce d'IL-4 par une population des cellules spléniques non B et non T (possiblement des basophiles) a été fortement inhibée chez les souris MIF/KO lors de l'infection. Une observation collatérale dans notre étude a été la surexpression du facteur de transcription T-bet chez des cellules T CD4 + naïves déficientes en MIF. Étant donné que les lymphocytes CD4+ déficients en MIf sécrètent plus d'IFN-y et moins d'IL-4 suite à leur stimulation in vitro, nos résultats suggèrent que le MIF module la capacité des lymphocytes CD4+ à sécréter des cytokines de type Th1. De plus, elle suggère aussi la présence d'une corrélation entre l'expression plus forte de T-bet et le phénotype Th1 accentué chez les lymphocytes CD4+ déficients en MIF. Par contre, cette régulation n'impliquerait pas la voie des récepteurs TLR étant donné qu'aucune modulation de ces récepteurs par le MIF n'a été observée. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : MIF, Plasmodium, IFN-y, IL-4, Lymphocyte

The pathology of multiple sclerosis (MS) is characterized by an inflammatory mononuclear infiltration in the white matter. There has been converging evidence of the oxidative stress playing a role in the onset and progression of MS. We postulated that the decreasing oxidative stress might help in the management of MS. We know that the induction of phase 2 enzymes decreases the oxidative stress. The experimental allergic encephalomyelitis (EAE) induced in the Lewis rats were used to test this hypothesis. The 24 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 7.5 g/kg of tetra-butylhydroxyanisole (BHA), a food preservative. All the animals were administered 100 µg of guinea pig myelin basic protein in their tails to induce EAE and examined daily in a double blinded fashion. On 29th day of the induction, the animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. All the animals, regardless of their diet status, developed symptoms of EAE on different days ranging from tail weakness to hind limb paralysis and all of them reached remission of acute EAE before the 28th day of induction. The non-BHA fed animals developed hind limb weakness in 8 animals and hind limb paralysis in 4 cases, while that of BHA fed group developed tail paralysis in 2, hind limb weakness in 2 and hind limb paralysis in 8 cases. The histology of the non-BHA group correlated well with the clinical symptoms of perivascular mononuclear infiltration. However, the BHA group revealed complete pathological recovery. Animals with BHA in the diet had significantly raised GSH, indicating the induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers show potential therapeutic benefits in EAE and should be examined for this role in MS.

<p>Transforming growth factor–β (TGF–β) signaling regulates a range of processes in a variety of cell types. Consequently, TGF–β plays a complex role in the progression of several types of cancers; it acts as a tumor suppressor in normal cells and early in tumor progression, yet it can promote tumor progression in later stages of cancer.</p><p>Among the cancers that TGF–β has been implicated in is glioblastoma multiforme (GBM), the most common primary brain neoplasm and one of the most lethal types of cancer. Because of its high mortality rate and the lack of effective treatments, discovering the molecular mechanisms that underlie GBM formation and growth is of great clinical interest. To this end, we investigated the function of a TGF–β target gene — the putative tumor suppressor N‐Myc downstream‐regulated gene 4 (NDRG4) — in GBM cell viability, proliferation and tumor formation. Contrary to the established roles of other NDRG family members, we found that NDRG4 expression is elevated in GBM and that NDRG4 is required for the survival of established GBM cell lines and primary GBM xenograft cells enriched for highly tumorigenic GBM cancer stem cells. Knockdown of NDRG4 expression results in G₁ cell cycle arrest followed by apoptosis that is associated with a decrease in the expression of XIAP and survivin. Finally, knockdown of NDRG4 expression in established GBM cell lines and GBM cancer stem cells results in decreased tumorigenicity following intracranial implantation of these cells into immunocompromised mice. Collectively, these data indicate that NDRG4 does not function as a tumor suppressor like other NDRG family members, but rather it is essential for GBM tumorigenicity and may represent a potential therapeutic target for this devastating disease.</p><p>In the second portion of this dissertation, we examine the TGF–β cytostatic signaling pathway in B lymphocytes. TGF–β–induced growth inhibition is the most extensively studied biological response to a TGF–β signal. Although in most cell types this response is mediated by Smad3– dependent regulation of c–Myc, p15<super>Ink4B</super>, and p21<super>Cip1</super> transcription, studies from Smad3 null mice suggest that TGF–β–induced growth inhibition in B lymphocytes occurs regardless of Smad3 status. We prove that this response does indeed occur independently of Smad3 in purified primary B lymphocytes and WEHI–231 cells. Consistent with this, p15<super>Ink4B</super> and p21<super>Cip1</super> are not noticeably induced by TGF–β in these cells, whereas Id3 and cyclin G2 are induced in a Smad3–independent manner. Finally, unlike the MAPK pathways we tested, the BMP–specific Smads 1 and 5 are activated in response to TGF–β in these cells, and this activation is dependent on ALK5 kinase activity. Collectively, these data indicate that TGF–β induces growth inhibition in B lymphocytes through a novel signaling pathway, and Smads 1 and 5 may help mediate this response.</p> / Dissertation

<p>The E-protein transcription factors E2A and HEB regulate various cell processes during the development of B and T lymphocytes, including cell differentiation, lineage commitment, recombination of immune receptor genes, proliferation, and survival. B cell development is dependent on E2A from the earliest stages whereas T cell development relies on the cooperative efforts of both E2A and HEB. Established work demonstrates that the timing and dosage of E-protein expression is critical for mediating these diverse functions. The goal of this dissertation is to develop and utilize new genetic tools to manipulate the timing and dosage of E2A and HEB expression in order to enhance our understanding of E-protein function. Here we develop two new mouse models to identify novel lineage and stage specific roles of E-proteins during B lineage commitment and thymocyte selection.</p><p>First, we have generated an E2A inducible mouse model to allow reversible regulation of E2A function and precise timing of induction at the protein level. This system was created by inserting a tamoxifen responsive region of the estrogen receptor ligand binding domain (ER) at the carboxyl end of the <italic>tcfe2a</italic> gene, encoding E2A, to generate E2AER fusion proteins. To our knowledge, the ER fusion system has not yet been tested from an endogenous locus in live animals. Using the E2AER system, we have demonstrated rapidly induced E2AER activity upon tamoxifen treatment that is capable of supporting B cell development in an <italic>ex vivo</italic> culture system. In addition to characterizing the kinetics and reversibility of this inducible system, we have utilized tamoxifen treatment of E2AER B cell progenitors to identify potential novel E2A target genes driving B lineage commitment.</p><p>Second, we have analyzed E-protein function during the double positive (DP) stage of alpha beta T cell development by using a Cre-loxp conditional deletion system. Here, E-protein dosage was manipulated by removal of both E2A and HEB, and the timing of deletion was controlled by using a CD4Cre transgene. During development, survival through the DP stage and initiation of differentiation to the subsequent single positive (SP) stage for generation mature alpha beta T cells is dependent on the production of a functional alpha beta T cell receptor (TCR). The mechanism that maintains cells at the DP stage prior to expression of a mature TCR remains unclear. In this study, we have shown that E2A and HEB together are required to maintain DP fate and regulate the transition to the SP stage. Loss of E2A and HEB in DP thymocytes was sufficient to trigger DP to SP differentiation, even in the absence of a TCR. Deletion of E2A and HEB allowed cells to bypass the requirement for a TCR-mediated positive selection signal. These findings identify E2A and HEB as key regulators enforcing thymocyte positive selection to ensure maturing T cells express a functional receptor.</p> / Dissertation

Abstract The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens, 3-amino-1-methyl-5 H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), and/or H2O2 was evaluated using single-cell electrophoresis (comet assay). The toxicity of these tested compounds on lymphocytes was not found. These compounds did not cause DNA damage at lower concentration of 0.1-10 &#x00B5;g/ml. At a concentration of 100 &#x00B5;g/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA damage. TA and its related compounds decreased the DNA damage induced by Trp-P-2 or PhIP at a concentration of 0.1-10 &#x00B5;g/ml. Moreover, the inhibition of H2O2-induced DNA damage increased with increasing concentrations up to 10 &#x00B5;g/ml. DNA repair enzymes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG) were used to examine the levels of oxidised pyrimidines and purines in DNA damage induced by H2O2, respectively. All the compounds at 10 &#x00B5;g/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicate that these compounds can enhance lymphocyte resistance towards DNA damage induced by food mutagens or H2O2. Keywords: Tannic acid; Human lymphocyte; Comet assay; Hydrogen peroxide

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Recent studies support the association between metabolic syndrome (MetS), a cluster of cardiovascular risk factors, and diastolic dysfunction. Disproportionate collagen accumulation, particularly cross-linking of collagen, plays a key role in translating interstitial fibrosis into mechanical chamber stiffness and diastolic dysfunction. Characteristic changes in the expression and activity of myocardial lysyl oxidase (LOX), a matrix modifying enzyme that catalyzes cross-linked collagen, are unclear in MetS. We established a diet-induced MetS model to study diastolic dysfunction by treating male C57BL/6 mice a high-fat high-simple carbohydrate (HFHSC) diet for 6 months. Despite blunted gene expression of LOX isoforms, MetS mice demonstrated significant increase in the ratio of protein expression of mature to proenzyme LOX, enhanced LOX activity, and increased cardiac cross-linked collagen compared with controls. This fibrotic response coincided with marked increase in left ventricular end-diastolic pressure and stiffness and impaired diastolic filling pattern. Our data demonstrate that diet-induced MetS alters the remodeling enzyme LOX, thereby increasing the amount of crosslinking and inducing diastolic dysfunction.Furthermore we examined the role of T-lymphocytes in myocardial LOX regulation in diet-induced fibrotic hearts. Female SCID mice which are devoid of functional T-lymphocytes and C57BL/6 mice were treated with HFHSC diet for 12 months. Similar to male C67BL/6, female HFHSC-fed C57BL/6 mice demonstrated significant increase in maturation and catalytic activity of myocardial LOX, cross-linking, ventricular stiffness and diastolic dysfunction. Whereas induction of LOX protein was minimal in SCID mice compared with wild-type counterparts. Correspondingly fibrillar cross-linked collagen formation and diastolic dysfunction were less prominent in SCID mice. Our results suggest a potential role of T-lymphocytes in induction of myocardial stiffness and diastolic dysfunction through modulation of LOX-dependent collagen maturation.Moreover we studied the role of leptin, an adipokine over-produced in MetS with fibrotic effects in non-cardiac tissues, as a key mediator of profibrogenic responses in the heart by administrating leptin to C57BL/6 and leptin-deficient ob/ob mice. With exogenous leptin administration ob/ob mice displayed passive diastolic filling dysfunction that coincided with increase in myocardial collagen compared with ob/ob controls. Our findings suggest profibrotic effects of leptin in the heart, primarily through predominance of collagen synthesis over degradation.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

No description available.