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Les lymphocytes T ?d seraient des effecteurs essentiels dans la réponse immunitaire aux stress induits notamment par les infections et la tumorigénèse. Plusieurs arguments dont leur localisation intra-épithéliale mais aussi leurs capacités effectrices multiples et rapides les caractérisent comme des acteurs primordiaux dans l’immunité anti-tumorale. Mon projet de thèse consistait à examiner le potentiel anti-tumoral des lymphocytes T ?d humains, Vd2 négatifs (neg), dans un contexte in vivo, grâce à l’utilisation d’un modèle murin. Des études antérieures menées au laboratoire démontraient une expansion de lymphocytes T Vd2neg dans la circulation sanguine de transplantés rénaux développant une infection à cytomégalovirus (CMV). Des clones T Vd2neg, isolés de ces patients, présentaient une forte réactivité in vitro contre des cellules infectées par le CMV mais aussi contre des cellules tumorales notamment d’origine colique (comme la lignée HT29). Un ligand commun induit par l’infection à CMV et la transformation tumorale, reconnu par les clones T Vd2neg serait à l’origine de cette double réactivité. La première partie de mon projet s’est concentrée sur l’étude du potentiel anti-tumoral de ces clones T Vd2neg in vivo, qui comprenait leur capacité à atteindre des cellules tumorales d’origines coliques (HT29) et à les lyser. Dans un modèle de xénogreffe dans des souris immunodéficientes, nous avons démontré que les clones TVd2neg, injectés dans le péritoine (i.p) pouvaient retarder la croissance de tumeurs solides HT29 sous-cutanées. D’après nos résultats, cette inhibition du développement tumoral proviendrait d’une action précoce et spécifique des cellules T Vd2neg et impliquerait le récepteur à chimiokine CCR3. Nos données suggèrent donc que des lymphocytes T Vd2neg, réactifs contre le CMV, pourraient migrer in vivo jusqu’au site d’injection des cellules tumorales et inhiber la croissance de la tumeur probablement grâce à leur acticité cytolytique. La deuxième partie de mon projet de thèse proposait d’approfondir l’étude du rôle des lymphocytes T Vd2neg contre les tumeurs coliques. Ainsi nous avons testé, in vivo, l’implication de lymphocytes T Vd1+ humains, une population représentative des épithéliums intestinaux, dans le cancer métastatique colorectal (CMC). Nous avons développé un modèle d’implantation orthotopique de cellules tumorales HT29 dans des souris immunodéficientes, qui mime le développement du CMC chez l’homme. Des tumeurs primaires intra-caecales et des métastases pulmonaires et hépatiques se développent chez les souris. De plus, nous avons pu suivre leur croissance grâce à l’introduction de la luciférase dans les HT29 et à une technique d’imagerie in vivo en bioluminescence. Nos résultats montrent qu’un traitement continu des souris par des injections de lignée T Vd1+ en i.p inhibe le développement des tumeurs primaires et retarde l’apparition des métastases à distance. Ces données soutiennent l’implication des lymphocytes T Vd2neg dans le contrôle des CMC. De façon intéressante, elles mettent en avant une implication anti-métastatique des cellules T Vd2neg. L’ensemble de nos travaux souligne le rôle des cellules T Vd2neg dans la réponse immunitaire contre les cancers colorectaux et étaye leur potentiel d’action lors de la progression des tumeurs vers des métastases, ouvrant ainsi des perspectives pour l’utilisation de ces cellules dans les thérapies des CMC. / Gamma delta (?d) T lymphocytes contribute to host immune competence uniquely especially during stress immune responses to infections and tumors. Because ?d T cells colonize epithelial surfaces, where they can exert rapid and pleiotropic effector functions, they are critical protagonists in anti-cancer response. During my Phd project we explored the anti-tumor potential of Vd2 negatives (neg) ?d T lymphocytes, in vivo using a mouse xenograft tumor model. A few years ago, studies in our laboratory showed an increase of peripheral blood Vd2neg ?d T lymphocytes in allograft recipients infected by cytomegalovirus (CMV). Interestingly, Vd2neg ?d T clones isolated from these patients showed a cytotoxic activity against CMV infected fibroblast in vitro. Moreover, they were able to kill colon cancer cells (HT29) in vitro, in contrast to normal epithelial cells. Cancer cell- as well as CMV infected cell- killing involved T cell receptor (TCR) engagement, independently of major histocompatibility complex (CMH) recognition, probably with a common ligand. The first part of my Phd project was undertaken to evaluate the in vivo tumor reactivity of anti-CMV Vd2neg clones, including their ability to inhibit tumor growth as well as their migratory potential toward colon cancer cells. In immunodeficient mice, we showed that systemic intraperitoneal (i.p) injections with human Vd2neg clones inhibited the growth of HT29 hypodermal tumors xenografts. Furthermore, our results demonstrated that Vd2neg T cells had an early and specific anti-tumor effect, and that such activity could be hampered in vivo using an anti-CCR3 antibody. Our study suggest that Vd2neg T cells with an anti-viral potential are able to reach a tumor site in vivo, and inhibit tumoral growth exercising a cytolytic activity. The second part of my Phd project proposed to get further insights on the role of Vd2neg T cells in the immune surveillance against colon cancer. To this aim, we tested, the involvement of human Vd1+ T lymphocytes, a substantial fraction of T cells in intestinal epithelia, in limiting tumor spread in vivo, using a mouse model of colorectal carcinoma (CRC). We sat up a physiological mouse model of CRC by orthotopic microinjection of HT29 colon cell, which mimics the natural history of human CRC. Indeed, primary colic tumors and pulmonary and hepatic distant metastases grew in mice. Furthermore, bioluminescence imaging was used to follow the outcome of luciferase expressing cancer cells. We showed that systemic treatment with human Vd1+ T lymphocytes could inhibit the growth of intracaecal HT29 tumors and led a substantial reduction of distant metastases. Our results are the first arguing for a crucial role of ?d T cells against CRC, specially in preventing the dissemination of colon cancer cells. Taken together, our results underline the role of of ?d T cells in theimmune response against colorectal cancer. Our findings put forward Vd2neg T cells as attractive candidates for novel anti-tumor immunotherapy protocols.

MicroRNAs (miRNAs) are short, non-coding RNAs involved in the regulation of several processes associated with inflammatory diseases and infection. Bacterial infection modulates miRNA expression to subvert innate immune response. In this study, we analyzed bacterial modulation of miRNAs in bone-marrow-derived macrophages (BMMs), in which activity was induced by infection with Porphyromonas gingivalis (Pg) through a microarray analysis. Several miRNA expressions levels were modulated 3 hours post infection (at a multiplicity of infection (MOI) of 25). A bioinformatics analysis was performed to further identify pathways related to the innate immune host-response pathways that are under the influence of the selected miRNAs. To assess the effects of the identified miRNAs on cytokines secretion (pro inflammatory TNF-α and anti-inflammatory IL-10), BMMs were transfected with selected miRNAs mimics or inhibitors. Transfection with mmu-miR-155 and mmu-miR- 2137 did not modify TNF-α secretion while their inhibitors increased it. Inhibitors of mmumiR-2137 and mmu-miR-7674 increased the secretion of the anti-inflammatory IL-10. In Pginfected BMMs, mmu-miR-155-5p significantly decreased TNF-α secretion while inhibitor of mmu-miR-2137 increased IL-10 secretion. In vivo, in a Pg-induced calvarial bone resorption mouse model, injection of mmu-miR-155-5p or anti-mmu-miR-2137 reduced the size of the lesion significantly. Furthermore, anti-mmu-miR-2137 significantly reduced inflammatorycell infiltration, osteoclast activity and bone loss. Bioinformatics analysis demonstrated that pathways related to cytokines and chemokines related pathways but also osteoclast differentiation may be involved in the observed effects. The study highlights the potential therapeutic merits of targeting mmu-miR-155-5p and mmu-miR-2137 to control inflammation induced by Pg infection. To assess the regenerative process in the same animal model, we aimed to compare the effect of Bone Morphogenic Protien 2 (BMP2), Platelets Rich Plasma (PRP), Leukocyte-Platelets Rich Fibrin (L-PRF), and Polygucosamine (PGIcNAc) on bone formation in critical size bone defects in mice. One-hundred-thirty-eight mice were divided into 23 groups (n=6), negative control, different combinations of the PGIcNAc with or without of BMP2, Collagen Sponge (SurgiFoam), PRP, and L-PRF. The 5mm defect, then, was allowed to heal. After six weeks, samples were analyzed for bone formation utilizing radiographs, H&E staining, alkaline phosphatase staining. Our results show that BMP2 were able to produce 90-95% healing of critical size defects after six weeks histologically and radiographically. However, SurgiFoam, PRP and L-PRF with or without PGIcNAc were able to close 60% of the original defect. This study supports that BMP2 is more effective for bone regeneration than SurgiFoam, PRP, L-PRF and PGIcNAc.

THESIS SUMMARY Food allergy is one of the frequent disorders and its incidence in paediatric as well as adult population is continuously rising, having doubled in the last two decades. Although wheat belongs to major food allergens and is a staple food in most diets, we have only little knowledge of wheat proteins causing IgE mediated hypersensitivity reaction. Diagnostic approaches of food allergy to wheat have a high sensitivity, but low specificity. Poor predictability and specificity may be associated with the insufficient purity of wheat extracts used in sIgE assays or with the lack of major allergens in these extracts. In the first step, we characterized 19 potential allergens recognised by IgE Abs of allergic patients, using proteomic techniques (1-DE, 2-DE, MALDI-TOF, QTOF and LCQDECA nLC-MS/MS ion trap technique). We identified these IgE-binding molecules such as: α-amylase inhibitors, β-amylase, profilin, serpin, β-D-glucan exohydrolase and 27K protein. To quantify sIgE in patient's sera we developed ELISA using the whole wheat extract and two commercially available α-amylase inhibitors. Second, we developed a procedure that allows isolation of wheat allergens from natural sources using Rotofor cell and HPLC. Twenty-seven potential wheat allergens have been successfully identified; of these, the...

O herpesvirus equino tipo 1 (EHV-1) é um importante patógeno que causa doença respiratória, abortamento e desordens neurológicas em equinos. O presente estudo foi realizado visando estabelecer um modelo murino de infecção pelo EHV-1 para investigar a resposta do hospedeiro frente à infecção viral e as alterações neurológicas causadas por esse agente. Camundongos das linhagens BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- e C57BL/6 CD8-/- foram inoculados por via intranasal com as estirpes brasileiras A4/72, A9/92 e A3/97 do EHV-1. Neste estudo, associou-se a histopatologia, a imunoistoquímica e o método de transcrição reversa seguida pela PCR quantitativa em tempo real para investigar a relação entre a infecção pelo vírus com o desenvolvimento de lesões e a resposta de citocinas pró-inflamatórias no SNC de camundongos das diferentes linhagens. As estirpes brasileiras A4/72 e A9/92 do EHV-1 causaram infecção aguda e letal nas diferentes linhagens de camundongos isogênicos. Os sinais clínicos e neurológicos, tais como perda de peso, pelos arrepiados, postura arqueada, apatia, descarga nasal e ocular, dispnéia, desidratação e sialorréia apareceram entre o 2º e 3º dpi. Essas manifestações foram acompanhadas pelo aumento da sensibilidade a estímulos externos, convulsões, recumbência e morte. O vírus foi consistentemente isolado do SNC, pulmões, fígado, baço e timo de todos os camundongos com sinais neurológicos. As alterações histopatológicas consistiram de leptomeningite, hemorragia focal, ventriculite, degeneração e necrose neuronal, neuronofagia, inflamação não supurativa, gliose multifocal e infiltração perivascular de células polimorfonucleares e mononucleares. A análise imunoistoquímica demonstrou que as estirpes A4/72 e A9/92 do EHV-1 replicaram-se nos neurônicos do bulbo olfatório, cortex cerebral e no hipocampo. Ao contrário, os camundongos inoculados com a estirpe A3/97 do EHV-1 não apresentaram perda de peso ou quaisquer sinais clínicos ou neurológicos; entretanto, o vírus foi isolado dos pulmões no 3º dpi. As estirpes A4/72 e A9/92 do EHV-1 apresentaram tropismo pelo tecido nervoso com capacidade de neuroinvasão e neurovirulência. A estirpe A3/97 do EHV-1 não foi neurovirulenta, apesar de ter sido reisolada do SNC de camundongos BALB/c nude infectados. Detectou-se aumento da expressão de mRNA para TNF-α, IL-6 e CCL2 no SNC dos camundongos infectados pelo EHV-1 com 2 e 3 dpi; entretanto, não houve expressão de mRNA para IFN-γ. Os camundongos com o fundo genético C57BL/6, que apresentam predominantemente resposta do tipo Th1, mostraram níveis mais altos de expressão de mRNA para TNF-α, IL-6 e CCL2, quando comparados com os BALB/c. A gravidade dos sinais observados em camundongos infectados pode ser correlacionada com o pico destas citocinas pró-inflamatórias (TNF-α e IL-6) e da quimiocina CCL2, que são produzidas logo após a infecção viral por células residentes da glia e/ou infiltrativas no SNC. Esses achados indicam que as diferentes linhagens de camundongos isogênicos são susceptíveis a infecção por estirpes neuropatogênicas do EHV-1 e poderiam servir como modelo para o estudo da patogênese e dos mecanismos que contribuem no desenvolvimento da mieloencefalopatia herpética equina. / Equid herpesvirus type 1 (EHV-1) is a major pathogen which causes respiratory disease, abortions and neurological disorders in horses. The present study was carried out to establish a murine model of EHV-1 infection and investigate host response against the virus and neurological disorders caused by this pathogen. BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- and C57BL/6 CD8-/- mice were intranasally inoculated with EHV-1 A4/72, A9/92 and A3/97 Brazilian strains. In this study, we combined histopathology, immunohistochemistry, and a quantitative real-time RT-PCR method to investigate the relationship between virus infection and the development of lesions and cytokine responses in the CNS of different strains of mice. Intranasal inoculation of EHV-1 A4/72 and A9/92 induced acute and lethal meningoencephalitis in mice. Clinical and neurological signs appeared between the 2nd and 3rd dpi and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was consistently recovered from the CNS and visceral organs of all mice with neurological symptoms. Histopathological changes consisted of leptomeningitis, focal hemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multi-focal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, cortical regions and hippocampus. In contrast, mice inoculated with the EHV-1 strain A3/97 showed neither weight loss nor apparent clinical or neurological signs of the disease; however, the virus was recovered from their lungs at 3 dpi. While EHV-1 strains A4/72 and A9/92 exhibited a high degree of tropism for the CNS with robust neuroinvasiveness and neurovirulence, the EHV-1 strain A3/97 was not neurovirulent despite being detected in the CNS of infected BALB/c nude mice. Increased mRNA levels of TNF-α, IL-6 and CCL2 were detected in the nervous tissue of EHV-1 infected mice at 2 and 3 dpi; however, IFN-γ mRNA was not consistently expressed. Mice with the background C57BL/6, which exhibit predominantly Th1-type responses, showed the highest levels of TNF-α, IL-6 and CCL-2 mRNA in the CNS, when compared to BALB/c mice. The severity of signs observed in infected mice could be correlated with the peak of these proinflammatory cytokines (TNF-α and IL-6) and the chemokine CCL2, which are produced early after viral infection by both cells infiltrating into the CNS from the periphery and/or glial resident cells. These findings indicate that several inbred mouse strains are susceptible to neuopathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to equine herpes myeloencephalopathy in horses.

Die schlechte Prognose der meisten disseminierten Tumorerkrankungen ist häufig in einer vorbestehenden oder erworbenen Resistenz gegenüber Zytostatika begründet. Da die meisten Zytostatika mit zellulären Strukturen interagieren, war lange angenommen worden, dass der antineoplastische Effekt unmittelbar durch massive Zellschädigung bewirkt wird. Hieraus folgte, dass Chemoresistenz auf Mechanismen beruhen müsse, welche das Zytostatikum an der Wechselwirkung mit seiner intrazellulären Zielstruktur hindern. Arbeiten der letzten Jahre haben jedoch gezeigt, dass die meisten Zytostatika indirekt über DNA-Schädigung ein relativ uniformes, genetisch kodiertes Zelltod-Programm auslösen, demzufolge postuliert wurde, dass auch Apoptosedefekte für "Multi-Drug-Resistenz" verantwortlich sein könnten. Allerdings ist der tatsächlich Beitrag zytostatika-induzierter Apoptose am Therapieerfolg nicht geklärt, wobei der Wahl geeigneter Testsysteme eine wesentliche Bedeutung für diese Kontroverse zuzukommen scheint. Gegenstand der vorliegenden Arbeit ist daher die Etablierung eines transgenen Lymphom-Modells, in welchem chemotherapeutische Effekte an spontan entstandenen Tumoren mit definierten genetischen Läsionen in ihrer natürlichen Umgebung untersucht werden können. Hierbei konnte gezeigt werden, dass Mutationen in apoptose-relevanten Genloci wie p53, INK4a/ARF oder bcl2 sowohl die Manifestation myc-transgener Lymphome dramatisch beschleunigen, als auch den Therapieerfolg kompromittieren. Neben Apoptose wurde darüberhinaus prämature Seneszenz, ein terminaler Zellzyklus-Arrest, als prognose-relevantes Chemotherapie-Effektorprogramm identifiziert. Damit dokumentiert die vorliegende Arbeit einen wichtigen Zusammenhang von Gendefekten, die während der Tumorigenese erworben wurden, und später evidenter Chemoresistenz, wobei manche Mutationen bereits vor Zytostatika-Exposition Resistenz begründen können. Die Identifikation und pharmakogenomische Charakterisierung potentiell resistenz-vermittelnder Gene und Mutationen in relevanten Testsystemen wird für die Entwicklung spezifischerer, aber weniger toxischer "targeted Therapeutics" von großer Bedeutung sein. / Intrinsic or acquired chemoresistance is the major cause for the adverse outcome of disseminated malignancies. The fact that most anticancer agents bind to subcellular targets prompted the assumption that drug-induced cytotoxicity must be a direct consequence of severe cellular damage. Hence, chemoresistance was thought to arise from mechanisms that prevent or disrupt the drug-target interaction. By contrast, more recent data suggested that DNA damage caused by most, if not all, anticancer agents may trigger a relatively uniform, genetically encoded cell death program. In turn, defects in the apoptotic machinery should account for multi-drug resistance as well. However, due to technical limitations of current test systems, it has been difficult to assess the overall contribution of apoptotic cell death to treatment outcome. In the studies presented here, a transgenic mouse lymphoma model was established in order to exploit drug responses of spontaneously developed malignancies growing at their natural sites but harboring defined genetic defects. Using this model, alterations in apoptosis-related gene loci such as p53, INK4a/ARF or bcl2 result in both dramatic acceleration of myc-driven lymphomagenesis and compromised treatment responses. Importantly, not only apoptosis, but premature senescence, a terminal cell-cycle arrest, was found to impact on treatment outcome. In essence, this work describes and important connection between cancer genes and cancer therapy, i.e. genetic defects acquired during tumorigenesis may already co-select for chemoresistance prior to any drug encounter. The identification and pharmacogenomic evaluation of resistance conferring candidate genes and mutations using adequate test systems is likely to play a key role in the development of novel, more specific but less toxic so called "targeted Therapeutics".

Macrophages belong to the mononuclear phagocyte system and they perform fundamental roles to maintain homeostasis in the organism. Macrophage development, survival, proliferation and functionality depend upon the colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), which signal through the CSF1 receptor (CSF1R). CSF1R is a type III tyrosine kinase receptor that is present in the plasma membrane of monocytes and macrophages. Mutations in Csf1r in mice produce the loss of many tissue macrophage populations and multiple developmental abnormalities. In humans, abnormal enhancement of CSF1R expression has been correlated to adverse prognosis in a subset of carcinomas; and mutations in the human CSF1R are associated with an autosomal-dominant neurodegenerative disease. CSF1R is encoded by the c-fms proto-oncogene and its expression is partially controlled by the fms-intronic regulatory element (FIRE). The FIRE sequence is highly conserved across species and contains binding motifs for multiple transcription factors, which are relevant for haematopoiesis. Previous results from murine Csf1r transgenes showed that FIRE is essential for driving Csf1r expression, and that interactions between FIRE and multiple myeloid transcription factors contribute to maximal regulatory activity. This project aimed to study the role of FIRE in its normal chromatin context, in vivo. A FIRE knockout (FIRE-/-) mouse model was generated using the CRISPR/Cas9 technology in mouse embryonic stem cells (ESCs) and in mice. In ESCs, the deletion severely compromised the differentiation of macrophages from embryoid bodies generated in vitro. In mice, the frequency of the FIRE- /- genotype in the progeny does not follow a Mendelian distribution and about 5% of the offspring developed hydrocephalus. Unlike Csf1r -/-mice, which die before weaning, most surviving FIRE-/- mice grew normally and were fertile. The impact of the mutation on macrophage populations is selective. FIRE-/- mice are not monocyte deficient (identified as F4/80+ Csf1r+ cells in peripheral blood), although these cells have reduced levels of Csf1r mRNA and do not bind porcine CSF1 Fc fusion protein. The development of peritoneal macrophages and Iba-1+ microglia was abolished, but Adgre1+ (F4/80+) macrophage populations in liver and spleen were unaffected. Csf1r was greatly reduced in bone marrow progenitors, but about 30% of these cells were able to differentiate into macrophages in vitro, upon exposure to recombinant human CSF1 (rhCSF1). This study shows that FIRE is essential for the development of a subset of tissue-resident macrophage populations. In FIRE-/- mice, potential compensation from additional regulatory elements within Csf1r might underlie the development of unaffected tissue-resident macrophages.

Le processus visuel débute lorsque les photorécepteurs transforment la lumière en un signal biochimique qui est ensuite traité et transmis via la rétine. Notre groupe s'intéresse à élucider les défauts génétiques et les mécanismes à l'origine de pathologies rétiniennes comme la cécité nocturne congénitale stationnaire (CNCS), conséquence d'un défaut de transmission du signal entre les photorécepteurs et les cellules bipolaires. Cette thèse apporte de nouvelles connaissances sur la physiologie de cette première synapse visuelle. Nous avons identifié quatre nouvelles mutations dans SLC24A1, un échangeur ionique intervenant dans l'homéostasie du calcium dans les bâtonnets, à l'origine de la CNSC de type Riggs. Nous avons également identifié LRIT3 comme étant un nouveau gène impliqué dans la forme complète de CNCS. Nous avons décrit un modèle de souris invalidé pour Lrit3 avec un phénotype visuel similaire à celui des patients. Nous avons confirmé la localisation de LRIT3 aux extrémités dendritiques des cellules bipolaires ON, suggérant un rôle dans la cascade de signalisation mGluR6. Nous avons montré que LRIT3 était nécessaire à la localisation fonctionnelle de TRPM1. Nous avons de plus démontré un rôle additionnel de LRIT3 dans la formation de la synapse du cône n'impactant probablement que faiblement les voies OFF. Nous avons également réussi à détecter LRIT3 par spectrométrie de masse, ouvrant la voie à l'identification de ses partenaires. La meilleur connaissance de la physiologie et de la physiopathologie rétinienne doit mener non seulement à un meilleur diagnostic et conseil génétique des patients mais également au développement de nouvelles approches thérapeutiques. / The first steps in vision occur when rod and cone photoreceptors transform light into a biochemical signal, which gets processed through the retina. Our group investigates genetic causes and mechanisms involved in inherited retinal diseases as congenital stationary night blindness (CSNB), which reflects a signal transmission defect between photoreceptors and bipolar cells. This thesis gives several insights on the retinal physiology at this first visual synapse. We identified four novels mutations in SLC24A1 underlying the Riggs-type of CSNB, which has a role in calcium balance in rods. We subsequently identified a novel gene, LRIT3, which is mutated in the complete form of CSNB. We delivered a knock-out mouse model lacking Lrit3 which displays a phenotype similar to patients. We confirmed the localization of LRIT3 at the dendritic tips of ON-bipolar cells, suggesting a role of LRIT3 in the mGluR6 signaling cascade. We showed that LRIT3 is necessary for the functional localization of TRPM1. We also revealed that LRIT3 has an additional role in formation of the cone synapse but with probably only a minor effect on OFF-pathway functionality. We finally succeeded in immunoprecipitating and detecting LRIT3 by mass spectrometry, opening the way for the identification of LRIT3 partners. Improving knowledge about retinal physiology and physiopathology will lead to a better diagnosis and genetic counseling of the patients and to the development of novel therapeutic approaches.

O herpesvirus equino tipo 1 (EHV-1) é um importante patógeno que causa doença respiratória, abortamento e desordens neurológicas em equinos. O presente estudo foi realizado visando estabelecer um modelo murino de infecção pelo EHV-1 para investigar a resposta do hospedeiro frente à infecção viral e as alterações neurológicas causadas por esse agente. Camundongos das linhagens BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- e C57BL/6 CD8-/- foram inoculados por via intranasal com as estirpes brasileiras A4/72, A9/92 e A3/97 do EHV-1. Neste estudo, associou-se a histopatologia, a imunoistoquímica e o método de transcrição reversa seguida pela PCR quantitativa em tempo real para investigar a relação entre a infecção pelo vírus com o desenvolvimento de lesões e a resposta de citocinas pró-inflamatórias no SNC de camundongos das diferentes linhagens. As estirpes brasileiras A4/72 e A9/92 do EHV-1 causaram infecção aguda e letal nas diferentes linhagens de camundongos isogênicos. Os sinais clínicos e neurológicos, tais como perda de peso, pelos arrepiados, postura arqueada, apatia, descarga nasal e ocular, dispnéia, desidratação e sialorréia apareceram entre o 2º e 3º dpi. Essas manifestações foram acompanhadas pelo aumento da sensibilidade a estímulos externos, convulsões, recumbência e morte. O vírus foi consistentemente isolado do SNC, pulmões, fígado, baço e timo de todos os camundongos com sinais neurológicos. As alterações histopatológicas consistiram de leptomeningite, hemorragia focal, ventriculite, degeneração e necrose neuronal, neuronofagia, inflamação não supurativa, gliose multifocal e infiltração perivascular de células polimorfonucleares e mononucleares. A análise imunoistoquímica demonstrou que as estirpes A4/72 e A9/92 do EHV-1 replicaram-se nos neurônicos do bulbo olfatório, cortex cerebral e no hipocampo. Ao contrário, os camundongos inoculados com a estirpe A3/97 do EHV-1 não apresentaram perda de peso ou quaisquer sinais clínicos ou neurológicos; entretanto, o vírus foi isolado dos pulmões no 3º dpi. As estirpes A4/72 e A9/92 do EHV-1 apresentaram tropismo pelo tecido nervoso com capacidade de neuroinvasão e neurovirulência. A estirpe A3/97 do EHV-1 não foi neurovirulenta, apesar de ter sido reisolada do SNC de camundongos BALB/c nude infectados. Detectou-se aumento da expressão de mRNA para TNF-α, IL-6 e CCL2 no SNC dos camundongos infectados pelo EHV-1 com 2 e 3 dpi; entretanto, não houve expressão de mRNA para IFN-γ. Os camundongos com o fundo genético C57BL/6, que apresentam predominantemente resposta do tipo Th1, mostraram níveis mais altos de expressão de mRNA para TNF-α, IL-6 e CCL2, quando comparados com os BALB/c. A gravidade dos sinais observados em camundongos infectados pode ser correlacionada com o pico destas citocinas pró-inflamatórias (TNF-α e IL-6) e da quimiocina CCL2, que são produzidas logo após a infecção viral por células residentes da glia e/ou infiltrativas no SNC. Esses achados indicam que as diferentes linhagens de camundongos isogênicos são susceptíveis a infecção por estirpes neuropatogênicas do EHV-1 e poderiam servir como modelo para o estudo da patogênese e dos mecanismos que contribuem no desenvolvimento da mieloencefalopatia herpética equina. / Equid herpesvirus type 1 (EHV-1) is a major pathogen which causes respiratory disease, abortions and neurological disorders in horses. The present study was carried out to establish a murine model of EHV-1 infection and investigate host response against the virus and neurological disorders caused by this pathogen. BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- and C57BL/6 CD8-/- mice were intranasally inoculated with EHV-1 A4/72, A9/92 and A3/97 Brazilian strains. In this study, we combined histopathology, immunohistochemistry, and a quantitative real-time RT-PCR method to investigate the relationship between virus infection and the development of lesions and cytokine responses in the CNS of different strains of mice. Intranasal inoculation of EHV-1 A4/72 and A9/92 induced acute and lethal meningoencephalitis in mice. Clinical and neurological signs appeared between the 2nd and 3rd dpi and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was consistently recovered from the CNS and visceral organs of all mice with neurological symptoms. Histopathological changes consisted of leptomeningitis, focal hemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multi-focal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, cortical regions and hippocampus. In contrast, mice inoculated with the EHV-1 strain A3/97 showed neither weight loss nor apparent clinical or neurological signs of the disease; however, the virus was recovered from their lungs at 3 dpi. While EHV-1 strains A4/72 and A9/92 exhibited a high degree of tropism for the CNS with robust neuroinvasiveness and neurovirulence, the EHV-1 strain A3/97 was not neurovirulent despite being detected in the CNS of infected BALB/c nude mice. Increased mRNA levels of TNF-α, IL-6 and CCL2 were detected in the nervous tissue of EHV-1 infected mice at 2 and 3 dpi; however, IFN-γ mRNA was not consistently expressed. Mice with the background C57BL/6, which exhibit predominantly Th1-type responses, showed the highest levels of TNF-α, IL-6 and CCL-2 mRNA in the CNS, when compared to BALB/c mice. The severity of signs observed in infected mice could be correlated with the peak of these proinflammatory cytokines (TNF-α and IL-6) and the chemokine CCL2, which are produced early after viral infection by both cells infiltrating into the CNS from the periphery and/or glial resident cells. These findings indicate that several inbred mouse strains are susceptible to neuopathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to equine herpes myeloencephalopathy in horses.

The immune system is a complex network of tissues, cells and proteins which protects us against infections and invading pathogens we encounter every day. Immunosenescence refers to age-related impairments in immune function which may contribute to increased prevalence and severity of infectious disease in the elderly. How and why ageing affects the immune system is not fully understood. Using a naturally aged mouse model, work in this thesis shows that the abundance of a rare type of lymphocyte, known as NKT cells, increased across multiple immune organs. Additionally, macrophage abundance was also altered in the lymph nodes of aged mice. Invariant NKT (iNKT) cells express an invariant T cell receptor (TCR) which recognises lipids presented on the CD1d molecule. iNKT cells can be activated and respond to invading pathogens either by recognition of antigens through TCR-CD1d interactions or cytokine-dependent means. Less is known about NKT-like cells, which also express NK cell-associated surface markers, such as CD49b, but lack an invariant TCR. Data within this thesis show that both iNKT and NKT-like cell populations are abundant in the spleen and liver of aged mice. iNKT and NKT-like cells can be divided into subpopulations based on their expression of surface markers or transcription factors, and data suggests that not all subpopulations of these cells are affected by age equally. For instance, flow cytometry showed that while spleen-derived iNKT cells are significantly increased in aged mice, within the iNKT cell population the percentage representation of CD4+ cells are significantly reduced with age. Additionally, data indicates that both iNKT and NKT-like cells from aged mice show compromised responses to in vitro stimulation compared to young controls. Using bone marrow chimeras, where either young cells are reconstituted within an aged mouse or old cells are reconstituted within a young mouse, provided the opportunity to determine whether the aged environment contributes to this diminished response. Data demonstrates that the aged environment plays at least a partial role in these age-related changes to response to stimulation, however the young environment seems unable to reverse these changes. Macrophages are phagocytes which are found within all organs of the body. Studies in this thesis show that CD169+ macrophages have diminished numbers in the lymph nodes of aged mice, but this did not seem to affect the capture of the model antigen, dextran. Further studies revealed ageing affects macrophage populations differently in the different tissues within the body. For example, macrophage numbers remain constant in the spleen with ageing, but appear to increase in density in the lungs. To conclude, ageing can cause dramatic changes to the numbers and function of different cells of the immune system across multiple organs. Furthering our understanding of the ageing immune system and the underlying mechanisms which cause age-related decline in immune function is important to design strategies to improve the quality of the lives of the elderly.