Le terme invadosome regroupe à la fois les podosomes, dans les cellules normales, et les invadopodes, dans les cellules transformées par l’oncogène Src et les cellules cancéreuses. Ces structures sont capables d’interagir avec et de dégrader la matrice extracellulaire (MEC). Ils sont aussi considérés comme des méchanosenseurs car ils sont capables de sentir la rigidité et la nature de la MEC. Mon travail de thèse s’est focalisé sur l’impact du microenvironnement matriciel sur la formation et l’activité des invadosomes. Au cours d’une première étude, nous avons démontré que les cellules endothéliales microvasculaires forment de façon constitutive des podosomes. L’utilisation de matrices de rigidités contrôlées, a permis la mise en évidence d’une corrélation entre l’augmentation de la rigidité augmentait et le nombre de cellules formant des podosomes ainsi que la taille de ces structures. En plus de la rigidité, d’autres propriétés de la MEC, telles que sa composition moléculaire et son organisation pourraient influencer la formation des invadosomes. Dans une seconde et troisième étude, nous avons pu montrer que seul le collagène fibrillaire de type I était capable d’induire la formation de microdomaines d’actine linéaires qui présentent comme les invadosomes, la capacité de dégrader la MEC. Au vu de leur morphologie originale, nous avons nommés ces structures des invadosomes linéaires (LIs). De façon intéressante, nous avons pu établir que la formation et l’activité de dégradation des LIs étaient indépendantes des intégrines β1 et β3. Au contraire, nous avons démontré que les récepteurs à domaine discoïdine (DDRs) contrôlent la formation et l’activité des LIs. De plus, les voies de signalisation classiques associées aux invadosomes classiques ne sont pas impliquées dans la formation des LIs. Une analyse par spectrométrie de masse des interactants de DDR1 dans un contexte collagène de type I fibrilllaire a permis de mettre en évidence des régulateurs clés et de révéler une voie de signalisation potentiellement impliquée dans la formation des LIs.Ainsi, ce travail de thèse a permis d’identifier la rigidité de la matrice comme un inducteur majeur des podosomes mais aussi la capacité intrinsèque des cellules microvasculaires à former ces structures. De plus, nous avons identifié un nouveau type d’invadosome, les LIs, qui sont associés à un nouveau type de récepteur concernant les invadosomes, les DDRs. / Invadosome is a global term including podosome, found in normal cells, and invadopodia observed in Src-transformed and cancer cells. These structures are specialized cell-matrix contacts able to interact with and degrade the extracellular matrix (ECM). They are considered as mechanosensors as they are able to sense the strength, the nature of the extracellular matrix. My PhD work essentially focuses on the understanding of how matrix microenvironment impacts on invadosome formation and activity. In a first study, we demonstrated that microvascular cell types constitutively form podosomes. Thus, using matrices of controlled rigidity, we found that an increase of stiffness was associated with an enhancement in the number of cells forming podosomes and podosome size. In addition to the matrix rigidity, other microenvironment properties, such as the molecular composition and the organization of the matrix are expected to influence the formation of invadosome.In a second and a third part of this wok, we show that fibrillar type I collagen induce the formation of linear actin microdomains which exhibit invadosome characteristics. In view of their original architecture, we named these new structures, linear invadosomes (LIs).Interestingly, we show that the formation and degradation activity of LIs are independent of β1 and β3-integrins but required discoidin domain receptors (DDRs). Moreover, all the signalling pathways known to induce classical invadosome are not required for the LIs induction. A mass spectrometry analysis of DDR1 partners emphasized key regulators and these results highlight a new potential signalling pathway involved in LIs formation. This work allowed us identifying matrix stiffness as a major inducer of podosomes but also the intrinsic capacity of microvascular cells to form these structures. Moreover, we identify a new type of invadosome, the Linear Invadosome associated with DDRs receptors.

L'apoptose spontanée des lymphocytes T entraine une lymphopénie marquée dans les rats BBDP (BioBreeding Diabetes Prone) conduisant au développement du diabète auto-immun de type I. Le phénotype de la lymphopénie dans ces rats est lié au locus lyp. L'allèle lyp contient une mutation du cadre de lecture à l'intérieur du gène qui code pour GIMAP5 (GTPase of immunity associeted nucleotide binding protein 5). Cette mutation produit une protéine tronquée de 11 Kd. Les mécanismes par lesquels, GIMAP5 assure la fonction de survie des lymphocytes T ne sont pas bien connus. Cependant, des études antérieures ont montré que l'apoptose précoce des cellules T matures était indépendante des caspases, mais est associée à la perte du potentiel de la membrane mitochondriale. D'autres études de surexpression ont montré que GIMAP5 localise dans les mitochondries et le réticulum endoplasmique. En utilisant un antisérurn de lapin contre GIMAP5, nos résultats ont montré que la protéine GIMAP5 endogène est associée à une fraction subcellulaire sédimentable distincte de la mitochondrie et du réticulum endoplasmique dans les lymphocytes T matures de rat normaux. Pour confirmer davantage ces données par la microscopie confocale, nous avons surexprimé rGIMAP5 étiqueté par myc à la partie N-terminale dans les fibroblastes Rat-2, la partie C-terminale transmembranaire étant intacte. Nos résultats suggèrent que GIMAP5 régule l'intégrité de la membrane mitochondriale dans une fraction subcellulaire différente des mitochondries.

<p>Macrophages are an important key effector cell in the immune system which can practically be found in every tissue. Macrophages have for a long time been considered a population of cells only responsible for pro-inflammatory responses and anti-microbial activities. But over the past decade, many have come to realize the amazing plasticity of macrophages in response to different stimulations. The anti-microbial and pro-inflammatory macrophage is known as classically activated macrophages but newly discovered phenotypes have been revealed named wound-healing macrophages and regulatory macrophages. Through systematic screening we have identified an inducible macrophage activation state which has both wound-healing and regulatory capabilities activated by the novel cytokine combination IL-4/IL-10 with or without TGF-β.</p>

Mechanisms involved in the establishment of HCMV latency are poorly understood, however, work in our laboratory has demonstrated the ULb' encoded protein, pUL138, as the first viral determinant to function in the establishment of HCMV latency in CD34+ hematopoietic progenitor cells (HPCs). This work characterizes the transcripts that encode pUL138, identifies three novel ULb' proteins (pUL133, pUL135, and pUL136) and represents the first demonstration of an internal ribosome entry site (IRES) mediated expression of pUL138. pUL138 is encoded on three polycistronic transcripts of 3.6-, 2.7- and 1.4-kb in length. pUL133, pUL135 and truncated pUL136, are expressed on the 3.6-, 2.7- and 1.4-kb transcripts, respectively, in addition to pUL138. We demonstrate that pUL138 expression is inducible from the IRES on the 3.6- and 2.7-kb transcripts under conditions of cellular stress, whereas pUL138 expression from the 1.4-kb transcript is inhibited under these same conditions. Differential utilization of the UL138 transcripts and their respective encoded proteins may regulate the outcome of viral infection in a cell type or cell context dependent manner. The interaction of these proteins during HCMV latency is the focus of ongoing research. In addition, this work represents preliminary data regarding the type I interferon (IFN) response during HCMV during productive infection in MRC5 fibroblasts and during the establishment of HCMV latency in CD34+ HPCs.

Master of Science / Department of Statistics / Christopher Vahl / Sample size estimation is generally based on assumptions made during the planning stage of a clinical trial. Often, there is limited information available to estimate the initial sample size. This may result in a poor estimate. For instance, an insufficient sample size may not have the capability to produce statistically significant results, while an over-sized study will lead to a waste of resources or even ethical issues in that too many patients are exposed to potentially ineffective treatments. Therefore, an interim analysis in the middle of a trial may be worthwhile to assure that the significance level is at the nominal level and/or the power is adequate to detect a meaningful treatment difference. In this report, the impact of sample size re-estimation on the type I error rate for the continuous end-point in a clinical trial with two treatments is evaluated through a simulation study. Two sample size estimation methods are taken into consideration: blinded and partially unblinded. For the blinded method, all collected data for two groups are used to estimate the variance, while only data from the control group are used to re-estimate the sample size for the partially unblinded method. The simulation study is designed with different combinations of assumed variance, assumed difference in treatment means, and re-estimation methods. The end-point is assumed to follow normal distribution and the variance for both groups are assumed to be identical. In addition, equal sample size is required for each group. According to the simulation results, the type I error rates are preserved for all settings.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Interferons (IFNs) comprise a group of antiviral cytokines; however, their expression at the porcine maternal-fetal interface and in fetal tissues has not previously been investigated. The purpose of this study was to analyze the expression of type I IFNs and their receptors in maternal and fetal tissues from sows infected with PRRSV. The approach was to use real-time RT-PCR to identify the expression of different subtypes of type I IFN genes. The results show that the constitutive gene expression of some subtypes including IFN-[alpha] and IFN-[alpha][omega] were detected in fetal lymphoid nodes (IFN-[alpha][omega]), placenta (several IFN-[alpha] subtypes and IFN-[omega]5) and particularly, thymus (multiple IFN-[alpha], IFN-[delta] and IFN-[omega]5). The results demonstrate that porcine type I IFNs are differentially expressed at the maternal-fetal interface and in fetal tissues in response to PRRSV infection.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Yongming Sang / Animal metabolic and immune systems integrate and inter-regulate to exert effective immune responses to distinct pathogens. The signaling pathway mediated by mechanistic target of rapamycin (mTOR) is critical in cellular metabolism and implicated in host antiviral responses. Recent studies highlight the significance of the mTOR signaling pathway in the interferon (IFN) response. Type I IFNs mediate host defense, particularly, against viral infections, and have myriad roles in antiviral innate and adaptive immunity. In addition to their well-known antiviral properties, type I IFNs also affect host metabolism. However, little is known about how animal type I IFN signaling coordinates immunometabolic reactions during antiviral defense. Therefore, understanding the interaction of mTOR signaling and the type I IFN system becomes increasingly important in potentiating antiviral immunity. Tissue macrophages (MФs) are a primary IFN producer during viral infection, and their polarization to different activation statuses is critical for regulation of immune and metabolic homeostasis. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model, we found that genes in the mTOR signaling pathway were regulated differently in PRRSV-infected porcine alveolar MФs at different activation statuses. Therefore we hypothesize that: 1) the mTOR signaling pathway involves host anti-PRRSV regulation; 2) mTOR signaling interacts with IFN signaling to modulate the antiviral response; and 3) different type I IFN subtypes (such as IFN-α1 and IFN-β) regulate mTOR signaling differently. We show that modulation of mTOR signaling regulated PRRSV infection in MARC-145 cells and porcine primary cells, in part, through regulating production and signaling of type I IFNs. In addition, expression and phosphorylation of two key components in the mTOR signaling pathway, AKT and p70 S6 kinase, were regulated by type I IFNs and PRRSV infection. Taken together, we determined that the mTOR signaling pathway, a key pathway in regulation of cell metabolism, also mediates the type I IFN response, a key immune response in PRRSV infection. Our findings reveal that the mTOR signaling pathway potentially has a bi-directional loop with the type I IFN system and implies that some components in the mTOR signaling pathway can serve as targets for augmentation of antiviral immunity and therapeutic designs.

Master of Science / Department of Statistics / Christopher I. Vahl / Estimation of sample size is an important and critical procedure in the design of clinical trials. A trial with inadequate sample size may not produce a statistically significant result. On the other hand, having an unnecessarily large sample size will definitely increase the expenditure of resources and may cause a potential ethical problem due to the exposure of unnecessary number of human subjects to an inferior treatment. A poor estimate of the necessary sample size is often due to the limited information at the planning stage. Hence, the adjustment of the sample size mid-trial has become a popular strategy recently. In this work, we introduce two methods for sample size re-estimation for trials with a binary endpoint utilizing the interim information collected from the trial: a blinded method and a partially unblinded method. The blinded method recalculates the sample size based on the first stage’s overall event proportion, while the partially unblinded method performs the calculation based only on the control event proportion from the first stage. We performed simulation studies with different combinations of expected proportions based on fixed ratios of response rates. In this study, equal sample size per group was considered. The study shows that for both methods, the type I error rates were preserved satisfactorily.

Introdução: O saco endolinfático tem sido apontado como o alvo das reações imuno-alérgicas da orelha interna. A prevalência de alergia em pacientes com Doença de Ménière foi estabelecida em torno de 41,6% para inalantes e 26,6% para alimentos, por Derebery em 2000, dados aumentados em relação à prevalência de alergia na população em geral, que, no Brasil, varia de 9% a 30% para inalantes e de 1% a 3% para alimentos. Objetivos: Avaliar a prevalência de reações de hipersensibilidade tipo I a inalantes e alimentos na população do setor de Otoneurologia do Hospital das Clínicas da Faculdade de Medicina de São Paulo e descrever os sintomas vestibulares dos pacientes. Casuística e método: Setenta e cinco pacientes com distúrbios do equilíbrio de origem periférica foram submetidos a questionário de caracterização clínica de sintomas cócleo-vestibulares e teste cutâneo (prick test) para 13 inalantes e 5 alimentos. Resultados: Vinte e cinco (33,3%) pacientes apresentaram prick test positivo a pelo menos um alérgeno inalante e 6 (8%) a pelo menos um alérgeno alimentar. Quatro pacientes apresentaram prick test positivo na ausência de sintomas alérgicos. Prevaleceu a queixa de tontura de caráter rotatório em proporções semelhantes entre os pacientes com prick test positivo e negativo. Conclusão: A prevalência de reações de hipersensibilidade tipo I a inalantes e a alimentos na população avaliada foi maior do que na população em geral. Os sintomas vestibulares não diferiram entre os pacientes da amostra, com prick test positivo ou negativo. No entanto, deve-se obter maior número de amostra para que os dados sejam confiáveis. / Introduction: The endolymphatic sac has been pointed out as the target of immuno-allergic reactions in the inner ear. The prevalence of allergy in patients with Ménières disease was established as approximately 41,6% for inhalants and 26,6% for food by Derebery in 2000, an increase in the data in relation to that of the prevalence of allergy in the general population, which in Brazil varies from 9% to 30% for inhalants and from 1% to 3% for food. Objectives: To evaluate the prevalence of reactions to type I hypersensitivity to inhalants and food in the population of the Otoneurological Section of the Clinics Hospital of the University of São Paulo Medicine School and to describe the vestibular symptoms of the patients. Method: Seventy-five patients with peripheral equilibrium disturbances who had answered a questionnaire of clinical characterization regarding cochlear-vestibular symptoms and undergone prick test for 13 inhalants and 5 types of food. Results: Twenty-five (33,3%) of the patients were positive for the prick test and for at least one allergen inhalant and 6 (8%) for at least one food allergen. Four patients were positive for the prick test in the absence of allergy symptoms. There was a prevalence of the complaint of rotatory dizziness in similar proportions among the patients with positive and negative prick test. Conclusion: The presence of type I hypersensitivity reactions to inhalants and food in the population evaluated was greater than in the general population. The vestibular symptoms did not differ among the patients in the sample, neither with positive or negative prick test results. However, a sample of greater number should be obtained for a higher confidence level of data results.

Aichi virus (AiV) is a small, nonenveloped RNA virus categorized to Picornaviridae. AiV infection causes mild gastroenteritis, but in neonates, AiV usually causes the risk of certain enterovirus-related clinical syndromes, such as fever, nausea, vomiting and diarrhea. The first case of AiV infection in Taiwan was diagnosed from a young patient with diarrhea in Kaohsiung Veterans General Hospital, and the AiV was successfully isolated. Antiviral innate immune system of our body plays the major role to defense virus invasion. Because AiV is an emerging picornavirus, the knowledge about its pathogenesis and the interaction with host innate immunity were totally absent. This study aims to investigate the mechanism of AiV regulating innate immune response. We first demonstrated that AiV is a type I IFN sensitive virus. IFN-£\2 treatment potently inhibited AiV replication. Real-time quantitative PCR data indicated that AiV induced only small amout of type I IFN gene expression, and the similar result was observed using IFN-£] luciferase reporter assay. In addition, the AiV triggered IFN-£] luciferase activity was progressively decreased in the late phase of infection. Immunoblotting assay showed that AiV evidently activated IRF-3 and IRF-7, the transcription factors of type I IFN induction. However, the retinoic acid inducible gene I (RIG-I) protein was cleavaged and its activity was downregulated by AiV. This data suggested that AiV triggered low level of type I IFN response may due to the negative feedback regulation of RIG-I activity. This immune evasion might be important for AiV replication in cells. Our study first reveals the status of innate immune response of AiV infection, and provides the basic virological theory for the development of anti-AiV drugs and vaccines in the future.