O presente estudo discute o papel dos miRNAs na fisiopatologia molecular de Esclerose Múltipla Recorrente Remitente (EMRR). O estudo demonstrou que em linfócitos T CD4+ de pacientes com EMRR em surto ocorre a diminuição da expressão do miR-15a e do miR16-1 em contraposição ao aumento de seu gene alvo BCL-2, um importante gene regulador da apoptose. Esses achados sugerem a participação desses miRNAs no controle da apoptose na EM. Para explorar essa associação, foi analisado a expressão global de miRNAs nas subpopulações de linfócitos T de pacientes com EMRR no estágio de remissão. O resultado dessa análise determinou de forma inédita o aumento significativo da expressão de 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) nos linfócitos T CD8+ de memória central. A análise in silico dos alvos desses miRNAs indicou que três vias canônicas, relacionadas à ativação da apoptose, eram enriquecidas com alvos preditos e validados experimentalmente desses miRNAs. Desse modo sugerimos a forte relação desses miRNAs no controle da apoptose nos linfócitos T dos pacientes com EMRR. A fim de aprofundar nossos estudos, selecionamos os miRNAs miR-21 e miR-24, para a realização de experimentos funcionais in vivo. Foi verificada a indução da expressão miR-21 somente nos linfócitos T CD4+ de modelo experimental da EM. Adicionalmente, experimentos in vitro demonstraram que a expressão do miR-21 e restrita as populações de células Th2 e Th17. Nesse caso, miR-21 parece ser regulado pelo fator de transcrição STAT3, sugerindo assim que o aumento da expressão do miR-21 verificada no modelo animal possa estar relacionada com a presença de linfócitos T CD4+ de perfil Th17 nesse tecido. Em resumo, o conjunto desses resultados demonstra a relevância dos miRNAs na fisiopatologia da EMRR, principalmente no controle da apoptose. / This study discusses the role of miRNA in the Molecular Pathophysiology of Relapse Remitting Multiple Sclerosis (RRMS). The study has shown that CD4+ lymphocytes from relapsed RRMS patients had lower expression of miR15a and miR-16-1 in contraposition of higher expression of the target gene BCL-2, a key regulator of apoptosis. These findings suggest the role of those on the control of apoptosis in MS. In order to explore this association, the global expression of miRNAs was analysed in T lymphocyte subpopulations from remission RRMS patients. The result of this analysis has demonstrated for the first time a significant higher expression of 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) in central memory T CD8+ lymphocytes. In silico analysis of the miRNAs targets indicates that three canonical pathways related to the activation of apoptosis were enriched with predicted and experimental validated gene targets for those miRNAs. In this way, we suggest the strong relation of these miRNAs in the control of apoptosis in the lymphocytes from RRMS patients. In order to intensify our studies we selected miR-21 and miR-24 to perform in vivo functional experiments. It was verified miR-21 induction only in T CD4+ lymphocytes from MS animal model. Additionally, in vitro experiments have demonstrated that miR-21 expression was restricted to Th2 and Th17 cell populations. In this way, miR-21 seems to be regulated by the STAT3 transcription factor, thereby suggesting that the increase of miR-21 expression observed in vivo could be related with Th17 CD4+ present in this tissue. In summary, this set of results showed the relevance of miRNAs in the RRMS pathophysiology, mainly in the control of apoptosis.

O presente trabalho refere-se ao estudo do papel dos microRNAs no controle pós-transcricional das células T de camundongos Non Obese Diabetic (NOD) modelo que reproduz o diabetes mellitus do tipo 1 (DM-1). Durante o desenvolvimento do trabalho, procurou-se esclarecer a hipótese de que os microRNAs controlam os níveis de determinados RNAs mensageiros (mRNAs) das células T durante a indução ou perda de tolerância imunológica. Portanto, a expressão alterada dos microRNAs estaria contribuindo com o processo da autoimunidade. Sendo assim, o objetivo do estudo foi identificar os perfis de expressão e as redes de interação entre um conjunto de microRNAs e seus respectivos mRNAs alvos nos timócitos e nos linfócitos T CD3+ periféricos durante o desenvolvimento do diabetes mellitus do tipo 1 (DM-1) em camundongos NOD. Para avaliar a expressão de genes codificadores de mRNAs, sendo estes possíveis alvos de microRNAs, utilizou-se a tecnologia de microarrays. O uso de programas de análise e para a construção das redes foi imprescindível. Acreditase que fenômenos complexos como a regulação pós-transcricional de células T e seu envolvimento no processo de tolerância imunológica, bem como o surgimento de doenças autoimunes, podem ser melhor compreendidos por meio da genômica funcional. Os resultados encontrados evidenciam uma expressão diferenciada de mRNAs e microRNAs em timócitos e linfócitos T CD3+ periféricos durante o desenvolvimento do diabetes mellitus do tipo 1 (DM-1). As diferenças nos perfis transcricionais encontradas envolvem expressão de genes (mRNAs) relacionados diretamente ao sistema imune, a diferenciação e ativação de linfócitos T e a apoptose, bem como a outros processos relacionados a resposta imune. Além disso, as redes de interação microRNA-mRNA encontradas no presente trabalho evidenciam interações já conhecidas e apresentam novas interações, mostrando a participação de um grupo de microRNAs que estão atuando no controle pós-transcricional do diabetes do tipo 1 em camundongos NOD, contribuindo com a melhor compreensão do controle genético-molecular das doenças autoimunes, principalmente do diabetes do tipo 1. / This study refers to the role played by microRNAs in the post-transcriptional control of T cells from non obese diabetic (NOD) mice model which reproduces the type 1 diabetes (T1D). During the study, it was tried to clarify the hypothesis that microRNAs control certain messenger RNAs (mRNAs) levels of the T cells during the induction or loss of immunological tolerance. Therefore, the altered expression of microRNAs might be contributing to the process of autoimmunity. Thus, the study aim was to identify the expression profiles and interaction networks between a set of microRNAs and their mRNA targets in thymocytes and peripheral CD3+ T lymphocytes during the development of type 1diabetes (T1D) in NOD mice. The microarray technology was used to evaluate the expression of mRNAs as possible targets of microRNAs involved in this process. The use of bioinformatics software to reconstruct the networks was essential. It was realized that complex phenomena as post-transcriptional regulation in T cells and their involvement in the immune tolerance process, as well as the emergence of autoimmune diseases can be better understood only by means of functional genomics. The results show differential expression of mRNAs and microRNAs in thymocytes and peripheral CD3+ T lymphocytes during the development of type 1 diabetes (T1D). The differences found in the transcriptional profiles involve mRNAs related to the immune system, differentiation and activation of T lymphocytes and apoptosis as well as other processes related to immune response. In addition, the microRNA-mRNA interaction networks obtained in this study evidence the predicted interactions as well as new ones, showing the participation of a group of microRNAs that may be acting in post-transcriptional control of type 1 diabetes in NOD mice contributing to a better understanding of the molecular genetic control of autoimmune diseases in specially type 1 diabetes.

Orientadores: Laura Sterian Ward, José Vassallo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T01:32:40Z (GMT). No. of bitstreams: 1 Cunha_LucasLeite_M.pdf: 2465982 bytes, checksum: a87a268c547961632aa1f42747034935 (MD5) Previous issue date: 2012 / Resumo: O câncer de tireoide é a neoplasia endócrina mais comum. O objetivo geral deste projeto é investigar o padrão de expressão gênica e protéica de tumores resistentes e suscetíveis à ativação do sistema imunológico, bem como pesquisar evidências de mecanismos de evasão tumoral da resposta imune. Foram investigados 398 pacientes cujas amostras de tecidos foram mantidas no banco de tecidos do Hospital A.C.Camargo, São Paulo, sendo 253 carcinomas papilíferos, 13 carcinomas foliculares e 132 tumores benignos. O perfil de expressão protéica dos tumores foi avaliado por imunoistoquímica e a infiltração de células do sistema imunológico foi caracterizada por meio de marcadores imunoistoquímicos. Tiroidite linfocítica crônica concomitante ao câncer foi mais freqüente entre as mulheres, em tumores menos agressivos que não apresentavam invasão extratireoideana ou metástases ao diagnóstico, e em tumores pequenos, menores que 2 cm. O teste de log-rank mostrou que a presença de Tiroidite linfocítica crônica concomitante ao câncer está associada ao maior tempo livre de doença. A infiltração de macrófagos foi mais freqüente entre as mulheres e foi associado à maior tempo de sobrevida livre de recidiva. A infiltração de linfócitos CD3+ se correlacionou à malignidade e está associada à presença de metástases ao diagnóstico. As infiltrações de linfócitos CD4+ e CD20+ foram relacionadas às características de melhor prognóstico e a infiltração de linfócitos CD8+ pode ser um marcador de melhor prognostico. A expressão da proteína CD56 foi mais freqüente entre os tecidos benignos e não está associada ao prognóstico dos pacientes com carcinoma diferenciado de tireóide. Por sua vez, a infiltração de linfócitos FoxP3+ se correlacionou a características de menor agressividade tumoral. A expressão de RNAm B7-H1 foi característica de tecidos malignos e a proteína B7-H1 apontou um comportamento mais agressivo dos carcinomas. Tanto a infiltração de linfócitos Th17 e quanto células supressoras derivadas da linhagem mielóide foram mais freqüentes entre os tumores malignos, ainda que somente a infiltração de Th17 se correlacionasse a características de melhor prognóstico. Nossos dados sugerem que o carcinoma diferenciado de tireóide seja ricamente infiltrado por um conjunto de diferentes células do sistema imunológico. Provavelmente esta infiltração é dependente do padrão de expressão gênica e protéica destes cânceres, padrão este que pode refletir a imunogenicidade destes tumores. Mais ainda, a associação entre a infiltração destas células e características de melhor prognóstico, sugere que exista uma resposta imunológica ativa paramentada contra o carcinoma diferenciado de tireóide capaz de exercer papel antitumoral / Abstract: Thyroid cancer is the most common endocrine malignancy. The objective of this project is to investigate the pattern of gene and protein expression of tumors resistant and susceptible to activation of the immune system as well as research evidence tumor evasion mechanisms of the immune response. We investigated 398 patients whose tissue samples were kept in the tissue bank ACCamargo Hospital, São Paulo, with 253 papillary carcinomas, 13 follicular carcinomas and 132 benign tumors. The protein expression profile of tumors was assessed by immunohistochemistry and the infiltration of immune cells was characterized by immunohistochemical markers. Chronic lymphocytic thyroiditis concurrent to cancer was more frequent among women, less aggressive tumors that did not have extra thyroid invasion or metastases at diagnosis, and small tumors smaller than 2 cm. The log-rank test showed that the presence of chronic lymphocytic thyroiditis concurrent to cancer was associated with longer relapse-free. Macrophage infiltration was more frequent among women and was associated with relapse-free survival. The infiltration of CD3+ lymphocytes correlates with malignancy and is associated with the presence of metastases at diagnosis. The infiltration of CD4+ and CD20+ were related to the better prognosis. The infiltration of CD8+ lymphocytes may be a marker of better prognosis. The CD56 protein expression was more frequent among benign tissues and is not associated with prognosis of patients with differentiated thyroid carcinoma. The infiltration of FoxP3+ lymphocytes correlated with less aggressive tumor characteristics. The mRNA expression of B7-H1 was characteristic of malignant tissues and B7-H1 protein could point to a more aggressive behavior of the differentiated thyroid carcinoma. Both the infiltration of Th17 lymphocytes and the myeloid derived suppressor cells were more frequent in malignant tumors, although only the infiltration of Th17 correlated with features of better prognosis. Our data suggest that differentiated thyroid carcinoma is enriched by a number of different immune cells. Probably this infiltration is dependent on the pattern of gene and protein expression of these cancers, a pattern that may reflect the immunogenicity of these tumors. Moreover, the association between these cells and infiltration characteristics of better prognosis, suggests that there is an active immune response against attired differentiated thyroid carcinoma can exert antitumor / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Les Lymphocytes T CD8 (LT CD8) sont des cellules du système immunitaire capables de reconnaître et de détruire des cellules infectées ou tumorales. De plus, les LT CD8 mémoires générés suite à une première rencontre avec un agent pathogène confèrent à l’organisme une protection efficace contre une réinfection. Cela a pour origine une modification des capacités effectrices et migratoires des LT CD8 mémoires par rapport aux LT CD8 naïfs. Ces fonctions améliorées des LT CD8 mémoires peuvent être régulées : des travaux préalables réalisés au sein de l’équipe ont montré que l’Interleukine-4 (IL-4), une cytokine sécrétée lors des réponses allergiques ou contre des pathogènes extracellulaires, inhibe certaines propriétés des cellules mémoires comme la sécrétion rapide de CCL5. Les effets de l’IL-4 sur les fonctions des LT CD8 mémoires sont cependant mal caractérisés : l’objectif de cette thèse a été de les identifier plus précisément. En utilisant une approche par micro-array, nous avons identifié une signature de gènes régulés par l’IL-4 dans les LT CD8 mémoires et montré que cette cytokine modifie l’expression de gènes impliqués dans la prolifération, la migration, et les fonctions effectrices des LT CD8 mémoires. Nous avons montré que l’IL-4 inhibe l’expression du récepteur de costimulation NKG2D à la surface des LT CD8 mémoires in vivo, s’accompagnant d’une diminution du signal costimulateur délivré par l’engagement de NKG2D. Nous avons également montré que la présence de certaines cytokines γc telles que l’IL-4 ou l’IL-21 lors de l’activation cellulaire affecte fortement les fonctions des LT CD8 effecteurs générés ainsi que leur différenciation en cellules mémoires. / Immunological memory is characterized by a secondary response that is faster and stronger than the primary response. For CD8 T cells this results from an increase frequency of antigen specific cells that display an improved response as compared to naïve cells. Memory CD8 T cells also display a new pattern of surface molecules that is associated with modified homing or activation properties. γc cytokines are known to affect both CD8 T cells functions and survival. Indeed, IL-4, a γc cytokine involved in Th2 responses induced in response to parasitic infections or allergies has been shown to reduce both IFNγ secretion and cytotoxicity of CD8 T lymphocytes. In the lab, we have demonstrated that IL-4 down-regulates ccl5 mRNA stores by inhibiting ccl5 transcription via a STAT6-dependent pathway. However, effects of IL-4 on CD8 T cells’ biology remain largely unknown. To identify such effects, we realised a microarray study that allowed us to identify the signature of genes affected by IL-4 in memory CD8 T cells. Among this signature, we identified genes involved in several functions of CD8 T cells, such as proliferation, migration, and effector functions. We then confirmed both in vitro and in vivo that NKG2D, a member of the NK receptors family involved in the modulation of the activation threshold of memory CD8 T cells, is down-regulated by IL-4, inhibiting NKG2D-dependant costimulation of memory CD8 T cells. Finally, we found that γc cytokines such as IL-4 and IL-21 affect the effector capacities of in vitro activated CD8 T Cells, as well as the differentiation of activated cells into memory cells.

A T helper cell clone, referred to as clone 9, was derived from an allogeneic mixed lymphocyte culture. Clone 9, as well as supernatant factor(s) derived from it, could help the cytotoxic T lymphocyte (CTL) responses of H-2 Db (Db) responder cells to alloantigens, or they could help the CTL responses of non- Db responder cells to Db alloantigens. Clone 9 cells or their factor(s) were active only when added during the first 24 hours of a five-day culture period. Clone 9 or its factor(s) could also synergize with interleukin-2 (IL-2)-containing medium in mounting cytotoxic responses to alloantigens. The helper activity in clone 9 supernatant was not due to IL-2 and it was specifically absorbed out by Db -spleen cells. The characterization of the Db -specific helper factor(ASHF) was facilitated by the isolation of a T hybridoma clone (clone 25), obtained from fusion of clone 9 cells with the T cell lymphoma, BW5147, and a B cell hybridoma that produced an IgM monoclonal antibody (clone 30 IgM) which bound ASHF. An additional monoclonal antibody (F23.1), which recognizes a determinant of the Vβ8 family of the T cell receptor, was also particularly useful for the characterization of ASHF. Analysis with these reagents showed that both clone 30 IgM and F23.1 immunoadsorbents could retain ASHF activity. Preabsorption of the ASHF with Db spleen cells prior to affinity purification over a clone 30 IgM column resulted in the absorption of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band on SDS-PAGE under reducing conditions. Furthermore, affinity purification of ASHF over the F23.1 immunoadsorbent, but not an irrelevant monoclonal antibody (mAb) column, also yielded a 50,000 MW molecule. Taken together, these findings suggest that the 50,000 MW molecule is a component of the ASHF and it is intimately related to the B chain of the T-cell receptor. The mode of action of clone 9 and its products in the induction bfCTL responses was also investigated. It was found that clone 9 and ASHF could help CTL responses by inducing IL-2 production in B6-stimulated cultures. In addition to ASHF, clone 9 cells also produced an additional factor(s) which participated in the induction of CTL responses. This additional factor(s) was referred to as IL-X. IL-X synergized with excess human recombinant IL-2 in the activation of CTL precursors (CTL-P) in the absence of antigenic stimulation. A model which involves the participation of ASHF, T helper cells, IL-2 and IL-X in the induction of CTL responses is proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Bien que des progrès aient été réalisés dans la lutte contre la transmission du VIH depuis sa découverte il y a bientôt 30 ans, aucun outil thérapeutique actuellement sur le marché ne permet d'éliminer totalement le virus en 2011. Une meilleure connaissance des mécanismes physiopathologiques au niveau des compartiments muqueux, qui sont les principales portes d'entrée de l'infection par le VIH et abritent la majorité des lymphocytes T de l'hôte, est donc indispensable pour espérer éradiquer la pandémie. L'objectif de notre travail a été de caractériser les relations hôte-HIV dans le compartiment pulmonaire et génital ainsi que de décrire la synergie immuno-virologique entre le VIH et le virus herpès simplex (VHS) favorisant la transmission virale.Nous avons observé que les lymphocytes T sont présents en plus faibles proportions dans les sécrétions pulmonaires et génitales que dans le sang et ces cellules ont plus fréquemment un phénotype mémoire et activé. Les lymphocytes T muqueux sont donc plus prompts à sécréter des virus que ceux du sang. L'infection par le VIH est également associée à une dérégulation de la réponse immune muqueuse de l'hôte pouvant favoriser sa réplication. De plus, la modification de l'environnement immunologique liée à la synergie entre le VIH et le VHS apparaît comme un phénomène qui pourrait avoir un impact dans la transmission de chacun des deux virus. Une vision intégrée de la réponse immunitaire muqueuse contre le VIH permettra d'identifier les mécanismes à l'origine de l'infection et de la transmission en tenant compte des spécificités du virus et de l'environnement local ainsi que des interrelations avec le compartiment circulant. Les futurs essais cliniques devront en outre tenir compte des caractéristiques des muqueuses et de l'infection par le VIH afin de développer des thérapies anti-infectieuses avec une efficacité optimale, y compris dans les pays du Sud où les populations sont les principales victimes du VIH. / Although scientific advances have occurred in the fight against HIV transmission since its discovery almost 30 years ago, no current therapeutic tool enables the complete elimination of this virus in 2011. A better understanding of the physiopathological mechanisms at the mucosal compartment levels, which are the main gateways of HIV infection and host the majority of body T lymphocytes, is therefore crucial to bring hopes for eradicating HIV pandemic. The aim of our work is to characterize the host-HIV relationships in the pulmonary and genital compartments as well as the immuno-virological synergy between HIV and the herpes simplex virus (HSV) favouring the viral transmission.We have observed small proportions of T lymphocytes were found in the pulmonary and genital secretions in comparison with blood; and these cells have a more frequent memory and activated phenotype. The mucosal T lymphocytes are therefore quicker to produce viral particles than the blood counterpart. HIV infection is also associated with a deregulation of the host mucosal immune response, which could favour its own replication. Moreover, change in the immunological environment related to the HIV/HSV synergy appears as a phenomenon that could have an impact on each of the two viruses transmission.An integrated view of the mucosal immune response against HIV will allow identifying the mechanisms leading to the infection and transmission, taking into account the viral and local environment specificities as well as the interrelations within the circulating compartment. In addition, future clinical trials will have to take into account the HIV infection, the mucosal characteristics in order to develop anti-infectious therapy with an optimal efficiency, and the developing countries where the populations are the main victims of HIV.

Orientadores: Liana Maria Cardoso Verinaud, Wilson Savino / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T20:13:37Z (GMT). No. of bitstreams: 1 Francelin_Carolina_D.pdf: 110859425 bytes, checksum: e99991048374fbaee764c7b0f509643f (MD5) Previous issue date: 2014 / Resumo: O timo é o órgão linfoide primário responsável pela geração de linfócitos T maduros. Para que isso ocorra, células precursoras de linfócitos T, provenientes da medula óssea, entram no timo e migram constantemente através do microambiente tímico ¿ o qual é composto por componentes linfoides e não linfoides. Esta migração intratímica é fundamental para que os precursores das células T encontrem os sinais necessários para sobrevivência, proliferação, diferenciação e geração de diversidade de repertório. Assim como os outros órgãos linfoides, o timo está sujeito a um rígido controle neuroendócrino, o qual impõe consequências diretas sobre o funcionamento do sistema imunológico através de neurotransmissores, hormônios e citocinas. Entretanto, ainda é pouco o que se sabe sobre as interações entre os componentes do timo e hormônios do eixo HPA. Neste trabalho, foram avaliados os compartimentos linfoide e estromal na atrofia tímica observada no modelo experimental da diabetes tipo I. Nesse estudo foi observado que camundongos diabéticos apresentaram redução nos níveis séricos e intratímicos de leptina e elevados níveis séricos e intratímicos de corticosteroide, acompanhando a queda dos níveis séricos de insulina. Diante das alterações hormonais, nós observamos: modificações nos componentes linfoides e estromais do timo, caracterizadas por redução no número de timócitos, aumento na secreção de elementos de matriz extracelular, contração da porção cortical do timo acompanhada por acúmulo de linfócitos no estágio pré - seleção positiva, aumento da apoptose de células epiteliais tímicas e timócitos e aumento na exportação de células T imaturas para os órgãos linfoides secundários. Sucintamente, após o estabelecimento da hiperglicemia e ausência de insulina circulante, o timo de animais diabéticos apresentou alterações morfológicas e em todos os tipos celulares e fatores solúveis que compõe o estroma tímico, culminando em alterações nas células presentes na periferia do sistema imune. Acreditamos que os dados gerados nesse estudo contribuem, s.m.j., para um melhor entendimento da deficiência na resposta imune em indivíduos diabéticos e do desenvolvimento do linfócito T na ausência de insulina / Abstract: Thymus is the primary lymphoid organ responsible for the generation of T lymphocytes. For this to occur, precursor cells of T lymphocytes from bone marrow enter the thymus and migrate continuously through the thymic microenvironment - which consists of lymphoid and non-lymphoid components. This intrathymic migration is essential for T cell precursors get contact with signs that promote survival, proliferation, differentiation and generation of diversity of repertoire. Like other lymphoid organs, the thymus is subject to a rigid neuro-endocrine control, which requires direct consequences on the functioning of the immune system through neurotransmitters, hormones and cytokines. However, it is still little known about the interactions between the components of thymus hormones and the HPA axis. In this study, we evaluated the alterations in lymphoid and stromal thymic compartiments in thymic atrophy during experimental model of diabetes type I. Here in, we found that diabetic mice exhibit a reduction in serum aand intrathymic levels of leptin yet, intrathymic and serum corticosteroid levels were high, followed by a drop in serum insulin levels. Given the hormonal changes, we observed: changes in lymphoid and non-lymphoid component of the thymus, characterized by reduction in the number of thymocytes, increased secretion of extracellular matrix elements, contraction of the cortical portion of the thymus accompanied by accumulation of lymphocytes in the pre stage - positive selection, increased apoptosis of thymic epithelial cells and thymocytes and increase in export of immature T cells to secondary lymphoid organs. Briefly, after the onset of hyperglycemia and lack of circulating insulin, thymus in diabetic animals showed alterations in all cell types that comprise the thymic microenvironment, resulting in abnormal cells present in the periphery of the immune system. We believe that the data generated in this study will contribute to a better understanding of the immune deficiency in diabetic individuals and the development of T lymphocytes in the absence of insulin response / Doutorado / Imunologia / Doutora em Genética e Biologia Molecular

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-19T15:00:56Z No. of bitstreams: 1 Ana Paula de Souza Mernick.pdf: 747300 bytes, checksum: 7cec9bdaa4c2bd9e6dfcc62d9bfdbd12 (MD5) / Made available in DSpace on 2016-05-19T15:00:56Z (GMT). No. of bitstreams: 1 Ana Paula de Souza Mernick.pdf: 747300 bytes, checksum: 7cec9bdaa4c2bd9e6dfcc62d9bfdbd12 (MD5) Previous issue date: 2014-12-16 / Periodontitis is an infectious disease characterized by the destruction of the supporting tissues of the teeth and therefore the dental loss. Neutrophils are the first cells to arrive in periodontal inflammation and have as function phagocytosis and cytokine release. After a few days, the neutrophils are replaced by lymphocytes, which give the chronic nature of periodontitis. The established treatment for periodontitis is oral hygiene care, scaling and root planing, and in more advanced cases, there may be the need for systemic antibiotics. In order to reduce the prescription of antibiotics, it has been researched the use of photodynamic therapy (PDT) as antimicrobial intervention, with great results. The objective of this work is to verify if the PDT interferes with the chemotaxis of neutrophils and T lymphocytes into the periodontal tissue. For such, 05 patients who had undergone prior treatment with periodontal good oral hygiene and periodontal pockets with residual surgical indication were allocated. PDT was applied under the following parameters: methylene blue (50 µg/ml) was applied to the bottom of the periodontal pocket and after 5 minutes, via transmucosal, was irradiated with diode laser 660 nm, P = 100 mW, t = 90 s per point 9 J/point, energy density: 22 J/cm2, power density: 250 mW/cm2. One week after PDT, the surgical removal of the periodontal pockets was performed and the samples sent for routine histology and immunohistochemistry for detection of neutrophil and lymphocyte. The samples were photographed and positive cells counted with the aid of ImageJ software. The data were evaluated statistically and no significant difference between groups (Mann-Whitney p = 0.5). It was concluded that PDT did not interfere with the chemotaxis of neutrophils and T lymphocytes in the periodontal tissue after 7 days. / A periodontite é uma doença infecciosa caracterizada pela destruição dos tecidos de suporte dos dentes e consequentemente à perda do elemento dental. Os neutrófilos são as primeiras células a chegar ao processo inflamatório periodontal e têm como função fagocitose e liberação de citocinas. Após alguns dias os neutrófilos vão sendo gradualmente substituídos por linfócitos, que dão o caráter crônico da periodontite. O tratamento estabelecido para a periodontite é a orientação de higiene oral, raspagem e alisamento radicular e em casos mais avançados, pode haver a necessidade de antibioticoterapia sistêmica. No sentido de reduzir a prescrição de antibióticos, tem-se pesquisando o uso da terapia fotodinâmica (PDT) como intervenção antimicrobiana, com ótimos resultados. Assim, o objetivo deste trabalho é verificar se a PDT interfere na quimiotaxia de neutrófilos e linfócitos T no tecido periodontal. Para tal, foram alocados 05 pacientes que passaram por tratamento periodontal prévio com boa higiene oral e presença de bolsas periodontais residuais com indicação cirúrgica. A PDT foi aplicada nos seguintes parâmetros: azul de metileno (50 µg/mL) aplicado no fundo da bolsa periodontal e após 5 minutos foi irradiado via transmucosa com laser de diodo, 660 nm, P= 100 mW, t= 90 s por ponto, 9 J/ponto, densidade de energia: 22 J/cm2, densidade de potência: 250 mW/cm2. Uma semana após a PDT foi realizada remoção cirúrgica das bolsas e as amostras encaminhadas para processamento histológico de rotina e imunohistoquímica para detecção de neutrófilos e linfócitos T. As amostras foram fotografadas e as células positivas contadas com auxílio do software ImageJ. Os dados foram avaliados estatisticamente e não houve diferença significativa entre os grupos (Mann-Whitney p= 0,5). Conclui-se que a PDT não interfere na quimiotaxia de neutrófilos e linfócitos T no tecido periodontal após 7 dias.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-19T15:16:35Z No. of bitstreams: 1 Erika Elisabeth Evangelista.pdf: 1020129 bytes, checksum: dee500eb8c5a17c94acdc64c0d81e2de (MD5) / Made available in DSpace on 2016-05-19T15:16:35Z (GMT). No. of bitstreams: 1 Erika Elisabeth Evangelista.pdf: 1020129 bytes, checksum: dee500eb8c5a17c94acdc64c0d81e2de (MD5) Previous issue date: 2014-12-11 / Periodontal disease is an inflammatory disease affecting the supporting tissues of the teeth, leading to loss of periodontal ligament and bone. The treatment involves scaling and root planning, and in more advanced cases there is the need for surgery and sometimes antibiotics. Photodynamic therapy is a therapeutic antimicrobial resource to aid in modulation of the inflammatory response triggered by the light. One of the main cells is the macrophage inflammatory process which acts in reciprocity with T lymphocytes. Thus the aim of this study was to evaluate the effect of photodynamic therapy in the chemotaxis of macrophages and activated T lymphocytes in the periodontal tissue. For such, patients who have undergone previous periodontal treatment with good oral hygiene and periodontal pockets residual requiring surgery were allocated. PDT was applied on the following parameters: methylene blue (50ug / ml) was applied to the bottom of the pocket and after 5 minutes via transmucosal the photosensitizer was irradiated with diode laser 660 nm, P = 100 mW, t = 90 s per point 9 J/point; energy density: 22 J/cm2, power density: 250 mW/cm2. One week after PDT, the surgical removal of the pockets was performed and samples sent for routine histology and immunohistochemical detection of activated T lymphocytes (CD45RO +) and macrophages (CD68 +). The samples were photographed and positive cells counted with the aid of ImageJ software. Data were evaluated statistically and no significant difference between groups (Mann-Whitney p = 0.9). We conclude that PDT does not interfere with chemotaxis of macrophages and activated T lymphocytes in periodontal tissue after seven days. / A doença periodontal é uma doença inflamatória que afeta os tecidos de suporte dos dentes, levando à perda de ligamento periodontal e de osso. O tratamento consiste em raspagem e alisamento radicular e nos casos mais avançados há a necessidade de cirurgias e, às vezes antibioticoterapia. A terapia fotodinâmica é um recurso terapêutico antimicrobiano que auxilia a modulação da resposta inflamatória desencadeada pela luz. Uma das principais células do processo inflamatório é o macrófago, que age em reciprocidade com os linfócitos T. Assim o objetivo deste trabalho foi avaliar o efeito da terapia fotodinâmica na quimiotaxia de macrófagos e linfócitos T ativados no tecido periodontal. Para tal foi realizado um estudo clínico transversal no qual foram alocados pacientes que passaram por tratamento periodontal prévio com boa higiene oral e bolsas periodontais residuais com indicação cirúrgica. A PDT foi aplicada nos seguintes parâmetros: azul de metileno (50ug/mL) aplicado no fundo da bolsa e após 5 minutos foi irradiado via transmucosa com laser de diodo, 660 nm, P= 100 mW, t= 90 s por ponto, 9 J/ponto, densidade de energia: 22 J/cm2, densidade de potência: 250 mW/cm2. Uma semana após a PDT foi realizada remoção cirúrgica das bolsas e as amostras encaminhadas para processamento histológico de rotina e imunohistoquímica para detecção de linfócitos T ativados (CD45ro+) e macrófagos (CD68+). As amostras foram fotografadas e as células positivas contadas com auxílio do software ImageJ. Os dados foram avaliados estatisticamente e não houve diferença significativa entre os grupos (Mann-Whitney p= 0,9). Conclui-se que a PDT não interfere na quimiotaxia de macrófagos e linfócitos T ativados no tecido periodontal após 7 dias.

The overall objective of this study was to understand the mechanisms that control antigen processing and binding of peptides to major histocompatibility complex (MHC) molecules. Towards this goal I investigated (a) the structural features of T cell-presented peptides with a focus on the role of recurrent hydrophobic residues in catalysis of helix formation by these peptides and (b) the biochemical events that determine the fates of the invariant chain molecule (Ii) in its various post-translational processing pathways. In the structural studies, I tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an α-helix can lead to helical nucleation on a hydrophobic surface.For a series of HPLC-purified peptides, including some T cell-presented peptides varying considerably in primary sequence, percentage helicity in the presence of lipid vesicles correlated with strip-of-helix hydrophobicity index (SOHHI), as shown by circular dichroism (CD) analysis. A prototypic helix peptide PH-1.0 (LYQELQKLTQTLK) was designed with a strong axial hydrophobic strip of 4 leucine residues. PH-1.0 formed about 38% helical structure in 10 mM phosphate buffer at pH 7.0 with di-O-hexadecyl phosphatidylcholine (DHPC) lipid vesicles, but no helical structure was detected when the peptide was in phosphate buffer alone. The helix-forming tendencies of 9 analogs of PH-1.0 with one or two amino acid variations from the parent peptide were examined in the presence of lipid vesicles and the results showed that (a) decreasing the strip-of-helix hydrophobicity by substituting even one of the four leucine residues in the axial hydrophobic strip with a less hydrophobic threonine residue reduced the helix-forming tendency of a peptide in the presence of lipid vesicles; (b) the placement of recurrent hydrophobic residues within the axial hydrophobic strip appeared to be critical for a peptide to be induced to form an α-helix by a hydrophobic surface; (c) there was an orientation preference for these peptides to interact with lipid vesicles and to form helical structure; (d) extra hydrophobic residues in other positions appeared to compete with the hydrophobic residues within the axial hydrophobic strip for interaction with the lipid vesicles and therefore to decrease the helix-forming tendency of peptides. For the biochemical studies of the function of Ii, a 17-residue peptide, Ii-3 (Ii 148-164), was synthesized. The CD analysis of Ii-3 showed mainly an α-helical conformation when the peptide was examined in the presence of lipid vesicles. [125I]-labeled Ii-3, after coupling at the N-terminus with a photoactivatable, heterobifunctional crosslinker N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), was able to bind to both α and β chains of class II MHC molecules, indicating that this region of Ii might cover the desetope of class II MHC molecules from the time of their synthesis until their charging with foreign peptides at an endosomal compartment. The biosynthesis of a chondroitin sulfate proteoglycan-form of Ii (CS-Ii) was examined in a class II MHC-negative cell line P3HR-1. [35S]sulfate-labeled microsomal membrane proteins of P3HR-1 were immunoprecipitated with anti-Ii monoclonal antibody and the results of SDS-PAGE analysis demonstrated that P3HR-1 could process Ii to CS-li in the absence of class II MHC molecules and the chondroitin sulfate identity of this molecule was confirmed by chondroitinase-ABC treatment. We conclude that there might be a class II MHC-independent pathway to process Ii to a chondroitin sulfate proteoglycan form as compared to the pathway in which Ii was associated with class II MHC and later cleaved by proteases residing in the endosomal compartment. In an effort to demonstrate in vitro that the class II MHC-associated Ii was eventually dissociated from class II MHC molecules by a proteolytic cleavage process, it was found that cathepsin B could completely remove Ii without damage to class II α and β chains. In order to identify those cleaved Ii fragments, three polyclonal anti-Ii peptide sera were produced by immunizing rabbits with keyhole limpet hemocyanin (KLH)-conjugated Ii peptides. Anti-Ii (146-169) was shown to be able to precipitate a p18 molecule only in cells expressing Ii. Anti-Ii (148-164 )and anti-Ii(78-92) were specific for their respective antigenic peptides as tested by enzyme-linked immunosorbent assay (ELISA).