Made available in DSpace on 2015-04-14T14:48:06Z (GMT). No. of bitstreams: 1 431360.pdf: 192175 bytes, checksum: ad3d53980e72c328f5516279100aec4a (MD5) Previous issue date: 2011-03-11 / A presente disserta??o, vinculada ? Linha de Pesquisa Criminologia e Controle Social, do Programa de P?s-Gradua??o em Ci?ncias Criminais da PUC/RS, pretende realizar uma an?lise da dupla tutela legislativa contra os il?citos contra a ordem econ?mica brasileira, especialmente sob o enfoque criminol?gico. Neste setor, a evolu??o dos estudos sociol?gicos europeus e norte-americanos acerca das quest?es que envolvem o fen?meno criminal, conduziu ? investiga??o de Edwin H. Sutherland sobre os delitos de colarinho branco, o qual trouxe enormes contribui??es para o estudo das ilicitudes ocorridas no ?mbito empresarial, mormente pelas pessoas de status social elevado que cometem delitos no exerc?cio de suas fun??es. De modo cr?tico, realizou-se um estudo interdisciplinar das fun??es dos entes formadores do Sistema Brasileiro de Defesa da Concorr?ncia, em especial, do Conselho Administrativo de Defesa Econ?mica (CADE) e dos mecanismos legais criados para disciplinar a mat?ria concorrencial. O objetivo foi realizar uma an?lise desses instrumentos que a Lei n. 8.884/94 disp?e para vislumbrar, ao final, a ocorr?ncia de um tratamento legal diferenciado aos crimes contra a ordem econ?mica. Neste ponto, conduziu-se uma investiga??o conjunta das leis administrativa e penal para propor uma an?lise aprofundada da efetiva aplicabilidade das san??es criminais aos homens de neg?cio que cometem il?citos penais anticoncorrenciais.

Made available in DSpace on 2015-04-14T14:48:08Z (GMT). No. of bitstreams: 1 437212.pdf: 127046 bytes, checksum: 06d6acb3be280ab146208ddb06349487 (MD5) Previous issue date: 2012-12-19 / The aim of this paper is to analyze models of contemporary Criminal Law foundations which are based on the idea of unlawful (Unrecht) or the idea of criminal sanction. In this context, it aims at investigating in which core must reside its foundations. Moreover, the research hypothesizes that the theories issued from unlawful are granted a retrospective foundation, while the theories issued from criminal sanction are granted a prospective foundation. These last theories would be not consistent because consequentialist and, this way, incompatible with the concept of foundation scaffolded in this research, aimed at the past or the present. In order to develop this study, a distinction among the concepts of meaning, foundation, function and structure of Criminal Law is established. Subsequently, a brief historical evolution from its foundations in the diverse theories of the delict (causal, neoclassic and final) to actual proposals is elaborated. Then, the study examines two specific theories on Criminal Law foundation, one issued from criminal sanction (the Claus Roxin`s functionalism) and another issued from unlawful (the onto-anthropological conception from Jos? Francisco de Faria Costa). In this process, the following theorical problems are faced: consequentialism, crime concept instrumentalization, boundaries between Criminal Law and Criminal Policies, qualitative distinction of criminal unlawfulness and other unlawfulnesses and definition of criminal legal interest and offensivity. Finally, this research proposes the foundation and the function of Criminal Law, concluding that its meaning must reside in the idea of criminal unlawful (Unrecht); its foundation, in the offense of relevant criminal legal interests, and its function, in the subsidiary protection of relevant criminal legal interests. Key / A presente pesquisa centra-se na an?lise de modelos ou de tentativas te?ricas de fundamenta??o do Direito Penal na Contemporaneidade que tenham por ponto de partida ou a no??o de il?cito (Unrecht) penal, ou a no??o de pena criminal. Nesse contexto, visa a investigar em qual destes n?cleos de valor o il?cito ou a pena deve situar-se o seu fundamento. Para tanto, tem por hip?tese que propostas te?ricas de fundamenta??o do Direito Penal a partir do il?cito conferem-lhe um fundamento retrospectivo, ao passo que propostas te?ricas de fundamenta??o do Direito Penal a partir da pena atribuem-lhe um fundamento prospectivo, ou seja, voltado ao futuro. Estas ?ltimas seriam inconsistentes porque consequencialistas e, dessa forma, incompat?veis com o conceito de fundamento sustentado nesta investiga??o, necessariamente voltado ao passado ou ao presente. A fim de realizar os objetivos aos quais se prop?e, o estudo ? inaugurado com uma distin??o entre os conceitos de sentido, fundamento, fun??o e estrutura do Direito Penal. A seguir, elabora-se uma breve evolu??o hist?rica do seu fundamento nas diversas teorias do delito (causal, neocl?ssica e final), chegando-se ?s propostas Contempor?neas. Ap?s, o trabalho volta-se ao exame anal?tico e cr?tico de duas espec?ficas tentativas te?ricas atuais de fundamenta??o do Direito Penal, uma que parte da pena (o funcionalismo teleol?gico-racional, de Claus Roxin) e outra que parte do il?cito (a concep??o onto-antropol?gica, de Jos? Francisco de Faria Costa). Durante tal percurso, adentra ainda no estudo dos seguintes problemas te?ricos: consequencialismo, instrumentaliza??o do conceito de crime, limites da rela??o entre Dogm?tica Penal e Pol?tica Criminal, diferencia??o material da ilicitude penal frente ?s demais ilicitudes e conceitua??o das no??es de bem jur?dico penal e de ofensividade. Percorrido esse caminho, s?o apresentados, ao final, o fundamento e a fun??o do Direito Penal sugeridos por esta investiga??o, concluindo-se que o seu lugar de sentido na Contemporaneidade deve residir na no??o constitucionalmente vinculada de il?cito (Unrecht) penal; o seu fundamento, na de ofensa a bens jur?dicos penalmente relevantes, e a sua fun??o, na de tutela subsidi?ria de bens jur?dicos penalmente relevantes.

A Interleucina 8 (IL-8) é uma quimiocina CXC angiogênica que tem papel importante no desenvolvimento e progressão de vários tumores malignos, incluindo o câncer de próstata (CaP). O polimorfismo de nucleotídeo único (SNP) -251 T/A da região promotora do gene da IL-8, relativo ao local de início da transcrição deste gene, está associado com a produção desta citocina. O efeito da IL-8 é mediado através de dois receptores de alta afinidade, CXCR1 e CXCR2. O presente estudo investigou a influência da variação dos genes IL-8 e CXCR2 na susceptibilidade e nas características clinicopatológicas do CaP em um grupo de brasileiros. Duzentos e um pacientes e 185 controles saudáveis foram selecionados neste estudo casocontrole. Amostras de sangue foram coletadas para extração de DNA; a tipagem da IL-8 -251 T/A e CXCR2 +1208 C/T foi realizada através da reação em cadeia da polimerase com sequência específica de primers (PCR-SSP), seguida pela eletroforese em gel de agarose. O risco associado entre os genótipos, a susceptibilidade do CaP e as características do tumor, foi estimado pelo odds ratio (OR), com intervalo de confiança de 95%, usando análise de regressão logística e ajustando para idade ao diagnóstico. Encontramos uma associação estatisticamente significativa entre o genótipo heterozigoto CT do gene CXCR2 +1208 e CaP. Este genótipo foi significativamente menos frequente em pacientes com estádio clínico T3-T4 comparado com T1-T2 (56.7% versus 80.5%). Nossos achados sugerem que os portadores do genótipo CT CXCR2 +1208 tiveram um efeito protetor para estádio avançado de CaP (CT versus CC: OR ajustado = 0.25; P = 0.02). Não foi encontrada associação significativa entre o polimorfismo -251 T/A da IL-8 e os parâmetros clinicopatológicos do CaP. Estes resultados indicam que o genótipo CT do CXCR2 +1208 é menos frequente em estádios avançado de CaP, sugerindo que este receptor de quimiocina tenha um papel na patogênese desta doença. / Interleukin-8 (IL-8) is an angiogenic CXC chemokine that plays an important role in both the development and progression of several human malignancies including prostate cancer (PC). A single nucleotide polymorphism (SNP) at -251 upstream of the transcriptional start site of the IL-8 gene has been shown to influence its production. The effects of IL-8 are mediated by two highly related chemokine receptors, CXCR1 and CXCR2. The present study investigated the influence of the IL-8 and CXCR2 gene variation on susceptibility and clinicopathological characteristics of PC in a group of Brazilian subjects. Two hundred and one patients and 185 healthy controls were enrolled in a case-control study. Blood was collected for DNA extraction; typing of IL-8 -251 T/A and CXCR2 +1208 C/T genes was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP), followed by agarose gel electrophoresis. Risk association between the genotypes, PC susceptibility and tumor characteristics was estimated by odds ratio (OR) and 95% confidence intervals (95% CI) using logistic regression analysis, after adjusting for age at diagnosis. A significant association was found between the heterozygous CXCR2 +1208 CT genotype and PC. The CXCR2 +1208 CT genotype was significantly less frequent in patients with clinical stage T3-T4 compared to T1-T2 (56.7 versus 80.5%). Our findings suggest that carriers of the CXCR2 +1208 CT genotype had a protective effect for advanced PC (CT versus CC: adjusted OR = 0.25; P = 0.02). No association was observed between the SNP for IL-8 -251 T/A and clinicopathological parameters of PC. These results indicated that the CXCR2 +1208 CT genotype is less frequent in advanced stages of PC, suggesting that this chemokine receptor plays a role in the pathogenesis of this disease.

O carcinoma espinocelular de boca (CEB) é a neoplasia maligna mais comum da cavidade oral, correspondendo à aproximadamente 94% dos casos dessa região. Apesar dos diversos estudos moleculares e celulares do CEB, a taxa de sobrevida dos pacientes é de aproximadamente 50%, devido principalmente ao tamanho do tumor, metástase em linfonodos regionais, grau de diferenciação das células e sítio anatômico. O microambiente tumoral do CEB, é extremamente complexo e diversificado, tendo como característica principal um estado inflamatório crônico imunossupressivo. Este microambiente é sustentado pela liberação de diferentes citocinas inflamatórias, como IL-6, TNF- - atividades exercidas tanto pelas células tumorais quanto pelas estromais. Dentre essas atividades, tem sido relatado na literatura que as citocinas inflamatórias são capazes de aumentar a migração e a capacidade de invasão das células tumorais. Entre as células estromais, os macrófagos são as mais abundantes e participam da manutenção do microambiente tumoral. De acordo com o estímulo, podem ser polarizados M1, com papel pró-inflamatório e antitumoral, e M2, com papel anti-inflamatório e pró-tumoral. O objetivo desta tese foi compreender o papel dos macrófagos no prognóstico de CEB e das citocinas inflamatórias IL-6, TNF- - linhagens celulares de CEB. Para verificar o papel dos macrófagos no prognóstico, foi realizada uma revisão sistemática na qual foram incluídos apenas os estudos que utilizavam amostra de pacientes com CEB e avaliavam o prognóstico com marcadores para macrófagos. Foi observado que maiores concentrações de macrófagos CD68+ e CD163+ estavam relacionados com pior prognóstico de pacientes com CEB, embora não tenha sido possível concluir qual região tumoral a presença destas células seja mais importante 7 para o desfecho. Para analisar o papel das citocinas inflamatórias IL-6, TNFILensaios in vitro utilizando duas linhagens celulares, SCC25 e Cal27, em condições promotoras de migração sob a influência dessas citocinas. Foi observado que a citocina IL-6 foi capaz de aumentar a velocidade de migração e a direcionalidade tanto da SCC25 quanto da Cal 27 e que esta melhora na capacidade migratória ocorreu através de um crosstalk entre a via de sinalização relacionada a IL6 (STAT3) e a via reguladora de migração celular, Rho GTPase Rac1. Estes dados reforçam o papel do microambiente tumoral no processo de progressão tumoral e sugerem potenciais alvos terapêuticos como a modulação do perfil da população de macrófagos e o papel de interleucinas no controle de invasão tecidual e metástase. / Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, corresponding to approximately 94% of the cases in this region. Despite the diverse molecular and cellular studies of OSCC, the patient survival rate is approximately 50%, mainly due to tumor size, regional lymph node metastasis, cell differentiation and anatomic site. The OSCC tumor microenvironment is extremely complex and diverse, with the main characteristic being an immunosuppressive chronic inflammatory state. This microenvironment is supported by the release of different inflammatory cytokines, such as IL-6, TNF- - and enhance the activities of both tumor and stromal cells. Among these activities, it has been reported in the literature that inflammatory cytokines are capable of increasing migration and invasiveness of tumor cells. Among stromal cells, macrophages are the most abundant and participate in the maintenance of the tumor microenvironment. According to the stimulus, macrophages can be polarized in M1, with pro-inflammatory and anti-tumoral role, and M2, with antiinflammatory and pro-tumoral role. Thus, the aim of this thesis was to evaluate the role of macrophages in the prognosis of OSCC and the influence of inflammatory cytokines IL-6, TNF- - OSCC cell lines. To assess the role of macrophages in the prognosis, a systematic review was conducted in which only studies using a sample of OSCC patients were evaluated and the prognosis was evaluated with macrophage markers. It was observed that higher concentrations of CD68 + and CD163 + macrophages were related to worse prognosis in patients with OSCC, although it was not possible to conclude which tumor region the presence of these cells is more important for the outcome. In order to analyze the role of the inflammatory cytokines IL-6, TNF- - atory 9 behavior of OSCC cells, in vitro assays using two cell lines, SCC25 and Cal27, were performed in migration-promoting conditions under the influence of these cytokines. It was observed that IL-6 was able to increase the speed migration and directionality of both SCC25 and Cal 27 and that this improvement in migratory capacity occurred through a crosstalk between the IL6-related signaling pathway (STAT3) and cell migration-related pathway, RhoGTPase Rac1. These data reinforce the role of the tumor microenvironment in the tumor progression process and suggest potential therapeutic targets such as the modulation of the profile of the macrophages population and the role of interleukins in the control of tissue invasion and metastasis.

O câncer é um problema de saúde pública mundial, apresentando acréscimo na sua incidência a cada ano. O seu processo de evolução ainda não foi completamente desvendado, dificultando a elaboração de terapias adequadas. Na busca por um melhor prognóstico, pesquisas recentes têm discutido o papel das citocinas inflamatórias, do nicho perivascular e das células-tronco nos mecanismos de desenvolvimento e manutenção dos tumores malignos. Os tumores de glândula salivar representam uma pequena porcentagem das patologias malignas da região de cabeça e pescoço, podendo ocorrer em adultos e em crianças. O diagnóstico dificilmente é precoce e a taxa de sobrevida é extremamente baixa comparada aos demais tumores da região. Assim, este estudo teve como objetivo estudar as células provenientes dos tumores de glândula salivar do tipo adenoide cístico (CAC) e adenocarcinoma NOS (AdNOS) quanto ao seu perfil imunofenotípico, quanto à existência ou não de células-tronco tumorais nessa população, bem como investigar possíveis modificações na ativação de STAT3, Akt e ERK (moléculas envolvidas em vias de sinalização de manutenção do tumor), quando em contato com fatores secretados por células endoteliais. Foram coletados 5 CACs e 4 AdNOS, no Hospital da Universidade de Michigan (Ann Arbor, MI, EUA), durante 2010 e 2012. As células foram isoladas e caracterizadas em citometria de fluxo em P0 e P7, demonstrando um perfil de células CD44+ALDH+Lin- variando de 0,33 a 3,19% e 0,36 a 2,00%, respectivamente, entre 5 linhagens avaliadas. Na avaliação por western blotting, a e-caderina, o Snail e a actina de músculo liso foram ausentes em todos os tipos tumorais, enquanto que a citoqueratina 20 (Ck20) foi presente apenas nos AdNOS. Comparando os tumores com suas metástases, a presença de Ck20, p63 e β-catenina foi semelhante, enquanto que citoqueratina 7, a vimentina e o Bmi-1 foram maiores nas metástases. Tanto os AdNOS quanto CACs apresentaram receptores para IL-6, IL-8 e EGF. Foi observado que mediadores solúveis liberados pelas células endoteliais foram capazes de fosforilar STAT3, Akt e ERK em todas as células salivares estudadas, no entanto, a proteína recombinante humana IL-6, isoladamente, não foi capaz de ativar Akt. Orosferas foram geradas em todos os tipos tumorais, demonstrando o potencial de auto-renovação celular. Um maior número de esferas foi observado nas células metastáticas em relação às primárias. Células CD44+ALDH+, comparadas com CD44-ALDH-, geraram mais esferas, quando plaqueadas em alta densidade (5.000 células). No entanto, o inverso foi encontrado, quando uma única célula foi utilizada para o ensaio (p>0,05). Devido à dificuldade de obtenção e manipulação de células de tumores de glândula salivar, ainda há muito que se investigar mecanisticamente. Considerando a fosforilação de STAT3 na presença de IL-6, semelhante ao verificado em outros tumores, o uso de anticorpos contra IL-6, talvez sejam uma opção no futuro. / Cancer is a public health problem worldwide, with an increase in incidence every year. The process of its evolution is still not completely understood, hindering the development of appropriate therapies. In the search for a better prognosis, recent reports have discussed the role of inflammatory cytokines, perivascular niche and stem cells in the mechanisms of development and maintenance of malignant tumors. The salivary gland tumors represent a small percentage of malignancies of the head and neck and can occur in both adults and children. Early diagnosis is difficult and the survival rate is extremely low compared to other tumors in the same region. Thus, this study aimed to study cells from the adenoid cystic carcinoma (ACC) and adenocarcinoma NOS (AdNOS) tumors of salivary gland regarding its immunophenotypic profile and the existence or absence of tumor stem cells in this population, as well as investigate possible changes in the activation of STAT3, Akt and ERK (molecules involved in signaling pathways of tumor maintenance), when exposed to factors secreted by endothelial cells. ACCs (n=5) and AdNOS (n=4) were collected at the Hospital of the University of Michigan (Ann Arbor, MI, USA), during 2010 to 2012. Cells were isolated and characterized by flow cytometry at P0 and P7, showing a profile of ALDH+CD44+Lin- ranging from 0.33% to 3.19% and 0.36% and 2.00%, respectively, between 5 cell lines evaluated. In the protein profile, e-cadherin, Snail and SMA were absent in all tumor types. Ck20 was present only in AdNOS. Comparing primary tumors and their metastases, the presence of Ck20, and p63 β-catenin was similar, while Ck7, vimentin and Bmi-1 were higher in metastases. Both AdNOS as ACCs had receptors for IL-6, IL-8 and EGF. It was observed that soluble mediators released by endothelial cells were able to activate STAT3, Akt and ERK phosphorylation in all cells studied. However, recombinant human IL-6 alone was not able to activate Akt. Orospheres were generated in all tumor types, indicating the potential for cellular self-renewal. Highest number of spheres was observed in metastatic cells compared to primary. ALDH+CD44+ cells compared to ALDH-CD44- generated more spheres when plated in high density (5,000 cells), however, the opposite was found when one single cell seed was evaluated (p> 0.05). There is doubt if these cell markers would be consider for a stem cell model in salivary tumors. Due to the difficulty of obtaining and manipulating salivary gland tumor cells, there is still much to investigate mechanistically. As the phosphorylation of STAT3, in the presence of IL-6, was similar to that observed in other tumors, the use of antibodies against IL-6, may be an option in the future.

Regulatory T cells (Treg) play a crucial role in controlling immune homeostasis. Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Treg. While several mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain unknown. The Treg pool consists of highly diverse subpopulations, indicating that there is a potential to optimise Treg-targeted therapies if disease-relevant mechanisms can be established. Microarray data from our lab suggests a marked upregulation of the integrin αv as well as the IL-33 receptor ST2 in Treg retrieved from the inflamed central nervous system (CNS) during experimental autoimmune encephalomyelitis compared to peripheral lymphoid organs. These two molecules were further investigated within this PhD project with the aim to understand their role in Treg function during chronic inflammatory disease. αvβ integrins have been reported to be needed for effector T cell migration to inflamed sites through binding of extracellular matrix components and are involved in TGF-β activation by a variety of cell types. Conditional knockout mice lacking the integrin αv specifically in Foxpγ+ Treg were generated to address the role of αv integrins on regulatory T cells in inflammatory disease. αv-/- Treg showed a deficiency in activating latent TGF-β, but were able to suppress responder T cell proliferation in vitro as well as in vivo. αv-/- Treg were also able to migrate to the inflamed CNS during EAE and resolve disease. However, αv-/- Treg were detected at significantly lower numbers and proportions in the inflamed gut during a curative T cell transfer model of colitis; this led to a quantitative impairment in the ability of αv-/- Treg to cure colitis when compared to wild-type (WT) Treg. Whether this is a deficit in migration, survival, proliferation, or Foxp3 stability, remains to be investigated. IL-33 acts as an alarmin and is best studied as a cytokine released upon tissue damage that induces a potent type 2 immune response by acting on a multitude of immune cells. Expression of the IL-33 receptor ST2 on Treg has recently been associated with positive metabolic parameters in visceral adipose tissue, protection from gut inflammation, and tissue-restorative function in other inflamed tissues such as injured muscle or lung. This project showed that in steady state, ST2+ Treg expressed high levels of several markers which have been associated with potent regulatory function. When stimulated in vitro, ST2+ Treg showed a better survival and expansion rate compared to their ST2- counterparts, even more so in the presence of IL-33. T-bet deficiency in Treg resulted in an increased ST2+ Treg pool, and T-bet-associated cytokine IFN-γ was found to antagonise IL-33-induced expansion of the ST2+ Treg pool in a T-bet-independent manner. When ST2+ and ST2- Treg were tested for their respective suppressive capacity in vivo, ST2+ Treg were able to suppress responder T cell expansion despite being found only at low numbers in secondary lymphoid organs compared to ST2- Treg. However, in a curative model of T cell transfer colitis, ST2+ Treg were less capable of controlling the ongoing immune response than ST2- Treg. A possible explanation for the superiority of ST2- Treg in this setting can be found in the fact that injected ST2- Treg acquired a distribution of ST2 expression reminiscent of WT Treg over the course of disease. On the other hand, an increased starting pool of ST2+ Treg as occurs in T-bet-/- Treg significantly enhanced the capacity of Treg to control colitis compared to WT Treg. In conclusion, both ST2- and ST2+ Treg are likely to have a distinct, non-redundant role in suppressing T cell activation in secondary lymphoid organs and controlling ongoing inflammation in peripheral tissue, respectively.

Many parasites have evolved to release products that inhibit host defence mechanisms such as enzymes in the mammalian host, in order to promote and sustain their survival within the host. The human filarial nematode Brugia malayi produces larval microfilariae, which circulate in the blood stream. Their most abundant secreted product is a serine protease inhibitor Bm-SPN-2. Serine protease inhibitors (Serpins) are reported to be involved in how the nematodes avoid host immune defences, and in the case of Bm-SPN-2, the protein was found to specifically inhibit the enzymatic activity of human neutrophil elastase and cathepsin G in a dose-dependent manner. More recently, these two enzymes have been linked to the activation of a major innate cytokine IL-33, which is stored as a full-length 270-aa protein in the cell nucleus, and released as an active C-terminal domain upon stimulation. As full-length (FL) human and murine IL-33 are not commercially available, soluble murine and human FL-IL-33 were produced in transfected HEK 293T cells, following mutation of the nuclear binding motif. In this form, IL-33 is no longer retained in the nucleus and can be purified as a soluble protein. It was confirmed that once cleaved, recombinant human IL-33 was able to induce significant IL-6 secretion by mast cells. Bm-SPN-2 was then shown to block human full-length IL-33 cleavage by inhibiting human neutrophil cathepsin G in a dose dependent manner, supporting the hypothesis that Bm-SPN-2 may act in vivo to prevent IL-33 activation and the promotion of the TH2 immune response. However, in the in vivo setting, it was unexpectedly found that IL-33R (ST2) gene deficiency did not enhance the survival of B. pahangi microfilariae. Furthermore, in the absence of IL-33R, murine immune responses to microfilariae were not significantly altered compared to wild-type BALB/c mice, other than in a significant increase in IL-33 expression. Hence while Bm-SPN-2 can act in vitro to forestall one of the key events in TH2 induction, this has not yet been shown to be crucial to the immune response to the parasite in vivo.

Vaccines are a highly effective means of preventing infectious disease. However, for many diseases of livestock the available vaccines are ineffective or sub-optimal. This is partly due to challenges surrounding the specific targeting of antigen presenting cells (APCs). In order to improve the delivery of protective antigens to host APCs, a novel lentiviral vector derived from visna / maedi virus (VMV) has been developed. Initial characterisation using an enhanced green fluorescent protein (eGFP) reporter transgene found that the novel VMV vector efficiently transduced a wide range of cell lines including cells of ovine, human, murine, bovine and caprine origin. In addition, the VMV vector was found to elicit sustained transgene expression for at least 4 weeks in rapidly dividing cell lines. One of the most important factors for acceptable vaccines is their safety. Therefore, in order to increase the bio-safety of the VMV vector, integration-defective and self-inactivating forms were produced. Integration-defective VMV lentiviral vectors (IDLVs) were found to produce 1-LTR circular episomes favourably over integrated provirus following the transduction of target feline and ovine cell lines. This led to a decrease in transgene expression over time in dividing cells. In contrast, in non-dividing cells transgene expression was maintained at a similar level to integration-competent VMV vectors. Self-inactivating (SIN) VMV vectors were constructed and found to have a significant decrease in LTR activity. Transgene expression was maintained by the insertion of an internal promoter derived from human cytomegalovirus (CMV) acting directly on the transgene. When self-inactivating and integration-defective modifications were incorporated into the same vector particle, a 4-fold decrease in transduction relative to the parent vector was observed. Ovine monocyte-derived dendritic cells (MDDCs) and macrophages (MDMs) were found to be efficiently transduced by the VMV vector, whereas lentiviral vectors derived from HIV-1 poorly transduced both of these primary cell populations. Following this work, the ability to deliver pathogen genes into APCs was studied using the Chlamydia abortus (C. abortus) major outer membrane protein (MOMP) as the transgene. C. abortus is the most common infectious cause of ovine abortion worldwide and MOMP has previously been shown to stimulate strong antibody responses after vaccination. Unexpectedly, the VMV vector encoding either eGFP or MOMP was found to induce apoptosis in MDDCs and MDMs using Annexin V staining. Apoptotic cells were detectable as early as 6 hours post-transduction of cells. Furthermore, release of the pro-inflammatory cytokine IL-1β was associated with the formation of late apoptotic cells. Apoptotic bodies produced post-transduction were able to be phagocytosed by immature MDDCs and the transgene efficiently cross-presented to T-cells. The ability of the novel VMV vector to induce a suitable recall immune responses was investigated using an in vitro model. Here, an autologous population of MDDCs were cultured with the apoptotic bodies produced post-transduction before the addition of autologous PBMC. Proteins from the apoptotic bodies were presented by the MDDCs to PBMC leading to a strong, antigen specific recall immune response against C. abortus MOMP. This was proven by the detection of cytokines IFNγ and IL-10 in the co-culture supernatant from PBMC activated by the MOMP transgene cross-presented by MDDCs. No release of IL-4 or IL-17A could be detected. These data presented in this thesis show the potential for improving delivery of antigens in livestock vaccines by the use of lentiviral vectors. In addition, this vector system provides a strong base for the study of other potential protective antigens in vitro.

Une série d’études récentes chez l’homme et dans les modèles murins a conduit à la mise en évidence d’une réponse immune, qui met en jeu des cellules de l’immunité innée et de l’immunité adaptative dans le tissu adipeux (TA) obèse. Nous avons mis en évidence un dialogue pro-inflammatoire entre les macrophages et les LT CD4+ dans le TA humain obèse mettant en jeu l’IL-1β, l’IL-17 et l’IL-22. Le pourcentage de Th17 est positivement corrélé au % d’HbA1c. De plus, la sécrétion des cytokines impliquées dans cette boucle pro-inflammatoire diminuent après une perte de poids induite par la chirurgie bariatrique. Par la suite, nous avons montré que l’IL-1β et l’IL-17 induisent des programmes transcriptionnels pro-inflammatoires concordant dans trois types de cellules non-immunitaires du TA, les pré-adipocytes, les cellules endothéliales et les adipocytes, et diminuent les gènes du métabolisme dans les adipocytes. Les effets d’IL-1β sont nettement plus prononcés que ceux de l’IL-17. Le milieu conditionné de cellules immunitaires CD45+ reproduit les réponses pro-inflammatoire et catabolique induites par les cytokines recombinantes dans les adipocytes, et ces réponses sont inhibées après la neutralisation de l’IL-17 et l’IL-1β. Ces résultats démontrent une implication pathologique de l'IL-1β et de l'IL-17 dans les altérations du TA induites par l'obésité. / A series of recent studies in humans and mouse models has led to the detection of an immune response, which involves cells of the innate immunity and adaptive immunity in obese adipose tissue (AT).We highlighted a proinflammatory crosstalk between macrophages and CD4+ T cells in obese human AT involving IL-1β, IL-17 and IL-22. Percentage of Th17 is positively correlated with HbA1c %. Moreover, the secretion of the cytokines implicated in this proinflammatory loop decreased after weight loss induced by bariatric surgery.Subsequently, we have shown that IL-1β and IL-17 induce proinflammatory transcriptional programs in three types of non-immune cells of the TA, preadipocytes, endothelial cells and adipocytes , and decrease metabolism genes in adipocytes. IL-1β effects are much more pronounced than those of IL-17. The conditioned medium of CD45+ immune cells reproduces the pro-inflammatory and catabolic responses induced by the recombinant cytokines in adipocytes, and these responses are inhibited after the neutralization of IL-17 and IL-1β.These results demonstrate a pathological involvement of IL-1β and IL-17 in the AT dysfunction induced by obesity.

O desenvolvimento do câncer cervical depende da exposição ao HPV, fator necessário, mas não suficiente. Outras bactérias, tais como ureaplasmas, têm sido associadas como cofatores. O objetivo deste estudo foi avaliar a presença de ureaplasmas em mulheres com lesão cervical, e observar alterações em PHK causadas pela infecção por ureaplasmas. 140 swabs vaginais foram coletados. O material foi submetido a PCR para a detecção de HPV, Mollicutes, U. urealyticum, U. parvum e seus sorotipos, e outras bactérias de importância ginecológica; e qPCR para U. urealyticum e U. parvum. Também foi realizada a infecção de ureaplasmas em PHK transformados com HPV. As células foram contadas e realizou-se a dosagem das citocinas IL1-β, IL-6 e TNF-α. HPV, Mollicutes, U. parvum, sorotipos 1 e 6 de U. parvum, T. vaginalis e G. vaginalis, além de alguns fatores socioeconômicos, foram associados com lesão cervical. Verificou-se maior carga de U. parvum entre mulheres com lesão. Houve diminuição do número de células e maior liberação de IL-6 e TNF-α nos grupos infectados. Com os resultados obtidos neste estudo, foi possível verificar uma associação entre os ureaplasmas e HPV no início das lesões cervicais, contudo mais estudos precisam ser realizados para aprimorar essa hipótese. / The development of cervical cancer depends on the exposure to HPV, necessary factor, but not enough. Other bacteria, such as ureaplasmas, have been associated as cofactors. The aim of this study was to evaluate the presence of ureaplasmas in women with cervical injury, and observe changes in PHK infected by ureaplasmas. 140 vaginal swabs were collected. The material was subjected to PCR for detection of HPV, Mollicutes, Ureaplasma urealyticum, U. parvum (and serotypes) and other bacteria gynecological importance; qPCR for U. urealyticum and U. parvum was made. PHK transformed by HPV was infected by ureaplasma. Cells were counted and it was done titration of IL1-β, IL-6 and TNF-α. HPV, Mollicutes, U. parvum, serotypes 1 and 6 U. parvum, T. vaginalis and G. vaginalis, and some socioeconomic factors were associated with cervical injury. Besides this, it was detected higher load U. parvum among women with injury. There was decrease in cell number and increased release of IL-6 and TNF-α in infected groups. With the results of this study, we found an association among HPV and ureaplasmas at the beginning of cervical lesions, but more studies are needed to enhance this hypothesis.