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Orientador: Raquel Franco Leal Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A retocolite ulcerativa (RCU) é uma das formas mais comuns das doenças inflamatórias intestinais (DII), e é caracterizada por períodos de remissão e atividade da doença, acompanhada de diarreia, dor abdominal, perda de peso e desnutrição. A RCU é uma doença de origem multifatorial que acomete indivíduos geneticamente susceptíveis, nos quais uma resposta imunológica inadequada resulta de complexas interações ambientais, microbianas e do sistema imune inato e adaptativo, levando à produção de citocinas pró-inflamatórias. Esta superprodução de citocinas prejudica o processamento estrutural proteico, deixando o lúmen do retículo endoplasmático (RE) repleto de proteínas mal formadas. O acúmulo de proteínas mal formadas no RE é chamado de estresse do retículo endoplasmático (ERE). A resposta ao acúmulo de proteínas mal formadas (unfolded protein response - UPR), iniciada pela ativação das vias IRE1/Xbp-1, PERK/eIF2? e ATF6, e foi previamente associada à inflamação intestinal em modelos experimentais, estudos de associação gênômica (GWAS). Além de promover a degradação de proteínas e apoptose, a ativação do ERE também induz a transcrição de genes pró-inflamatórios. Dessa forma, o objetivo desse projeto foi investigar e caracterizar a ocorrência da ativação do ERE em mucosa intestinal de pacientes com RCU. Nossos resultados mostram que, através de técnicas de hibridização in situ e de imunohistoquímica, o ERE está ativado em pacientes com RCU por meio das vias PERK/eIF2? e IRE1/Xbp-1, mas não via ATF6. A análise transcricional confirmou a ativação de PERK/eIF2?, bem como o aumento da expressão de genes relacionados a UPR. Também foi observado que os genes pró-apoptóticos e de autofagia (Bax e ATG6L1, respectivamente), bem como os de citocinas pró-inflamatórias estavam modulados em pacientes com RCU. Além disso, foi observado que o ERE é ativado não apenas nas células epiteliais intestinais, mas também nas células da lâmina própria da mucosa colônica de pacientes com RCU. Como conclusão, tivemos que as vias de ERE e UPR estão ativadas na mucosa do cólon de pacientes com RCU. O aumento dos marcadores de apoptose e autofagia em pacientes com RCU reforçam ainda mais que o ERE está ativado, uma vez que são mecanismos dependentes, ativados para contrabalançar o dano ao tecido. Esses achados fornecem novos paradigmas sobre os mecanismos moleculares que mantêm o processo inflamatório na RCU Abstract: Ulcerative colitis (UC) is one of the most common forms of inflammatory bowel disease (IBD), and is characterized by periods of remission and disease activity, accompanied by diarrhea, abdominal pain, weight loss and malnutrition. UC is a disease of multifactorial origin that affects genetically susceptible individuals, in which an inadequate immune response results from complex environmental, microbial and innate and adaptive immune system interactions, leading to the production of pro-inflammatory cytokines. This overproduction of cytokines impairs structural protein processing, leaving the lumen of the endoplasmic reticulum (ER) full of malformed proteins. The accumulation of malformed proteins in the ER is called endoplasmic reticulum stress (ERE). The response to the accumulation of malformed proteins (unfolded protein response - UPR), initiated by the activation of the IRE1/Xbp-1, PERK/eIF2? and ATF6 pathways, and was previously associated with intestinal inflammation in experimental models, gene association studies (GWAS). In addition to promoting protein degradation and apoptosis, ERE activation also induces transcription of pro-inflammatory genes. Thus, the aim of this project was to investigate and characterize the occurrence of ERE activation in the intestinal mucosa of UC patients. Our results showed that, through in situ hybridization and immunohistochemistry techniques, ERE is activated in UC patients through the PERK/eIF2? and IRE1/Xbp-1 pathways, but not via ATF6. Transcription analysis confirmed the activation of PERK/eIF2?, as well as the increased expression of UPR-related genes. We observed that pro-apoptotic and autophagy genes (Bax and ATG6L1, respectively), as well as pro-inflammatory cytokines were modulated in patients with UCR. Furthermore, ERE is activated not only in intestinal epithelial cells, but also in cells of the lamina propria of the colonic mucosa of UC patients. In conclusion, we found that the ERE and UPR pathways are activated in the UC colonic mucosa. Increased markers of apoptosis and autophagy further reinforce that ERE is activated, as they are dependent mechanisms activated to counteract tissue damage in UC. These findings may provide new paradigms on the molecular mechanisms that maintain the inflammatory process in UC Doutorado Ciências da Cirurgia Doutor em Ciências FAPESP 2020//04407-1 CAPES 88882.435110/2019-01

Introduction: The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Methods: Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. Results: A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. Conclusions: The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

Orientador: Joyce Maria Annichino Bizzacchi Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Alopecia androgenética (AAG) é caracterizada por uma perda padrão de cabelo, e atualmente, entre os novos tratamentos, podemos citar o uso do plasma rico em plaquetas (PRP). A resposta ao PRP é controversa, e isto poderia estar relacionado ao número de plaquetas e aos níveis de fatores de crescimento presentes no produto. O objetivo deste projeto foi analisar o número de plaquetas e os níveis de fatores de crescimento (PDGF-AA, EGF e VEGF) presentes no PRP fresco e liofilizado, e sua correlação com os parâmetros de crescimento capilar em pacientes diagnosticados com AAG, usando o TrichoScan como método objetivo de avaliação, em dois desenhos de estudo. No primeiro, quinze pacientes receberam aplicação de PRP fresco. Os resultados mostraram aumento significativo na contagem de fios (p = 0,0018) e na porcentagem de fios anagênicos (p = 0,0070). Porém, estes resultados não foram correlacionados com a quantificação plaquetária e/ou quantificações dos fatores de crescimento no PRP. No segundo estudo, foi utilizado o PRP liofilizado em vinte e dois pacientes (onze no grupo tratamento e onze no grupo controle), com três injeções mensais. Porém, o resultado clínico observado não foi significativo em nenhum dos parâmetros avaliados (p = 0,206 para contagem de fios, e p = 0,398 para porcentagem de fios anagênicos). A contagem de plaquetas e a concentração dos fatores de crescimento presentes no PRP liofilizado também não foram correlacionados com a resposta obtida. Esta falta de correlação com os dois tipos de PRP aponta que outros mecanismos ou outros fatores de crescimento não quantificados neste projeto podem estar envolvidos nessa resposta Abstract: Androgenetic alopecia (AGA) is characterized by a pattern hair loss, and currently, among the new treatments, we can mention the use of platelet rich plasma (PRP). The response to PRP is controversial, and this could be related to the number of platelets and levels of growth factors present in the product. The objective of this project was to analyze the number of platelets and levels of growth factors (PDGF-AA, EGF and VEGF) present in fresh and lyophilized PRP, and their correlation with the parameters of hair growth in patients diagnosed with AAG, using TrichoScan as an objective evaluation method, in two different studies. In the first study, fifteen patients received application of fresh PRP. The results showed a significant increase in hair count (p = 0.0018) and in the percentage of anagen hair (p = 0.0070). However, these results did not correlated with platelet quantification and/or quantification of growth factors in PRP. In the second study, lyophilized PRP was used. However, the observed clinical result was not significant in any of the evaluated parameters (p = 0.206 for hair count, and p = 0.398 for percentage of anagen hairs). The platelet count and concentration of growth factors present in the lyophilized PRP also did not correlated with the response obtained. This lack of correlation with the two types of PRP indicates that other mechanisms or other growth factors not measured in these studies may be involved in this response Mestrado Fisiopatologia Médica Mestra em Ciências

Osteoarthritis (OA) is the most prevalent joint disease and a common cause of joint pain, functional loss, and disability. In addition to macroscopic features, such as cartilage degradation with subchondral bone remodeling, hypertrophy of the joint capsule, and osteophytes formation, several cellular and molecular alterations are present in OA, which lead to a chronic low-grade inflammation. Inflammatory mediators observed in OA joints are thought to be the downstream effectors of the pathogenesis of the disease. Although cytokines are among the most extensively studied mediators of inflammation, such as IL-1β and TNF, there has been an increase in studies showing the contribution of chemokines and adipokines in OA progression. This fact is supported by recent progress, which has considerably improved knowledge of the factors involved in the development of OA and the mechanisms responsible for its progression. Therefore, the aim of this chapter is to discuss the involvement of the inflammatory response in OA maintenance, focusing on the main inflammatory markers observed in studies with OA.