Orientador: Eduardo Hochuli Vieira / Resumo: A proposta deste estudo foi avaliar o processo de manutenção de volume dos biomateriais vidro bioativo e beta tricálcio-fosfato (β-TCP) associados ou não ao osso autógeno na técnica de levantamento do seio maxilar de humanos. Treze seios maxilares foram enxertados com β-TCP, sete com β-TCP associado ao osso autógeno, onze com vidro bioativo, nove com vidro bioativo associado ao osso autógeno e onze com osso autógeno (Grupo Controle). Após 7 a 14 dias, tomografias computadorizadas de feixe cônico (TCFC) foram solicitados para mensurar o volume inicial do enxerto (T1). Decorridos 6 meses de reparação óssea, novos exames tomográficos foram realizados para avaliar o volume final (T2) e verificar as alterações volumétricas dos materiais. Após a análise dos resultados, considerando as alterações volumétricas em um período de 6 meses, evidenciou-se um comportamento semelhante entre o enxerto autógeno e os substitutos ósseos, não sendo observada diferença estatisticamente significante entre os materiais analisados. / Abstract: The purpose of this study was to evaluate the process of bone maintenance and remodeling with the use of bioactive glass and beta-tricalcium phosphate (β-TCP) associated or not with autogenous bone in the human maxillary sinus technique. Thirteen maxillary sinuses were grafted with β-TCP, seven with β-TCP associated with autogenous bone, eleven with bioactive glass, eleven with bioactive glass associated with autogenous bone and eleven with autogenous bone (Control Group). After 7 to 14 days, computed tomography (CBCT) were requested to measure the initial graft volume (T1). After 6 months of bone repair, new tomographic examination were performed to evaluate the final volume (T2) and to verify the volumetric changes of the materials. After analyzing the results, considering the volumetric changes over a 6-month period, a similar behavior was observed between the autogenous graft and the bone substitutes, and no statistically significant difference was observed between the analyzed materials. / Doutor

O monitoramento de imunossupressores, como os inibidores de calcineurina ou de mTOR, é essencial para evitar desfechos clínicos desfavoráveis, em receptores de transplante renal. Polimorfismos em genes envolvidos na farmacocinética têm sido associados com variabilidade na resposta a imunossupressores, porém o papel de polimorfismos em genes envolvidos na farmacodinâmica é pouco conhecido. O objetivo deste estudo foi investigar a influência de polimorfismos de MTOR, PPP3CA, FKBP1A, FKBP2 e FOXP3, genes envolvidos na farmacodinâmica de imunossupressores, sobre a resposta clínica a tacrolimo e everolimo, em receptores de transplante renal. Foram incluídos 269 pacientes do ensaio clínico original (NCT01354301), realizado no Hospital do Rim e Hipertensão da UNIFESP, e randomizados em três esquemas imunossupressores: tacrolimo 0,05 mg/kg/dia com everolimo 1,5 mg/dia (TAC5/EVR); tacrolimo 0,1 mg/kg/dia com everolimo 1,5 mg/dia (TAC10/EVR); e tacrolimo 0,1 mg/kg/dia com micofenolato de sódio (TAC10/MFS). Foram coletados dados clínicos e laboratoriais, tais como o monitoramento de imunossupressores e desfechos de eficácia de segurança. Os polimorfismos nos genes MTOR (c.4731G>A, c.1437T>C, c.2997C>T); PPP3CA (c.249G>A); FKBP1A (n.259+243936T>C); FBKP2 (c.-2110G>T) e FOXP3 (c.-23+2882A>C, c.-22-902A>G) foram analisados por PCR em tempo real. As frequências alélicas dos polimorfismos estudados foram similares às da população global do projeto 1000genomes. O tratamento com everolimo e tacrolimo em maior dose (TAC10/EVR) foi associado com menor taxa de filtração glomerular estimada (TFGe) e maior creatinina sérica. Enquanto que o tratamento com tacrolimo e micofenolato de sódio (TAC10/MFS) foi associado com maior número de episódios de infecção por citomegalovirus, no 1° ano pós-transplante. Com relação aos desfechos de eficácia, os portadores do genótipo CC de MTOR c.1437T>C e FOXP3 c-23+2882A>C apresentaram maiores concentrações de creatinina sérica, no 12° mês (p<0,05). O polimorfismo FOXP3 c.-23+2882A>C foi associado com maior probabilidade de creatinina sérica aumentada (OR=1,75; IC95%=1,07-2,86; p=0,025). Os resultados da análise de regressão logística mostraram que o alelo MTOR c.4731G (genótipos AG+GG) foi associado com maior risco de rejeição aguda (OR=3,37; IC95%=1,10-10,30; p=0,033). Os portadores do alelo c.4731G apresentaram maior incidência cumulativa de episódios de rejeição, no 1° ano pós-transplante. Com relação aos desfechos de segurança, a variante FKBP2 c.-2110G>T (genótipo GG) foi associada com maior risco de leucopenia (OR=7,10; IC95%=1,81-27,87; p=0,025). O polimorfismo FKBP1A n.259+24936T>C (alelo C) foi associado com maior risco de constipação (OR=2,52; IC95%=1,13 - 5,61; p=0,024), enquanto que os polimorfismos FOXP3 c.-22-902A>G (alelo A) e c.-23+2882A>C (alelo A) foram associados, respectivamente, com maior risco de epigastralgia (OR=2,15; IC95%=1,01-4,56; p=0,047) e náuseas e/ou vômitos (OR=2,38; IC95%=1,05-5,38; p=0,038). O risco de apresentar dislipidemia foi maior nos portadores dos genótipos FKBP2 c.-21110GG (OR=1,92; IC95%=1,01-3,69; p=0,049) e FOXP3 c.-22-902GG (OR=2,06; IC95%=1,08-3,92; p=0,028). Em conclusão, os polimorfismos de genes MTOR, FKBP1A, FKBP2 e FOXP3 influenciam na função renal do enxerto e estão associados com risco de rejeição aguda e de eventos adversos, em receptores de transplante renal. / The monitoring of immunosuppressive drugs, such as calcineurin and mTOR inhibitors, is essential to avoid undesirable kidney transplant outcomes. Polymorphisms in pharmacokinetics-related genes have been associated with variability in the response to immunosuppressive drugs, but the role of polymorphisms in pharmacodynamics-related genes is little known. The aim of this work was to investigate the influence of polymorphisms in MTOR, PPP3CA, FKBP1A, FKBP2 and FOXP3, genes involved in the pharmacodynamics of immunosuppressive drugs, on the clinical response to tacrolimus and everolimus in kidney transplant recipients. Two-hundred seventy-five kidney transplant recipients were included in this study, among the enrolled in the original clinical trial (NCT01354301) carried out at the Hospital do Rim e Hipertensão/UNIFESP, and randomized in three immunosuppressive treatments: tacrolimus 0.05 mg/kg/day with everolimus 1.5 mg/day (TAC5/EVR); tacrolimus 0.1 mg/kg/day with everolimus 1.5 mg/day (TAC10/EVR); and tacrolimus 0.1 mg/kg/day with sodium mycophenolate (TAC10/MFS). Clinical and laboratory data, including immunosuppressive drug monitoring, efficacy and safety outcomes, were recorded. Polymorphisms on the MTOR (c.4731G>A, c.1437T>C, c.2997C>T); PPP3CA (c.249G>A); FKBP1A (n.259+243936T>C); FBKP2 (c.-2110G>T) and FOXP3 (c.-23+2882A>C, c.-22-902A>G) genes were analyzed by real-time PCR. Allelic frequencies of the studied polymorphisms were similar to those of the global population reported by the 1000genomes project. Treatment with everolimus and high-dose tacrolimus (TAC10/EVR) was associated with lower estimated glomerular filtration rate (eGFR) and higher serum creatinine. Meanwhile treatment with tacrolimus and sodium mycophenolate (TAC10/MFS) was associated with higher number of cytomegalovirus infections, at 1-year post-transplantation. With regard to the kidney efficacy outcomes, the carriers of the CC genotype of MTOR c.1437T>C and FOXP3 c.-23+2882A>C had higher serum creatinine, at month 12 (p<0.05). The FOXP3 c.-23+2882A>C polymorphism was associated with high likelihood of increased serum creatinine (OR=1.75, 95%IC=1.07-2.86, p=0.025). The results of the logistic regression analysis showed that the allele MTOR c.4731G (AG+GG genotypes) was associated with higher risk of acute rejection (OR=3.37, 95%IC=1.10-10.30, p=0.033). The carriers of the c.4731G allele showed higher cumulative incidence of acute rejection episodes at 1-year post-transplantation. With regard to kidney safety outcomes, the FKBP2 c.-2110G>T variant (GG genotype) was associated with higher risk of leucopenia (OR=7.10, 95%IC=1.81-27.87, p=0.025). The FKBP1A n.259+24936T>C (C allele) polymorphism was associated with higher risk of constipation (OR=2.52, 95%IC=1.13-5.61, p=0.024), whilst FOXP3 c.-22 902A>G (A allele) and c.-23+2882A>C (A allele) were associated, respectively, with higher risk of epigastric pain (OR=2.15, 95%IC=1.01-4.56, p=0.047) and nausea and/or vomiting (OR=2.38, 95%IC=1.05-5.38, p=0.038). The risk of developing dyslipidemia was higher in carriers of the genotypes FKBP2 c.-21110GG (OR=1.92, 95%CI=1.01-3.69, p=0.049) and FOXP3 c.-22-902GG (OR=2.06, 95%CI=1.08-3.92, p=0.028). In conclusion, the polymorphisms in the MTOR, FKBP1A, FKBP2 and FOXP3 genes influence renal graft function and are associated with risk of acute rejection and adverse events in renal transplant recipients.

With recent research being conducted in categorizing and analyzing the human microbiome, evidence has now linked the human microbiome to a range of diseases. Dysbiosis of the human gut microbiome exists in colon cancer, obesity, and Clostridium difficile infections. The use of fecal microbiota transplants has been proven effective in treating recurrent C.difficile infections by restoring gut microbiota. More needs to be done to establish fecal microbiota transplants procedures, effectiveness, and safety. Once established, fecal microbiota transplants may play a role in modulating other diseases linked to human gut microbiome dysbiosis.

abstract: The need of organs for transplantation has become an increasing medical need due to a limited donor organ supply. Many organs fail within 10 years due to acute and chronic rejection. Acute or antibody mediated rejection leads to decreased long term graft survival and increases the need for a repeat transplant. In prior work, reducing endothelial heparan sulfation and blockade of chemokine-glycosaminoglycan (GAG) interaction with Myxomavirus-derived protein, M-T7, reduced aortic and renal graft vascular inflammation and rejection. Conditional endothelial Ndst1 deficiency and inhibition of chemokine-GAG interaction reduces early allograft damage and suggest new therapeutic options for graft rejection. Here acute renal rejection was examined in grafts with conditional endothelial N-deacetylase-N-sulfotransferase-1 knockout (Ndst1-/-) and in wildtype (WT) C57Bl6/J grafts treated with saline, M-T7, antisense oligonucleotides (ASO) for Ndst1 or a scrambled ASO control. Viruses have a highly adaptive ability to evade hosts defense and immune response. The immunomodulatory proteins derived from viruses provide potential therapeutic uses to alleviate this need for organs. The Myxoma virus derived protein M-T7 is a promising therapeutic for reducing kidney transplant rejection. Orthotopic transplantations in mice are extremely difficult and costly because they require a highly trained microsurgeon. This kidney to kidney subcapsular and subcutaneous transplant model is a practical and simpler method that requires fewer mice, one kidney can be used for transplants in 6 or more mice and there is much lower morbidity, pain and mortality. Heterotopic transplantation of allografts is a simple model for preliminary testing of treatments for early inflammation, ischemia, and graft rejection. Subcapsular kidney transplantation provides a first step approach to test virus-derived proteins as potential treatments to reduce transplant rejection and inflammation. This project reports on a broadly applicable platform on which to rapidly and conveniently test new treatments for transplant rejection. This finding will significantly lower the barrier to entry for labs which are interested in translating their laboratory findings to animal models of organ transplantation which is a complex surgical procedure, and thus accelerate the bench-to-bedside translation of novel, putative treatments for transplant rejection as an initial screening tool. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2020

La préservation statique à froid (SCS) est l’étalon-or de la préservation des organes après une greffe. En raison de la pénurie d’organes et de l’augmentation du nombre de patients figurant sur la liste d’attente, le recours aux organes provenant des donneurs à critères élargis, lesquels sont très sensibles au syndrome d’ischémie-reperfusion (IRS), ce qui entraîne une non-fonction primaire (PNF) ou un dysfonctionnement précoce (EAD), est de plus en plus fréquent.Cette recherche avait pour but d’étudier et d’identifier de nouvelles stratégies pour améliorer la qualité de la préservation des organes - d'atténuer les séquelles de l'IRS en utilisant la machine de perfusion hépatique à différentes températures et à différentes périodes d'utilisation après le prélèvement de l'organe ou en ajoutant une hémoglobine extracellulaire en tant que transporteur d'oxygène pendant le SCS.Deux modèles différents ex-vivo ont été analysés : L’un chez le petit animal avec des foies de rats normaux et stéatosiques, pour la perfusion hypothermique (HMP) et SCS avec le transporteur d'oxygène et au niveau préclinique, des foies humains stéatosiques récusés, pour la perfusion normothermique (NMP).Les résultats ont confirmé de manière significative l'intérêt de l’HMP dans la phase pré-ischémique du SCS et celui de l'utilisation de l'hémoglobine extracellulaire en améliorant la fonction hépatique, le maintien de l'anatomie des hépatocytes et en réduisant des marqueurs du stress oxydatif, de l'apoptose et de l'inflammation. Egalement, l'utilisation de NMP a permis d'analyser les foies sévèrement stéatosiques pouvant être récupérés pour une transplantation dans un avenir très proche.Cette recherche met en évidence de nouvelles approches en matière de préservation d'organes susceptibles d'augmenter le pool d'organes et d'améliorer les résultats en transplantation hépatique.Mots-clés : greffe de foie, stockage froid dans le froid, perfusion dans une machine à foie, lésion de reperfusion par ischémie, transporteur d'oxygène. / Static cold storage (SCS) is the gold standard of organ preservation after being procured for transplantation. Due to the organ shortage and the increase of number of patients in the waiting list have pushed the use organs from extended criteria donors which are very susceptible to the ischemia reperfusion syndrome (IRS) leading to primary non-function (PNF) or to early allograft dysfunction (EAD).This research was aimed to study and identify new strategies to improve the quality of organ preservation -liver, to attenuate the IRS sequels by using the liver perfusion machine (LPM) at different temperatures and times of usage after the organ procurement or by adding an extracellular hemoglobin as an oxygen carrier during SCS.Two different ex-vivo models were analyzed: small animal -normal and steatotic rat livers, for hypothermic perfusion (HMP) and SCS with the oxygen carrier and preclinical -steatotic discarded human livers, for normothermic perfusion (NMP).The results significantly confirmed the benefit of the HMP in the preischemic phase of SCS and that of the use of the extracellular hemoglobin by improving the liver function, maintenance of the hepatocytes anatomy and by a reduction of the oxidative stress, apoptosis and inflammation markers. Also, the use of NMP permitted to analyze the severely steatotic livers that can be rescued for transplantation in the very near future.This investigation unveils new approaches in organ preservation that could increase the pool of organs and improve the results in liver transplantation. Key words: liver transplantation, static cold storage, liver machine perfusion, ischemia reperfusion injury, oxygen carrier.

Orientador: Elcio Marcantonio Junior / Resumo: O objetivo desse estudo foi de avaliar retrospectivamente a taxa de sobrevivência de implantes com diferentes tipos de conexão protética instalados na região posterior de maxila, em osso nativo ou em seios previamente enxertados com diferentes biomateriais. Foram avaliados 310 implantes que foram instalados em 103 pacientes sendo que 87 implantes foram instalados associados a seios maxilares enxertados (56 cone morse - CM e 31 hexágonos externos-HE) em 37 pacientes e 223 implantes foram instalados em áreas de osso nativo (112 CM e 111 HE) em 76 pacientes. Foram realizadas análises clínicas peri-implantares: 1)Sangramento à sondagem; 2)Profundidade de sondagem; 3)Nível da margem periimplantar; 4)Presença de mobilidade ou supuração e análise do nível ósseo radiográfico. Dois implantes foram perdidos, um CM em osso nativo e um HE em seio enxertado, o que forneceu uma taxa de sobrevivência de 99.35 %. Foi verificado que os implantes do tipo CM apresentaram menores profundidades de sondagem e maior nível ósseo periimplantar em comparação aos implantes HE tanto em área enxertada como em área de osso nativo (p<0.05). Não houve diferenças estatisticamente significativas entre os implantes instalados em áreas de osso nativo e enxertado em nenhum parâmetro avaliado. Implantes HE instalados em áreas de osso nativo apresentaram maiores taxas de peri-implantite. Pode-se concluir que implantes de plataforma do tipo CM reduzem a perda óssea periimplantar, porém a instalação de implantes em ... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Orientador: Maysa Furlan / Banca: Angela Regina Araujo / Banca: Hosana Maria Debonsi / Resumo: As espécies das famílias Celastraceae mostram o acúmulo de substâncias de interesse biológico, com destaque para a friedelina, com potentes efeitos anti-inflamatórios, analgésicos e gastroprotetores, e os triterpenos quinonametídeos, promissores agentes antitumorais. Entretanto, para que sejam utilizados como fármacos, novas metodologias e/ou técnicas para obtenção dos mesmos em larga escala se fazem necessárias. Nesse contexto, um sistema heterólogo composto pela levedura Saccharomyces cerevisiae, foi modificado geneticamente com a inserção de plasmídeos (vetores) contendo genes oriundos de Maytenus ilicifolia que codificam a enzima friedelina sintase, a qual leva a produção de friedelina. A introdução dos genes heterólogos pode perturbar as vias metabólicas do sistema heterólogo e, além do metabólito alvo, outras classes e/ou substâncias de interesse biológico e/ou comercial podem ser produzidas. Para tanto, as leveduras foram cultivadas em meio sintético completo (0,67% de base para levedura nitrogenada sem adição de aminoácidos e 2% de glicose), suplementado com aminoácidos, bases e ácido p-aminobenzóico, sem adição de uracila. Para os estudos de variabilidade metabólica, análises por cromatografia gasosa acoplada a espectrometria de massas foram realizadas utilizando os extratos etanólicos, clorofórmicos e hexânicos obtidos do sistema heterólogo com e sem modificação genética. Com o auxílio de técnicas metabolômicas foi possível a identificação de uma variedade de substâ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species of the Celastraceae family shows the accumulation some compounds of biological interest, with emphasis on friedelin, with potent anti-inflammatory, analgesic and gastroprotective effects, as well as quinonemethide triterpenes, promising antitumor agents. However, in order to be used as drugs, new methodologies and / or techniques for obtaining them in large scale are necessary. In this way, a heterologous system of Saccharomyces cerevisiaeyeast was genetically modified with the insertion of plasmids (vectors) containing genes from Maytenus ilicifoliathat encode the enzyme friedelin synthase, looking for the production of friedelin by metabolic engineering. The introduction of the heterologous genes may disrupt the metabolic pathways of the heterologous system and, in addition to the target metabolite, other classes and / or compounds of biological and / or commercial interest may be produced. For this, yeasts were grown in complete synthetic medium (0.67% yeastnitrogen basewithout addition of amino acids and 2% glucose), supplemented with amino acids, bases and p-aminobenzoic acid, without addition of uracil. For metabolic variability studies, gas chromatographic analysis coupled with mass spectrometry were performed using ethanolic, chloroform and hexane extracts, obtained from the heterologous system with and without genetic modification. With the aid of metabolomic techniques it was possible to identify avariety of substances including various fatty acids and derivatives from 1to 5, as well as amides, such as (Z)-9-ocatadecenamide (7), terpenoid precursors such as squalene (8) and 2,3-oxidosesqualene (10), the triterpenes; friedelin (21) and lupeol (15), and alsosteroids ergosterol (14) and lanosterol (19). Due to perturbations in metabolic pathway of mevalonate, different substances were also observed, both in yeasts with the empty vector, plasmid considered as control... / Mestre

A blood transfusion may have important immunomodulatory effects and may carry certain risks which could be detrimental to the kidney transplant patient. The aim of this project is to examine the potential risks associated with post-transplant blood transfusions in kidney transplant recipients. We carried out a retrospective cohort study of all adult kidney transplant recipients at The Ottawa Hospital from 2002 to 2018 inclusive. We examined the risks for kidney transplant rejection, graft loss, death, infections and venous thromboembolic events (VTE) associated with the receipt of red blood cell transfusions (RBCTs) administered after kidney transplant. We calculated hazard ratios (HR) using Cox proportional hazards model with RBCT as a cumulative, time-varying exposure. Out of a total study population of 1,258 kidney transplants recipients, 37% received at least one RBCT. The receipt of a RBCT was not significantly associated with the risk for rejection, however it was associated with an increased risk for graft loss, death, infection and VTE. Important biases such as reverse causation and unmeasured confounding may account for some of these findings. That being said, our findings suggest clinicians should be judicious in their use of RBCT in kidney transplant patients.

Introduction: Penile transplantation, in its infancy, has the potential to reestablish functional outcomes for men with penile loss and disfigurement. However, significant bioethical considerations are pertinent, and systematic discussions are necessary to safely progress implementation. Aim: To determine the attitude of health practitioners toward the penile transplant and identify the key aspects of concern pertinent to the operation and clinical care. Methods: Health care professionals from the United States responded to either email invitation, web link, or social media post on Facebook to complete a questionnaire investigating perceptions and attitudes toward penile transplantation. Main Outcome Measures: Respondents' attitude toward penile transplantation, their own perceived important functions of the penis, and concerns about performing a penile transplantation. Respondents' previous exposure to visceral transplants, to penile disfigurement, and information about penile transplants were used as independent factors in analysis. Results: Among 412 health care professionals who responded to the questionnaire, 95.9% were in favor of visceral organ transplant, but only 64.3% were in favor of penile transplantation. The results showed that 61.3% of respondents first learned about the penile transplant from mass media, whereas only 37.5% had been exposed through a scientific journal, formal lecture, or a professional colleague. Younger health professionals and those exposed through professional forums surrounding penile transplantation were more likely to be in favor of the procedure (P <.001). The most important functions of the penis were identified by respondents as being sexual function (role in sexual activity) and gender identity (being a man) with rates of 86.4% and 85.3%, respectively (P <.001). Barriers identified by respondents included the use of immunosuppression and the potential subsequent effect on healthcare resource utilization. Reading an excerpt about penile trauma in war during the questionnaire improved acceptance of penile transplantation (P =.05). Conclusion: Penile transplantation is accepted by most health professionals surveyed. Younger respondents and those informed through professional outlets are more favorable toward penile transplantation. Anticipated limitations include the risk of immunosuppression, lack of available donors, and the effect on healthcare utilization. Najari B, Flannigan R, Hobgood J, et al. Attitudes Toward Penile Transplantation Among Urologists and Health Professionals. Sex Med 2018;6:316–323.

Hypertension guidelines recommend out of office blood pressure (BP) measurement especially 24- hour ambulatory measurement (ABPM), to diagnose and manage hypertension but this is not routinely performed in kidney transplant units. This study was to determine if 24-hour ABPM, compared with office BP in kidney transplant recipients, would be more informative regarding BP management, and if pulse wave analysis (PWA) would assist in risk stratification. This study included patients older than 18 years, with working graft kidney for >12 months, and without problems affecting BP measurement and interpretation. After performing office BP measurements, a 24-hour ABPM with additional capability of calculating pulse wave velocity (PWV),augmentation index and central BP was undertaken. Patients were assessed for controlled hypertension, uncontrolled hypertension, masked hypertension, nocturnal hypertension, white coat hypertension, and dipping BP status. Data were analysed using standard statistical tests. Of 30 patients, 15 were Black Africans and 15 were of Mixed Ancestry with a mean age of 48.9 years. Seventeen patients were males and 36.7% had controlled hypertension, 30% uncontrolled hypertension, 6.7% white coat hypertension and 33.3% masked hypertension, of whom 70% had isolated nocturnal hypertension. 70% had a non-dipping, 26.7% a reverse dipping and only 3.3% had a normal dipping BP pattern. The mean difference between brachia! systolic BP and central systolic BP was 10.4 mm Hg, whereas PWV and augmentation index were similar to healthy populations. CONCLUSION: In kidney transplant recipients, 24-hour ABPM was superior to office BP in defining hypertensive status that qualified for modification of therapy but PWA did not contribute to risk assessment.