Le cancer de la prostate est le deuxième plus fréquent cancer chez les hommes. La France occupe le troisième rang du taux d’incidence. La curiethérapie bas-débit dose (LDR) est une option de traitement largement utilisée. Au cours de la curiethérapie LDR, des graines radioactives sont implantées en permanence dans la prostate afin de délivrer une dose thérapeutique de façon locale dans la zone cancéreuse tout en épargnant les organes voisins à risque (OAR). Malgré son taux de réussite élevé (75% à 91%), les effets secondaires restent élevés. La dose délivrée à la tumeur dépend des positions d’implantation des graines, ce qui implique que la planification est essentielle. Les systèmes cliniques de planification fournissent automatiquement les positions d’implantation. Cependant, cette prédiction est basée sur un modèle dosimétrique simplifié où le corps humain est considéré comme un volume d’eau. Un autre facteur des erreurs est l’apparition d’un oedème de la prostate impliquant un changement volumétrique. L’oedème peut entraîner une sous-estimation du D90 par exemple il est de 13.6% pour un changement volumétrique de 20%. De plus, l’oedème varie (10% à 96%) entre les patients. Aujourd’hui le mécanisme exact de l’apparition de cet oedème reste inconnu. Nous proposons un système de traitement inverse pour la curiethérapie prostate qui tient compte d’une personnalisation précise de la dosimétrie et de l’oedème. Ces travaux peuvent également être utilisés dans d’autres contextes cliniques, tel que la curiethérapie haut-débit, mais également être adapté pour traiter d’autres organes. Dans le futur, nos travaux porteront sur la personnalisation du modèle biomécanique de la prostate proposé à chaque patient en utilisant des mesures d’élasticité via l’élastographie. En raison des limitations inhérentes à la FEM, l’incorporation du modèle biomécanique de l’oedème dans le système de planification du traitement est coûteuse en temps de calcul. Une méthode alternative, serait de proposer un modèle sans maillage afin d’améliorer la simulation de l’oedème. / Prostate cancer is the second most common cancer in men. Two-thirds of the cases are diagnosed in developed countries and France is ranked third in incidence rate. Low-dose-rate (LDR) brachytherapy is a widely used treatment option. During LDR brachytherapy, radioactive seeds are implanted permanently in the prostate to deliver a therapeutic dose locally in the cancerous region while sparing the organs at risk (OARs). Despite its high success rate (75% to 91%), the side-effects (sexual and urinary problems) remain high. The dose delivered to the tumor depends on the implantation positions of the seeds, which implies that treatment planning is essential. Clinical inverse planning systems automatically provide optimal implantation positions. However, this prediction is based on a simplified dosimetric model where the human body is considered an infinite volume of water. Another important factor that induces treatment errors is the occurrence of prostate edema during brachytherapy that involves volumetric changes of the organ. Edema can lead to a significant underestimation of the D90, for example, by 13.6% for a volumetric change of 20%. Moreover, the edema magnitude varies considerably (10% to 96%) between patients. Today the exact mechanism of edema formation remains unknown. We propose in this thesis a system of inverse treatment for prostate brachytherapy which considers a precise personalization of the dosimetry but also of the prostate edema. This work can also be used in other clinical contexts, such as high-dose-rate brachytherapy, but also be adapted to treat other organs. In the future, our work will focus on the study of the ability to adapt the proposed prostate biomechanical model to each patient using elastic measurements via prostate elastography. Due to the inherent limitations of FEM, the incorporation of the biomechanical model of edema into the treatment planning system is costly in computation time. An alternative method would be to propose a new meshless model to improve the simulation of edema during intraoperative planning.

Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T18:28:56Z (GMT). No. of bitstreams: 1 Vega_SalomonHuancahuire_M.pdf: 1822310 bytes, checksum: 4712d36f0c1a5963a20bb4649d8aa879 (MD5) Previous issue date: 2009 / Resumo: As enzimas PLA2 provenientes de veneno de serpentes são intensamente estudadas devido a que os envenenamentos constituem um dos principais problemas de saúde em muitos paises. Por outro lado, estas toxinas ajudam a revelar aspectos desconhecidos da fisiologia celular e tisular. Neste trabalho, apresentamos a purificação e a caracterização bioquímica e farmacológica de duas miotoxinas fosfolipases A2: BbTX-II e BbTX-III, a partir de veneno de Bothrops brazili. As duas proteínas foram isoladas e purificadas usando um procedimento simples e rápido envolvendo duas etapas cromatográficas, exclusão molecular em Sephadex G-75 e HPLC de fase reversa (C18). A eletroforese de ambas miotoxinas mostrou massas relativas em torno de 13 e 27 kDa (para monômeros e dímeros respectivamente). Espectrometria de massa por MALDI-TOf confirmou a pureza das proteínas e mostrou que possuem massas moleculares em torno de 13,8 kDa. A análise de aminoácidos mostrou alto conteúdo de aminoácidos básicos e hidrofóbicos, assim como 14 resíduos de Cys. BbTX-III apresentou atividade PLA2 na presença de um substrato cromogênico, mostrando comportamento sigmoidal, principalmente à baixas concentrações. Atividade máxima foi alcançada em pH 8 e entre 35-45 oC. BbTX-III mostrou-se completamente dependente de Ca2+ e, na presença dos íons Mg2+, Mn2+, Cd2+ e Zn2+, a atividade enzimática foi reduzida a níveis similares aos observados na ausência de Ca2+. A análise de composição de aminoácidos mostrou alta presença de Lys, His e Arg (pI 8,46). A presença de 14 resíduos de cisteína sugere a formação de 7 pontes dissulfeto. O estudo de homologia da seqüência da PLA2 BbTX-III mostrou que existem posições extremamente conservadas nas PLA2. S(1), L(2), E(4) 7 a 10 (QMIL), Y(21). Os resíduos conservados Y(28), G(30), G(32), D(49), H(48) e Y(52) estão direta ou indiretamente ligados com a catálise. Além disso, BbTX-III apresentou algumas mutações: K(35) -> G(35), R(51) -> Y(51) e D(118) -> A(118) que estão estrategicamente posicionadas para a expressão da atividade catalítica. Apesar destas substituições, as atividades farmacológicas e a atividade catalítica são mantidas. O efeito neurotóxico de BbTX-III foi analisado in vitro na preparação neuromuscular biventer cervicis de pintainhos. O resultado mostrou que a toxina é menos potente quando comparada com venenos crotálicos. BbTX-III demonstrou efeito miotóxico local in vivo através da liberação de creatina quinase (CK) e, efeito inflamatório, através de edema de pata. Como a BbTX-III produziu forte efeito inflamatório, a hidrólise de fosfolipídios poderia ser relevante neste fenômeno. BbTX-II foi caracterizada como uma PLA2 K49 (cataliticamente inativa) em função das características físico-químicas evidenciadas: massa de 13,68 kDa, 121 resíduos de aminoácidos, caráter básico (pI 8.73) e alto grau de homologia seqüencial na sua estrutura primária, quando comparada com outras PLA2B K49 procedentes de veneno de serpentes botrópicas. O alinhamento com outras seqüências completas de PLA2 BK49 mostrou a presença de algumas mutações importantes. Assim, as substituições Y?N(27), N?P(58) e L?F(114) não modificaram os efeitos biológicos aqui estudados, revelando que poderiam estar relacionados com outras atividades. Os resíduos N(28), K(111), L(32) poderiam contribuir com a interrupção da catálise. Esta nova PLA2 K49 BbTX-II mostrou miotoxicidade local in vivo, atividade inflamatória e letalidade, corroborando que se enquadra dentro da família de proteínas PLAB2B K49. Os estudos de neurotoxicidade revelaram um efeito neurotóxico in vitro na preparação biventer cervicis de pintainho (20 µg/ml). BbTX-II e BbTX-III mostraram ser miotoxinas com atividade edematogênica e neurotóxica, independentemente de apresentarem atividade catalítica (BbTX-III) ou não (BbTX-II) Apoiando a existência de regiões moleculares distintas à catalítica responsáveis pelos efeitos farmacológicos. Os efeitos farmacológicos da toxina BbTX-III (PLA2B D49) provavelmente tenham uma estrita relação entre a atividade enzimática e a ligação da toxina com micro-domínios na membrana plasmática onde sua atividade seja maximizada e cause danos relevantes na organização da membrana. No caso da BbTX-II (PLAB2 K49) possivelmente a combinação de aminoácidos aromáticos/hidrofóbicos e positivamente carregados da região C-terminal seja a responsável de alterar a integridade da membrana plasmática. / Abstract: The enzymes PLA2 coming of venom snake are studied intensely due to that the poisonings constitute one of the main problems of health in many countries. On the other hand, these toxins help to reveal unknown aspects of the cellular and tissue physiology. In this work, we presented the purification and biochemical and pharmacological characterization of two myotoxic phospholipases A2: BbTX-II and BbTX-III from Bothrops brazili venom. The two proteins were isolated and purified using a simple and fast procedure involving two chromatographic steps, molecular exclusion in Sephadex G-75 and reverse-phase HPLC (C-18 column). Both myotoxins showed around 13 and 27 kDa (for monomers and dimers, respectively) relative mass. MALDI-TOf mass spectrometry confirmed the purity of the proteins showing molecular masses around 13,8 kDa. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 cysteine residues. BbTX-III presented PLA2 activity in the presence of a chromogenic substrate, showing sigmoidal behavior, mainly at low concentrations. Maximum PLA2 activity was reached at pH 8 and 35-45 oC. Maximum activity required Ca2+ and, in the presence of Mg2+, Mn2+, Cd2+ and Zn2+, was reduced at similar levels as observed in the absence of Ca2+. Amino acids analysis of composition showed high presence of Lys, His and Arg (pI 8,46). The presence of 14 cystein residues suggested the formation of 7 disulfide bridges. Sequence homology of the PLA2 BbTX-III revealed positions extremely conserved in PLA2 S(1), L(2), E(4) 7 to 10 (QMIL), Y(21). The conserved residues Y(28), G(30), G(32), D(49), H(48) and Y(52) are direct or indirectly linked to the catalysis. Besides, BbTX-III presented some mutations: K(35) -> G(35), R(51) -> Y(51) and D(118) -> A(118) that are strategically positioned for the expression of catalytic activity. Despite these substitutions, the pharmacological and catalytic activities are maintained. The neurotoxic effect of BbTX-III was analyzed in vitro at chick biventer cervicis muscle preparation. Our results showed that the blockage of the muscle contraction was lower when compared with crotalic venoms. BbTX-III demonstrated in vivo myotoxic local effect through the liberation of creatine kinase (CK) and inflammatory effect through paw edema. As BbTX-III produced strong inflammatory effect, the phospholipids hydrolysis could be relevant in this phenomenon. BbTX-II was characterized as PLA2 homologous K49 (catalytically inactive), because its chemical and physical evidenced characteristics: mass of 13,68 kDa, 121 amino acids residues, basic character (pI 8.73) and high sequential homology in its primary structure, when compared with other PLA2 K49 from venom of Botrhops serpents. The alignment with other complete sequences of PLA2 homologous K49 showed the presence of some important mutations. Substitutions Y->N(27), N->P(58), and L->F(114) did not modify the biological activities here studied, revealing that it could be related to other activities. The residues N(28), K(111), L(32) could contribute with the interruption of the catalysis. This new PLA2 K49 BbTX-II showed in vivo myotoxic local effect, inflammatory and lethality activities, evidencing it was fitted to the family of proteins PLA2 K49 homologous. Beside BbTX-II revealed in vitro neurotoxyc effect at chick biventer cervicis muscle preparation (20 µg/mL). BbTX-II and BbTX-III showed to be myotoxins to activity edematogenic and neurotoxyc independently of present catalytic activity (BbTX-III) or no (BbTX-II) Supporting the existence of molecular areas different to the catalytic responsible for the pharmacological effects. The pharmacological effects of the toxin BbTX-III (PLA2 D49) they probably have a strict relationship between the enzymatic activity and binding of the toxin with micro-domains in the plasmatic membrane where the activity is maximized and cause relevant damages in the organization of the membrane. In the case of the BbTX-II (PLA2 K49) possibly the combination of amino acids aromatics/hidrophobics and positively loaded of the area C-terminal it is the responsible of altering the integrity of the plasmatic membrane. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Marcos Hikari Toyama / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T11:46:22Z (GMT). No. of bitstreams: 1 Oliveira_SimoneCristinaBuzzo_M.pdf: 2132823 bytes, checksum: b98ccca4e06a6ca0155f84135393b9b5 (MD5) Previous issue date: 2007 / Resumo: Lectinas são proteínas que se ligam de forma específica e reversível a carboidratos. Estão distribuídas pelos mais diversos organismos e apresentam atividades de interesse em pesquisas biológicas e médicas. Neste trabalho, duas isoformas de lectina da alga marinha vermelha Bryothamnion triquetrum foram purificadas por cromatografia de troca iônica seguida por uma de fase reversa. As frações foram nomeadas de BTLD1 e BTLD2 e ambas apresentaram massa molecular aparente em SDSPAGE de aproximadamente 9,0 kDa. A análise dos aminoácidos de ambas as lectinas revelaram moléculas de caráter básico e as regiões Nterminal das lectinas foram seqüenciadas e comparadas entre si e com lectinas de outras algas, apresentando grande similaridade com hypninA1, hypninA2 e BTL. Além disso, o dicroísmo circular revelou que a estrutura secundária das proteínas é predominantemente de arranjo aleatório. A lectina BTLD2, a isoforma mais abundante do extrato da alga, apresentou ação antibacteriana contra a bactéria grampositiva Clavibacter michiganensis subsp. michiganensis (Cmm), mas não foi eficiente contra a bactéria gramnegativa Xanthomonas axonopodis pv. passiflorae (Xap). Também foram utilizados dois modelos de fosfolipases A2, uma isolada do veneno total de Crotalus durissus cascavella que é cataliticamente ativa do tipo Asp 49 (D49), e uma outra PLA2 cataliticamente não ativa do tipo Lys49 (K49), isolada do veneno de Bothrops jararacussu, para ensaios biológicos. A atividade farmacológica e biológica de ambas as PLA2s foi significativamente afetada pela coincubação das mesmas com BTLD2. A formação de heterodímeros de BTLD2 com a PLA2 de Crotalus durissus cascavella ou com a PLA2 de Bothrops jararacussu sugere a formação de um complexo estável em solução com uma massa molecular de aproximadamente 23,0 kDa. A BTLD2 também foi capaz de aumentar significativamente a atividade enzimática da PLA2 de C. d. ascavella em comparação com a PLA2 cataliticamente ativa isolada. As PLA2s de C. d. cascavella e B. jararacussu mostraram forte atividade antibacteriana contra bactérias Cmm e tiveram pouco efeito contra a bactéria Xap. Entretanto, o complexo BTLD2: PLA2 D49 ou K49 mostram um aumento da atividade antibacteriana, principalmente contra a bactéria Xap. As PLA2s induziram atividade edematogênica em pata de ratos e também foram capazes de induzir uma forte agregação plaquetária usando o sistema de plaquetas lavadas. Em ambos os sistemas, o complexo BTLD2: PLA2 mostrou uma atividade menor do que os respectivos controles. Concluindo, os resultados apresentados mostram que esta nova lectina isolada de alga vermelha possui uma evidente capacidade de interação com moléculas de PLA2, tanto cataliticamente ativas quanto inativas, com a formação de heterodímeros. Portanto, esta interação é capaz de modificar significativamente a estrutura terciária da PLA2, uma vez que o complexo mostrou atividade enzimática aumentada além de possivelmente alterar a estrutura de outras regiões moleculares da enzima. É importante verificar que a atividade farmacológica das PLA2s não tem uma correlação direta com a atividade catalítica das mesmas, envolvendo outras regiões que permitem a sua interação com receptores cuja região está distante do sítio catalítico da PLA2 / Abstract: Lectins are proteins that bind specifically and reversibly to carbohydrates. They are widely distributed among living organisms and show many activities of biological and medical interest. In this work, two isoforms of lectins isolated from the red marine alga Bryothamnion triquetrum were purified by ion exchange followed by reverse phase chromatographies. The fractions named BTLD1 and BTLD2 showed apparent molecular mass of approximately 9.0 kDa in SDSPAGE. Both lectins probably have a basic character, as indicated the amino acid analyses using the Compute pI/Mw tool at the ExPASy server. The Nterminal sequences were compared between both lectins and also to other lectins, showing similarity with hypninA1, hypninA2 and BTL. The circular dichroism indicated that the secondary structure of the proteins are predominantly random coiled. Additionaly, BTLD2 (the more abundant lectin isoform in the alga extract) showed antibacterial activity against the grampositive bacteria Clavibacter michiganensis subsp. michiganensis (Cmm) but was not effective against the gramnegative bacteria Xanthomonas axonopodis pv. passiflorae (Xap). A possible modulation of phospholipase activity by BTLD2 was also studied using two phospholipases A2, one isolated from Crotalus durissus cascavella venom that is Asp49 (D49) catalytically active, and other PLA2 Lys49 (K49) catalytically non active from Bothrops jararacussu venom. The pharmacological and biological activities of PLA2s were significantly change by incubation with BTLD2. The heterodimer formation of BTLD2 with Crotalus durissus cascavella or Bothrops jararacussu PLA2s appears to be a stable complex in solution with a molecular mass of approximately 23 kDa. BTLD2 significantly increased the enzymatic activity of the PLA2 from C.d. cascavella compared to the PLA2 alone. Both PLA2s (catalytically active and nonactive) showed strong antibacterial activity against Cmm with little effect against Xap. However, the BTLD2: PLA2 D49 or K49 complexes showed increase in antibacterial activity, particularly against Xap. Moreover, both PLA2s induced edematogenic activity in rat paw and strong platelet aggregation in washed platelets system. Interestingly, addition of BTLD2 with consequent formation of the BTLD2: PLA2 complex decreased both PLA2induced edema and platelet aggregation. Taken toghether, these results show that this new lectin isolated from a red alga and named BTLD2 has the capacity to interact with catalytic or noncatalytic PLA2, forming heterodimers. Therefore, this interaction possibly modify the PLA2 tertiary structure; as the complex BTLD2: PLA2 increase the enzymatic activity of PLA2 (i.e., the phospholipase activity) but at the same time decrease pharmacological and biological PLA2 activities not related to catalysis (i.e., platelet aggregation and edema). This suggests that the PLA2 structure is possibly modified in other sites of the enzyme rather than in the catalytic site. It can be concluded that the PLA2s has several binding sites for different molecules comprising the catalytic site responsible for the phospholipase activity and at least one more pharmacological site responsible for the effects observed after interaction with BTLD2. Therefore, the pharmacological activity of PLA2s has no direct correlation with the catalytic activity, involving other binding sites that allow receptor interaction whose position might be far from the catalytic site of PLA2 / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: João Luiz Pinto e Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T12:21:28Z (GMT). No. of bitstreams: 1 Spaggiari_CristinaWenderholm_M.pdf: 3837300 bytes, checksum: f5468a274aeb11e7eb6c2e781445d81d (MD5) Previous issue date: 2008 / Resumo: O objetivo deste trabalho foi avaliar a efetividade e a segurança da Drenagem Linfática Manual no tratamento de mulheres com edema de membros inferiores no terceiro trimestre de gravidez. Foi realizado um estudo não controlado tipo antes e depois com 20 gestantes inscritas nos programas de Assistência Pré-natal do Centro de Atenção Integral à Saúde da Mulher da Unicamp. Todas apresentavam diagnóstico clínico de edema nos membros inferiores a partir de 28 semanas de gravidez. Um Termo de Consentimento Livre e Esclarecido foi assinado por cada gestante antes do agendamento das sessões de drenagem aplicadas pela fisioterapeuta pesquisadora. Medidas de cirtometria, peso e pressão arterial foram aferidas antes a após a massagem. Todas responderam a um questionário qualitativo antes e após cada sessão, que teve duração aproximada de 40 minutos. As consultas, coincidiram com os retornos médicos pré-natais. O procedimento da drenagem obedeceu a técnicas padronizadas escolhidas pela pesquisadora. Os dados foram armazenados em um banco de dados do programa Excel. A análise estatística utilizou o teste t de Student para as variáveis contínuas de distribuição normal e de Wilcoxon pareado para os dados de distribuição não-normal. Todos os testes foram avaliados à nível de significância de 5%. O resultado da análise das medidas cirtométricas de todos os locais das extremidades inferiores, antes e após a drenagem, mostrou redução significativa das circunferências das pernas. Igualmente, observou-se que a pressão arterial materna não se alterou significativamente após cada sessão. A comparação dos sintomas associados ao edema como dor, formigamento, sensação de peso e inchaço, analisados através da escala visual analógica, com a percepção das mulheres após a DLM mostrou melhora estatisticamente significativa. O grau de satisfação das mulheres na resolução do edema e sintomas associados medidos até a última sessão foi significativo. A conclusão do estudo foi que a Drenagem Linfática Manual proporcionou redução do edema e sintomas associados nos membros inferiores de mulheres a partir da 28º semanas de gravidez, sem alterar significativamente a pressão arterial, proporcionando alto grau de satisfação às pacientes / Abstract: The objective of the study was to evaluate the effectiveness and safety of the lymphatic manual drainage (LMD) in the treatment of women with edema of legs in the third quarter of pregnancy. It was conducted a non controled study before/after with 20 pregnant women registered in the program of Prenatal Care at the Center of Integral Attention to the Woman's Health (CAISM) of Unicamp. All participants had at least 28 weeks of gestation and clinical diagnosis of edema of legs. After signed an informed consent form the women initiate a drainage sessions of 40 minutes each by a physiotherapist who conducted the study. The extremities¿ circumference measures, weight and arterial blood pressure were taken before and after massage. In the aggregate it was applied a qualitative questionnaire before and after each session. The sessions were scheduled at the same days of prenatal consultations. The procedure of the drainage followed the standard technique chosen by the researcher. The statistical analysis included the Student t test for the variables with normal distribution and Wilcoxon test for paired samples for the abnormal distribution data. The significance was established at 5%. The results showed that LMD provoked a significant reduction of the extremities circumferences. In addition, there were not changes at women blood pressure after the sessions. The visual analogue scale (VAS) of the edema associated symptoms such as pain, tingling, weight sensation, and swelling showed significant improvement after the LMD. There was a significant degree of the women's satisfaction in the resolution of the edema and associated symptoms, measured at the last session. In conclusion, the LMD causes an effective reduction of the edema and the associated symptoms on women's inferior members from the 28th week of pregnancy without modification of the women arterial blood pressure and with high degree of patients' satisfaction / Mestrado / Ciencias Biomedicas / Mestre em Tocoginecologia

OBJETIVO: Determinar a efetividade de manobras padronizadas de drenagem linfática manual (DLM) na redução do edema facial, na distância interincisal máxima ativa e no quadro álgico de pacientes submetidos à cirurgia de Enxerto Ósseo Alveolar (EOA). METODOLOGIA: Esta pesquisa analisou 51 indivíduos com fissura labiopalatina reparada, entre 10 e 15 anos de idade, submetidos ao EOA no Hospital de Reabilitação de Anomalias Craniofaciais-USP, divididos em 2 grupos, grupo tratamento (n=29, 12 homens e 17 mulheres) e grupo rotina (n= 22, 15 homens e 7 mulheres). No grupo tratamento foram aplicadas manobras de DLM padronizadas diferentemente daqueles do grupo rotina. Foram realizadas nos dois grupos avaliações do edema facial por meio das medidas da linha 1 (distância da base da asa nasal ao tragus) e linha 2 (distância da base da asa nasal à implantação inferior do pavilhão auricular), na hemiface operada nos períodos pré-operatório (Pré), segundo (2ºPO) e quarto pós-operatório (4ºPO). Também foram realizadas as medidas da distância interincisal máxima ativa nos mesmos períodos. A Escala Analógica da Dor (EAD) foi aplicada no 1º, 2º, 3º e 4º períodos pós-operatórios. Adicionalmente, no grupo tratamento, após a terapia de DLM, foram realizadas perguntas sobre dor, relaxamento e sono. RESULTADOS: Houve redução do edema facial no grupo tratamento do 2ºPO para o 4ºPO o que não ocorreu no grupo rotina. As medidas da distância interincisal apresentaram aumento entre o 2ºPO e 4ºPO nos dois grupos estudados. Na avaliação da EAD, o grupo tratamento mostrou ausência de dor no 3ºPO enquanto o grupo rotina somente no 4ºPO. No grupo tratamento, a totalidade dos indivíduos relataram que a dor diminuiu, e que se sentiram mais relaxados após a DLM, nos 3 períodos avaliados. Relataram também em muitos casos que a DLM teve efeito facilitador na indução ao sono durante a DLM. CONCLUSÕES: As manobras padronizadas de DLM aplicadas da maneira proposta produziram redução significante do edema facial, aumento da distância interincisal máxima ativa, redução do quadro álgico no grupo tratamento comparativamente ao grupo rotina. Esses dados reforçam a hipótese de que a DLM proposta foi efetiva contribuindo de maneira importante na recuperação do paciente submetido à cirurgia de EOA. / OBJECTIVE: To determine the effectiveness of standardized manual lymphatic drainage (MLD) maneuvers in reducing facial swelling, maximum interincisal active distance and pain of patients undergoing surgery for Alveolar Bone Grafting (ABG). METHODS: This study examined 51 individuals with repaired cleft lip and palate, aged 10 to 15 years old, who underwent ABG at the Hospital for Rehabilitation of Craniofacial Anomalies- USP, divided into two groups: treatment group (n = 29, 12 men and 17 women) and routine group (n = 22, 15 men and 7 women). In the treatment group MLD standardized maneuvers were performed differently from those of the group routine. In both groups evaluation of facial swelling was performed by measuring the distance from the ala nasi to the tragus (line 1) and the distance from the ala nasi to the inferior portion of the ear auricle (line 2), in the operated side, preoperatively, and at the second and the fourth postoperative (PO) day. The maximum active interincisal distance was measured during the same periods. The Pain Analog Scale (PAS) was applied at the first, second, third and fourth days after ABG. Additionally, patients from the treatment group, were asked about pain, relaxation and sleep after MLD therapy. RESULTS: There was a reduction of facial edema in the treatment group from the 2nd to 4th PO what was not observed the routine group. Interincisal distance increased between the 2nd and 4th PO in both groups. PAS evaluation has shown that patients from the treatment group present no pain already at 3rd PO. In the routine group this occurred only at 4th PO. All the subjects from treatment group reported decrease of pain, and patients reported felt more relaxed after the MLD, at the three postoperative evaluated. Many patients reported that MLD had a facilitative effect on inducing sleep during MLD. CONCLUSIONS: The standardized MLD maneuvers applied in the proposed manner produced significant reduction of facial swelling, increase of the maximum active interincisal distance, reduction of the pain in the treatment group as compared to the routine group.These Abstract data reinforce the hypothesis that the proposed MLD is a effective procedure for the recovery of the patients undergoing ABG surgery.

L’œdème maculaire est une complication majeure de nombreuses pathologies rétiniennes induisant la cécité. Les traitements actuels ne sont pas curatifs. Ce sont des injections intraoculaires de corticostéroïdes et d'anti-VEGF. Les mécanismes d'action de ces médicaments sont mal connus dans l'œil car ces thérapies ont été initialement développées pour des pathologies non-oculaires (tels que l'asthme et les maladies auto-immunes pour les corticostéroïdes et les cancers pour certains anti-VEGF). De plus, les effets bénéfiques de ces molécules sont contrebalancés par des effets secondaires graves (glaucomes et cataractes pour les corticostéroïdes) et la résistance au traitement. Le projet vise à élucider les mécanismes d'action oculaires des corticostéroïdes et anti-VEGF; dans le but d'identifier de nouvelles cibles thérapeutiques pour le traitement de l’œdème maculaire. Ce travail a porté sur : 1) La compréhension des mécanismes des corticostéroïdes et de leurs récepteurs (le récepteur aux glucocorticoïdes ou GR et le récepteur aux minéralocorticoïdes ou MR) suite à l’injection intravitréene de corticostéroïdes ou d’aldostérone chez le rat. Nous avons ainsi : -1.1. Identifier les protéines partenaires du GR et du MR avant et après traitements par une approche protéomique (immunoprécipitations endogènes suivies d’une analyse par spectrométrie de masse). -1.2. Identification les gènes cibles dont l'expression est modulée par les corticostéroïdes ou l’aldostérone par ARN-sequencing. 2) L'identification des mécanismes d’action des aux anti-VEGFs et leur comparaison avec des molécules de même nature physico-chimique. 3) L’identification des gènes différentiellement exprimés entre la rétine périphérique et la macula (zone d’acuité visuelle susceptible à la formation d’œdèmes) chez le singe (modèle qui possède, comme l’homme, une macula). Ceci afin de comprendre pourquoi la macula est particulièrement sensible. 4) L’analyse et la comparaison de ces différents traitements. Ces études ont permis d’identifier les cofacteurs des récepteurs aux corticostéroïdes, ainsi que la dynamique de leurs interactions avant et après traitement à la corticostérone et à l’aldostérone; ainsi que les gènes dont l’expression est régulée par les corticostéroïdes et les anti-VEGF. L’analyse croisée des données, des analyses bioinformatiques ainsi que l’étude bibliographique des protéines et gènes identifiés nous ont permis d’établir une liste restreinte de cibles potentielles des traitements anti-œdémateux, dans le but final de potentialiser l’effet de ces traitements, trop souvent associés à des effets secondaires qui limitent considérablement leur(s) action(s) bénéfique(s) sur la fonction visuelle dans le traitement de l’œdème maculaire. / Macular Edema is a major complication of numerous retinal pathologies that lead the blindness. The available treatments (intra-ocular injection of corticosteroids and anti-VEGF) do not cure this disease. The ocular mechanisms of action of these drugs are not very well understood. In fact, these molecules have been initially developed for non-ocular pathologies, such as asthma, auto-immune diseases (for corticosteroid) and cancers (for anti-VEGF). In addition, the beneficial effects of these drugs are counteracted by their side effects (cataracts and glaucoma for corticosteroids) as well as resistance to treatment. The project aims at unraveling the ocular mechanisms of action of corticosteroids and anti-VEGF; in order to identify new therapeutic targets for the treatment of macular edema. The project focused on: 1) The understanding of the mechanisms of the corticosteroids and their receptors after an intravitreal injection of Corticosterone or Aldosterone in the rat model. We therefore: 1.1. Identified the partner proteins of the glucocorticoid and mineralocorticoid receptors before and after treatment, using a proteomic approach. 1.2. Identified the target genes which expression is modulated by the corticosteroid using the RNA-Sequencing technique. 2) The identification of the mechanisms of action of anti-VEGF molecules and their comparison with molecules that have similar physico-chemical structure. 3) The identification of differentially expressed genes between the retina and the macula in the monkey model. This aimed at understanding why the macula is particularly sensitive to edemas. 4) The analysis and comparison of these treatments. We were able to identify co-factors for corticosteroids receptors as well as the dynamics of these interactions (before and after treatment), as well as the genes which expression is regulated by the corticosteroids or anti-VEGF. The analysis of the bio-informatic data as well as the bibliographic study of the identified proteins and genes allowed us to establish a list of partiMacularly interesting genes that are potentially therapeutic targets for the treatment of macular edema.

Cette thèse a pour objet l’étude de nouvelles méthodes de traitement d’image appliquées à l’analyse d’images numériques du fond d'œil de patients diabétiques. En particulier, nous nous sommes concentrés sur le développement algorithmique supportant un système de dépistage automatique de la rétinopathie diabétique. Les techniques présentées dans ce document peuvent être classées en trois catégories: (1) l’évaluation et l’amélioration de la qualité d’image, (2) la segmentation des lésions, et (3) le diagnostic. Pour la première catégorie, nous présentons un algorithme rapide permettant l’estimation numérique de la qualité d’une seule image à partir de caractéristiques extraites de la vascularisation et de la couleur du fond d'œil. De plus, nous démontrons qu’il est possible d’augmenter la qualité des images et de supprimer les artefacts de réflexion en fusionnant les informations extraites de plusieurs images d’un même fond d'œil (images capturées en changeant le point d’attention regardé par le patient). Pour la deuxième catégorie, deux familles de lésion sont ciblées: les exsudats et les microanévrysmes. Deux nouveaux algorithmes pour l’analyse des images du fond d'œil sont proposés et comparés avec les techniques existantes afin de démontrer leur efficacité. Dans le cas des microanévrismes, une nouvelle méthode basée sur la transformée de Radon a été développée. Dans la dernière catégorie, nous présentons un algorithme permettant de diagnostiquer la rétinopathie diabétique et les œdèmes maculaires en analysant les lésions détectées par segmentation d’image; à partir d’une seule image, notre algorithme permet de diagnostiquer une rétinopathie diabétique et/ou un œdème maculaire en ~ 22 secondes sur une machine à 1,6 GHz avec 4 Go de RAM; de plus, nous montrons les premiers résultats de notre algorithme de détection d'œdème maculaire basé sur des images du fond d'œil multiples, qui peut éventuellement permettre d’identifier le gonflement de la macula même si aucune lésion n’est visible. / In this Ph.D. thesis, we study new methods to analyse digital fundus images of diabetic patients. In particular, we concentrate on the development of the algorithmic components of an automatic screening system for diabetic retinopathy. The techniques developed can be categorized in: quality assessment and improvement, lesion segmentation and diagnosis. For the first category, we present a fast algorithm to numerically estimate the quality of a single image by employing vasculature and colour-based features; additionally, we show how it is possible to increase the image quality and remove reflection artefacts by merging information gathered in multiple fundus images (which are captured by changing the stare point of the patient). For the second category, two families of lesion are targeted: exudate and microaneurysms; two new algorithms which work on single fundus images are proposed and compared with existing techniques in order to prove their efficacy; in the microaneurysms case, a new Radon transform-based operator was developed. In the last diagnosis category, we have developed an algorithm that diagnoses diabetic retinopathy and diabetic macular edema based on the lesions segmented; starting from a single unseen image, our algorithm can generate a diabetic retinopathy and ma cular edema diagnosis in _22 seconds on a 1.6 GHz machine with 4 GB of RAM; additionally, we show the first results of a macular edema detection algorithm based on multiple fundus images, which can potentially identify the swelling of the macula even when no lesions are visible.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-25T12:32:10Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Angelo-tese doutorado.pdf: 2038180 bytes, checksum: be401d99f221bbdf4a8d892feb579538 (MD5) / Made available in DSpace on 2017-07-25T12:32:10Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Angelo-tese doutorado.pdf: 2038180 bytes, checksum: be401d99f221bbdf4a8d892feb579538 (MD5) Previous issue date: 2016-11-24 / Introdução: A incidência de hidrocefalia pós craniectomia descompressiva(CD) em pacientes com traumatismo cranioencefálico(TCE) é entre 0-45% segundo a literatura. A hidrocefalia traz prejuízos ao prognóstico neurológico e demanda reconhecimento clínico precoce. Existem diversas variáveis radiológicas e clínicas descritas com associação ao risco de hidrocefalia. Para estudar a influência desses fatores conduzimos um estudo retrospectivo, observacional em um centro terciário de atendimento a pacientes com TCE com foco principal na análise do volume de herniação transcraniana (VHTC) após CD. Métodos: selecionamos 50 pacientes que realizaram CD para TCE entre janeiro de 2014 e janeiro de 2015. Hidrocefalia foi reconhecida e definida na presença de critérios radiológicos de Gudeman, indicação de derivação ventricular, e na mensuração do Índice de Evans modificado maior que 33%. Analisamos as seguintes variáveis: Idade, Sexo, Escala de Coma de Glasgow à admissão, reatividade pupilar, índice de Zunkeller, presença de higroma, VHCE, diâmetro da craniectomia e distância da craniectomia em relação à linha média. Regressão logística foi utilizada definindo o desfecho com ou sem hidrocefalia como medida de análise. Resultados: 17 pacientes desenvolveram hidrocefalia (34%). VHCE após CD (p<0.001), Higroma subdural (p<0.001) ), Escala de coma de Glasgow abaixo de 6( p=0.015), sinais de herniação uncal(p=0.042) e maior valor no índice de Zumkeller(p=0.04) foram associados com o desenvolvimento de hidrocefalia pós-CD. Regressão logística demonstrou que entre essas variáveis as que foram consideradas como fatores de risco independente são o VHTC (OR 11.08; 95%IC 2.10,58.4; p=0.004) e a presença de higroma (OR 49.59; 95%IC 4.1,459;p=0.002). Conclusões: Observamos uma forte associação entre a severidade do TCE, o volume de herniação cerebral transcraniana e presença de higroma subdural com o desenvolvimento de hidrocefalia. Pacientes com esses achados devem ser acompanhados rigorosamente visando evitar prejuízo clínico. / In patients undergoing decompressive craniectomy(DC) for traumatic brain injury(TBI) there has been reported an incidence of hydrocephalus between 0-45%. Hydrocephalus affects long term survival and needs a prompt and correct diagnosis. There are several radiological and clinical features described in association with development of hydrocephalus. For study the influence of these factors we conducted a retrospective observational single-center cohort study in a tertiary care center with special attention to the transcalvarial brain herniation volume(TCH) after DC. Methods: We selected 50 patients that underwent DC after closed head injury between january 2014 and January 2015. Hydrocephalus was defined as a modified frontal horn index greater than 33%, Gudeman CT scan criteria or insertion of ventriculoperitoneal Shunt. Variables we analyzed were: age, post-resuscitation Glasgow Coma Scale (GCS) score, pupil reactivity, Zunkeller index, presence of hygroma, TCH volume, craniectomy diameter and distance of craniectomy from midline. Logistic regression was used with hydrocephalus as the primary outcome measure. Results: 17 patients developed hydrocephalus(34%). TCH volume after decompression ( p<0.001), subdural hygroma ( p ), lower admission Glasgow Coma Scale score ( p=0.015), unilateral pupil reactivity(p=0.042) and higher Zumkeller index(p=0.044) were significant risk factors for hydrocephalus after decompressive craniectomy. Logistic regression analysis showed that factors independently associated with the development of hydrocephalus was the TCH volume (odds ratio 11.08; 95%CI 2.10, 58.4; p = 0.0046), and presence of hygroma (odds ratio 49.59; 95%IC 4.1, 459; p=0.002). Conclusions: There is a clear association between severity of TBI, TCH volume and subdural hygroma with the development of hydrocephalus. Clinicians should follow closely patients with those findings in order to avoid late deterioration.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22119号 / 医博第4532号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森 孝一, 教授 Shohab YOUSSEFIAN, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22320号 / 医博第4561号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 大森 孝一, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM