The insulin/IGF-1 signaling pathway plays a pivotal role in life span regulation in diverse organisms. In Caenorhabditis elegans, a PI 3-kinase signaling cascade downstream of DAF-2, an ortholog of the mammalian insulin and insulin-like growth factor-1 (IGF-1) receptor, negatively regulates DAF-16/forkhead transcription factor. DAF-16 then regulates a wide variety of genes involved in longevity, stress response, metabolism and development. DAF-16 also receives signals from other pathways regulating life span and development. However, the precise mechanism by which DAF-16 directs multiple functions is poorly understood. First, in Chapter II, we demonstrate that JNK is a novel positive regulator of DAF-16 in both life span regulation and stress resistance. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate life span and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into thc nucleus. Our findings define a novel interaction between the stress response pathway (JNK) and the master regulator of life span (DAF-16), and provide a mechanism by which JNK regulates longevity and stress resistance. Next, in Chapter III, we focus on the downstream targets of DAF-16. Here, we used a modified chromatin immunoprecipitation (ChIP) method to identify direct target promoters of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and randomly selected 33 targets for further analysis. The expression of majority of these genes is regulated in a DAF-16-dependent manner. Moreover, inactivation of more than 50% of these genes significantly altered DAF-16-dependent functions such as longevity, fat storage and dauer diapause. Our results show that the ChIP-based cloning strategy leads to greater enrichment of DAF-16 target genes, compared to previous studies using DNA micro array or bioinformatics. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream target genes. In summary, we identified the JNK signaling pathway as a novel input into DAF-16 to adapt animals to the environmental stresses. We also revealed a large number of novel outputs of DAF-16. Taken together, these studies provide insight into the complex regulation by DAF-16 to control diverse biological functions and eventually broaden our understanding of aging.

Dysfunction of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to the cognitive deficits of many neurodegenerative diseases and psychiatric disorders. Cognitive symptoms of Alzheimer's disease can be treated with NMDA receptor antagonists or drugs targeting the cholinergic system; however, there are no effective treatments for cognitive deficits of schizophrenia or Huntington's disease. With the discovery of a potent and selective allosteric modulator of the NMDA receptor, there is the possibility of new treatments based on NMDA receptor functional-enhancement through neuroactive steroids, closely related in structure to the endogenous neurosteroid, cerebrosterol. The aim of this thesis was to examine steroidal modulation of the NMDA receptor both in vitro and in vivo. In chapter 2, NMDA receptor enhancement of both the synthetic and endogenous neuroactive steroids was assessed in neurons maintained in cell culture using calcium imaging techniques. Sulphation of the steroids greatly increased the efficacy of NMDA receptor enhancement compared to the unsulphated steroids. Chapters 3 and 4 investigate the potential for neuroactive steroids to treat cognitive impairments of Huntington's disease. Using a mouse model, tests were selected that were analogous to those in which patients are impaired; however, no impairments were found in the mouse model. Chapter 5, therefore, used a different model of cognitive impairment – namely, rats with a set-shifting impairment, as is seen in many psychiatric and neurological disorders, including Huntington's disease – to assess the effect of the synthetic steroid administration. Unfortunately, the rats did not show the expected impairment. The lack of reliable animal models compromised testing the efficacy of these promising NMDA receptor positive allosteric modulators. Nevertheless, the promising in vitro results suggest that there could still be therapeutic potential. In addition, the compound is a useful research tool for exploring NMDA receptor function in health and disease.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation, and they are: (i) discovery of piperidine derivatives as T-type calcium channel inhibitors for the treatment of epilepsy and neuropathic pain and as protein disulfide isomerase inhibitors for the treatment of influenza viral infection; (ii) discovery of peptide-based calcitonin gene-related peptide receptor antagonists for the treatment of inflammatory pain; and (iii) synthesis of substituted 6-(dimethylamino)-2-phenylisoindolin-1-ones for the inhibition of luciferase. T-type calcium channels are important regulators of nervous system, and upregulated T-type calcium channel activities have been found to link to various types of neurological disorders, such as epilepsy and neuropathic pain. To discover novel T-type calcium channel blockers, a series of 1,4-disubstituted piperidine derivatives were designed and synthesized. Among them, compound 1-4 was found to be a good T-type calcium channel inhibitor with an IC₅₀ of 1 nM for Ca[subscript v]3.2 inhibition. It also showed 86% suppression of seizure induced death in mice and good in vivo analgesic effects on both thermal and mechanical pain thresholds in Spared Nerve Injury rat models. Therefore 1-4 can potentially be used as a T-type calcium channel blocker in the treatment of epilepsy and neuropathic pain. Influenza is a respiratory viral infection. Since viruses rely on host cell proteins for their entry, survival and replication, development of drugs targeting host cell proteins has identified as an effective strategy in controlling viral infections. We synthesized a series of 1,4-disubstituted piperidine derivatives for the inhibition of protein disulfide isomerase enzyme and influenza. Among them, 1-29 was found to possess strong anti-influenza activity (EC₅₀ = 2.5 µM). This suggests the potential use of piperidine scaffold in designing anti-influenza drugs in future. Calcitonin gene-related peptide (CGRP) receptor antagonism has been identified as a successful approach for the treatment of inflammatory pain. Therefore, a novel class of peptide antagonists of CGRP receptor was synthesized and screened for their binding affinities to the CGRP receptor and their analgesic effects on inflammatory-induced pain in rats. Among them, peptide 2-3 showed a higher binding affinity towards the CGRP receptor than previously reported peptide antagonists and exhibited analgesic effects up to 2 h in both Aδ and c-fiber pain tests. Therefore 2-3 indicates its potential use as a CGRP receptor antagonist in the treatment of inflammatory pain. Firefly luciferase is commonly used as a reporter in cells expressing a luciferase gene or its enzymatic activity under the control of a promoter of interest to assess its transcriptional activity. It has been found that some molecules such as molecules with carboxylic acid moiety can directly inhibit luciferase activity in cells. However, it is suggested that carboxylic acid moiety of the compounds may also be associated with side reactions in cells. Therefore, to study whether carboxylic acid moiety causes side effects, we designed two probe molecules, 3-1 and 3-2. Synthesis of probe molecule 3-2 is discussed. Synthesis of probe molecule 3-1 and further investigation of its luciferase inhibition will therefore be useful to understand the toxicity of carboxylic acid containing drugs in future.

Made available in DSpace on 2015-03-26T13:42:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 852162 bytes, checksum: 18350a1fa68989163ff6494b7784f27a (MD5) Previous issue date: 2012-07-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Fragile X syndrome is the most common inherited cause of intellectual disability with an estimated prevalence of 1 in 3600 males and 1 in 4000 to 6000 females, where it causes moderate to severe intellectual and social impairment together with syndromic features including large ears and head, long face and macroorchidism. It is most often is caused by expansion of a CGG trinucleotide repeat array in the 5' UT region of the FMR-1 gene at Xq27.3. This locus corresponds to a fragile site which can be observed in the metaphase chromosome following selective culture conditions. In this study we analyze two brothers with clinical indicative of fragile X syndrome. Peripheral blood lymphocytes were studied after 96 hrs culture periods at 37°C in RPMI 1640 (supplemented with 5% fetal serum bovine, 20 mM HEPES, 2mM glutamine and 1% M-PHA), LymphoGrow® and PB-MAX®. Two bottles for each medium was prepared for cultures with antagonists, and in each one 0,25 μg/mL fluorodeoxyuridine (FUdR) or 8 μg/mL methotrexate (Mtx) were added 72 hrs after the start of culture. Cultures were harvested after 95 hrs and 45 minutes using standard methods following exposure to 0,1 μg/mL colcemid for 15 minutes. Slides were stained with Giemsa 5% for fra(X) screening and G banding was employed for confirmation of the Xq27.3 fragile site. Lymphocytes growing in RPMI 1640, LymphoGrow and PB-MAX responded equivalently to the addition of antagonists. According to the F test at the level of 5% probability, there were no significant differences between the average number of metaphases in the three mediums in combination with FUdR. Similarly, in the three mediums methotrexate treatment was very toxic and resulted in insufficient number of analyzable cells. No fra(X) were detected in the cultures with this treatment. A total of 208 metaphases for brother 1 and 205 for brother 2 were scanned of which 21% and 20%, respectively, were positive for fra(X). These results of fragile X expression in brothers are in agreement with the estimate expression in affected males ranging from 10-40% and corroborate those of other studies which reported that men of the same family tend to have similar frequencies of fra(X). The procedures for induction of fragile site and cytogenetic analysis showed to be adequate in order to identify the presence of FRAXA in the analyzed brothers with clinical suspicion of fragile X syndrome. / A Síndrome do X frágil é a principal causa de retardo mental hereditário com prevalência estimada de 1 em cada 3600 homens e 1 em cada 4000 a 6000 mulheres. Essa síndrome provoca comprometimento intelectual e social moderado a severo, associado com características como cabeça e orelhas grandes, rosto alongado e macroorquidismo. A causa mais frequente dessa desordem é a expansão do trinucleotídeo CGG localizado na região 5' UT do gene FMR-1, em Xq27.3. Esse loco corresponde a um sítio frágil que pode ser visualizado em cromossomos metafásicos sob condições seletivas de cultura. Neste estudo analisamos dois irmãos com indicativo clínico da Síndrome do X frágil. Linfócitos oriundos de sangue periférico foram avaliados após 96 h de cultura a 37°C em meio RPMI 1640 (suplementado com 5% de FBS, 20 mM de HEPES, 2 mM de glutamina e 1% M-PHA), LymphoGrow® e PB-MAX®. Para as culturas com antagonista, dois tubos de cada meio disponível foram preparados, e em cada um, 0,25 μg/mL de fluorodeoxiuridina (FUdR) ou 8 μg/mL de metotrexato (Mtx) foram adicionados 72 h após o início da cultura. As culturas foram colhidas após 95 h e 45 min utilizando-se métodos de rotina, após exposição a 0,1 μg/mL de colcemide durante 15 min. As lâminas foram coradas com Giemsa 5% para rastreamento de cromossomos fra(X), e a técnica de bandeamento G foi empregada para a confirmação do sítio frágil na região Xq27.3. Os linfócitos cultivados nos meios RPMI 1640, LymphoGrow e PB-MAX responderam de forma equivalente à adição dos antagonistas. Não houve diferença significativa pelo teste F a 5% de probabilidade entre o número médio de metáfases observado nos três meios em combinação com FUdR. Do mesmo modo, nos três meios o tratamento com metotrexato foi muito tóxico e resultou em número insuficiente de células analisáveis. Nessas culturas com Mtx não foram detectados cromossomos fra(X). Um total de 208 metáfases para irmão 1 e 205 para o irmão 2 foram analisadas, das quais 21% e 20%, respectivamente, foram positivas para X frágil. Esses dados estão de acordo com a estimativa de expressão em homens afetados que varia de 10-40% e corroboram com os de outros estudos que reportaram que homens da mesma família tendem a apresentar frequências similares de fra(X). Os procedimentos para a indução de sítio frágil e análise citogenética mostraram-se adequados para identificação de FRAXA nos irmãos analisados com indicativo clínico da Síndrome do X frágil.

A doença de Chagas, descrita há quase 100 anos por Carlos Chagas, continua sendo um problema de saúde pública na América Latina, com 15 milhões de infectados e 28 milhões de indivíduos susceptíveis. Vários mecanismos fisiopatológicos foram propostos para a progressão da miocardiopatia chagásica crônica, entre eles a denervação simpática. O uso do betabloqueador na insuficiência cardíaca congestiva diminuiu morbidade e mortalidade. Entretanto, nenhum trabalho conclusivo incluiu a miocardiopatia chagásica como etiologia. OBJETIVO: Avaliar o papel do carvedilol na sobrevida e remodelamento miocárdico na miocardiopatia chagásica experimental. MATERIAL E MÉTODOS: Foram avaliados 55 Hamsters Sirius, divididos em grupo controle, grupo infectado com Trypanosoma cruzi com 105 formas tripomastigotas via intraperitoneal e grupo infectado, tratado inicialmente com carvedilol 10 mg/Kg/dia e após 6 meses com 15 mg/Kg/dia em dose única por gavagem. Foi analisado o peso do animal; os diâmetros ventriculares, função sistólica e diastólica obtidas pelo ecocardiograma; a freqüência cardíaca, a duração do QRS, a presença de extra-sístoles e o ritmo cardíaco usando o eletrocardiograma e a avaliação da mortalidade. Após 12 meses os animais sobreviventes foram sacrificados e realizada a quantificação da fração do volume de colágeno intersticial e perivascular no miocárdio. RESULTADO: A razão dos diâmetros diastólico e sistólico pelo tamanho da tíbia não mostrou diferenças estatísticas, apesar de maiores nos grupos infectados. A fração de encurtamento também não mostrou diferença estatística, apesar de menor nos grupos infectados. A fração do volume de colágeno do ventrículo esquerdo e perivascular mostrou maior acúmulo nos grupos infectado e carvedilol em relação ao controle de forma significativa (p<0,001), mas sem diferença nos grupos infectados tratados ou não. A mortalidade foi maior nos grupos infectado e carvedilol (p = 0,001) e não ocorreu diferença entre estes grupos após 12 meses. Na fase aguda (até 100 dias) o grupo carvedilol apresentou melhor sobrevida (p = 0,001) em relação ao infectado. CONCLUSÃO: O carvedilol não mostrou atenuação no remodelamento miocárdico e demonstrou benefício na mortalidade na fase aguda da doença nesse modelo de miocardiopatia chagásica crônica experimental. / Chagas\' disease was described 100 years ago by Carlos Chagas and nowadays it is a public health problem in Latin America yet. There are about 15 million infected people and another 28 million at risk to be infected. Several pathophysiologic mechanisms are suggested to be responsible for the progression of chronic Chagas\' cardiomyopathy. Among them the sympathetic denervation seems to be an important issue. The use of beta blockade regarding heart failure has been proving to improve morbidity and mortality. However, none of these papers included Chagas\' cardiomyopathy as etiology. OBJECTIVES: To evaluate the role of carvedilol upon survival and myocardial remodeling in a Chagas\' cardiomyopathy animal model. MATERIAL AND METHODS: 55 Hamsters Sirius were studied and divided into control group, intraperitoneously infected group with Trypanosoma cruzi and infected group treated with carvedilol 10 mg/Kg/day by gavage which was increased to 15 mg/Kg/day after 6 months. Ventricular diameters, systolic and diastolic left ventricular function were evaluated by 2D-echocardiogram. The heart rate, QRS lasting, premature ventricular complex and cardiac rhythm were analyzed by ECG. We evaluated clinical parameters as the case for body weight. The survival curve was also studied. After 12 months the survivors were sacrificed and the myocardial interstitial and perivascular collagen volume fraction were analyzed. RESULTS: The ratio of diastolic or systolic diameters over tibia length did not show statistical differences although it was larger on infected groups. The fractional shortening also did not show statistical differences although it was decreased on infected groups. The left ventricular collagen volume fraction at the interstitial and perivascular area showed a higher accumulation on infected groups compared to control (p<0.001) but no differences were observed between infected and carvedilol treated animals. The mortality was higher in the infected groups compared to control (p = 0.001) but no differences were observed between infected and carvedilol group during the total time of the experiment. However, when we divided in acute phase (until 100 days) the carvedilol significantly attenuated mortality compared to infected group (p = 0.001). CONCLUSION: Carvedilol did not show benefits regarding myocardial remodeling or total mortality and it did demonstrate a mortality reduction on acute phase of the disease in this experimental model of Chagas\' cardiomyopathy.

A hipertensão arterial pulmonar é uma síndrome clínica e hemodinâmica, caracterizada pelo aumento de resistência vascular na microcirculação. A vasoconstrição presente na doença ocorre principalmente devido à disfunção endotelial, induzindo a um estado pró-trombótico onde a participação das plaquetas é inequívoca. A trombina, principal agonista da ativação plaquetária, exerce seus efeitos nas células por meio de receptores ativados por proteases. Através da citometria de fluxo, este trabalho teve por objetivo analisar (1) a expressão do receptor ativado por protease do tipo 1 (PAR-1) na membrana de plaquetas, em seu estado íntegro ou clivado, (2) a atividade, mediante a formação de agregados entre plaquetas e leucócitos e, plaquetas e monócitos após estímulo de receptores ativados por proteases, além (3) da expressão de selectina-P na membrana plaquetária após estimulação. Foram estudados 30 pacientes portadores de hipertensão arterial pulmonar sob tratamento ambulatorial no Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo. A idade destes pacientes variou de 11 a 78 anos, e a média da pressão sistólica da artéria pulmonar foi de 89+29 mmHg. Em 30% dos pacientes, o número de plaquetas esteve abaixo de 150x103/l, e em 40%, o nível do hematócrito apresentou-se acima de 50%. No presente estudo, não foi observada diferença significante na expressão do PAR-1 íntegro em plaquetas de pacientes comparativamente aos controles (p = 0,2). Em contrapartida, as plaquetas dos pacientes apresentaram menor quantidade de receptor clivado (p = 0,01), sugerindo internalização destes receptores. Com relação à avaliação da atividade de PAR-1 através da formação de agregados entre plaquetas e leucócitos e, plaquetas e monócitos após estimulação por agentes capazes de atuar sobre o receptor de trombina, não foram observadas diferenças entre pacientes e controles, ou seja, em ambos os casos houve a formação de agregados (p = 0,2 e p = 0,4, respectivamente). Em relação aos leucócitos, o SFLLRN, estimulou o receptor independentemente do seu estado, íntegro ou clivado, nos pacientes (p < 0,05), enquanto que nos indivíduos normais, a resposta só foi observada quando o receptor íntegro foi estimulado por trombina (p < 0,05). Tanto nos pacientes estudados, como nos controles, a estimulação do PAR-1 plaquetário, promoveu aumento da expressão de selectina-P na superfície plaquetária (p < 0,0001), embora não houvesse diferença entre os grupos (p = 0,9). Estes resultados demonstram que as plaquetas dos pacientes não são refratárias à liberação de seu conteúdo granular, e encontram-se aptas a responder aos estímulos tanto quanto as plaquetas dos controles. Este estudo reforça a importância da terapia antiplaquetária em pacientes com hipertensão arterial pulmonar. / Pulmonary hypertension is a clinical and hemodynamic syndrome, characterized by the increase of vascular resistance in lungs, generally through various mechanisms, involving vasoconstriction and remodeling of the arterial wall. Endothelial dysfunction in pulmonary arterial hypertension leads to a prothrombotic status, in which platelet participation seems to be unequivocal. Thrombin is the most potent platelet activator and exerts its effects on cells, through protease-activated receptors. Flow cytometry procedure was employed to assess (1) platelet protease-activated receptor 1 (PAR-1) expression, in both uncleaved and cleaved forms, (2) PAR-1 activity, through platelet-leukocyte and platelet-monocyte aggregates formation in response to the thrombin receptor stimulus, and finally (3) platelet P-selectin expression after stimulus. Thirty patients with pulmonary arterial hypertension (age 11 to 78 years) under treatment at the Heart Institute, University of São Paulo were enrolled. The systolic pulmonary arterial pressure was 89+29 mmHg. The platelet count was < 150x103/l in 30% of the patients, and the hematocrit level was > 50% in 40% of the patients. In the present study, there was no relevant difference in the level of intact platelet protease-activated receptor 1 expression in patients and controls (p = 0.2). On the other hand, the expression of cleaved receptors was decreased in patients (p = 0.01) platelets, what suggests internalization. There was no difference on platelet-leukocyte, and platelet-monocyte aggregates in response to the thrombin receptor stimulus between patients and controls, in other words, in both cases there was the aggregates formation (p = 0.2). However, aggregates formation in patients appeared to occur predominately in response to an agent (SFLLRN) capable to stimulate the receptor, independent of its state, intact or cleaved (p < 0.05). Otherwise, in healthy individuals, the response occurred especially when the intact receptor was stimulated by thrombin (p < 0.05). In these patients, similarly to the controls, platelet protease-activated receptor 1 stimulation induced membrane P-selectin expression (p < 0.001), although there was no difference between the groups (p = 0.9). These findings suggest that platelets from patients are not refractory to its granular content secretion and are capable to respond to stimulus as the controls platelets. This study re-enforce the importance of anti platelet therapy in pulmonary arterial hypertension patients.

INTRODUÇÃO: A importância do parênquima pulmonar na piora funcional da asma tem sido recentemente investigada. Embora a ativação da enzima óxido nítrico sintase induzida (iNOS) amplifique a responsividade e o remodelamento das vias aéreas induzidos pela inflamação crônica, seu efeito no parênquima pulmonar não foi previamente estudado. OBJETIVO: Avaliar a influência do óxido nítrico derivado da iNOS na mecânica pulmonar, na inflamação e no processo de remodelamento no tecido pulmonar periférico de cobaias com inflamação pulmonar alérgica. MÉTODOS: Os animais foram submetidos a sete inalações com doses crescentes de ovalbumina (1~5 mg/mL) ou soro fisiológico por 4 semanas. As cobaias receberam 1400-W (inibidor específico de iNOS, intraperitoneal) ou veículo por 4 dias, iniciando 30 minutos antes da sétima inalação. Após 72h da sétima inalação, os animais foram anestesiados, exsanguinados e fatias de tecido pulmonar periférico foram retiradas e suspensas em banho orgânico de Krebs, e a resistência e elastância tecidual foram avaliadas em condição basal e após desafio com ovalbumina. Após, as fatias de tecido pulmonar periférico foram submetidas à avaliação histopatológica. RESULTADOS: Os animais expostos às inalações com ovalbumina apresentaram valores maiores de porcentagem de aumento da resistência e da elastância tecidual em relação ao basal após desafio com ovoalbumina no banho (p<0.05). Houve aumento no número de eosinófilos (p<0.001), nas células iNOS positivas (p<0.001), na deposição de fibras elásticas e colágenas (p<0.05), na densidade de actina (p<0.05) e na expressão de 8-epi-PGF2a (p<0.001) no septo alveolar. A administração de 1400-W reduziu todos estes parâmetros funcionais e morfológicos (p<0.05). CONCLUSÕES: Neste modelo experimental, o bloqueio específico da iNOS atenuou a constrição, a inflamação e o remodelamento no parênquima pulmonar. Estas alterações podem estar relacionadas aos efeitos do óxido nítrico na modulação da via do estresse oxidativo. O presente estudo sugere que a inibição específica da iNOS pode amplificar as estratégias terapêuticas utilizadas na abordagem de doenças inflamatórias crônicas pulmonares. / INTRODUCTION: The importance of lung parenchyma in functional asthma impairment has been recently addressed. Although the inducible nitric oxide synthase (iNOS) activation amplifies chronic inflammation-induced airway responsiveness and remodeling, its effect on lung parenchyma has not been previously investigated. OBJECTIVE: To evaluate the influence of iNOSderived NO in the pulmonary mechanics, inflammation, and remodeling processes in peripheral lung tissue of guinea pigs with pulmonary allergic inflammation. METHODS: Animals were submitted to seven ovalbumin exposures with increasing doses (1~5 mg/mL) or saline for 4 weeks. The guinea pigs received 1400-W (iNOS-specific inhibitor, intraperitoneal) or vehicle for 4 days, beginning 30 minutes before the 7th inhalation. At 72h after the 7th inhalation, animals were anesthetized, exsanguinated and peripheral lung tissue strips were retreat and suspended in a Krebs organ bath, and the tissue resistance and elastance were evaluated at baseline condition and after ovalbumin challenge. After that, strips were submitted to histopathological evaluation. RESULTS: The ovalbumin-exposed animals presented greater values of percentage of increase of tissue resistance and elastance related to baseline after ovalbumin challenge in the bath (p<0.05). There were increase in the number of eosinophils (p<0.001) and iNOSpositive cells (p<0.001), in collagen and elastic fiber deposition (p<0.05), in actin density (p<0.05) and in 8-epi-PGF2a expression (p<0.001) in the alveolar septa. The 1400-W administration reduced all these functional and morphological parameters (p<0.05). CONCLUSIONS: In this experimental model, the iNOS-specific blockage attenuated constriction, inflammation, and remodeling in the lung parenchyma. These alterations may be related to NO effects in the modulation of the oxidative stress pathway. The present study suggests that specific iNOS inhibition can amplify the therapeutics strategies used in the management in chronic inflammatory lung diseases.

O ergosterol é um componente responsável por manter a integridade e a fluidez das membranas de Leishmania spp. A partir de uma metodologia que consiste em seleção por superexpressão gênica, foram isolados nove diferentes loci de L. (L.) major relacionados com a resistência a dois inibidores da via de biossíntese do ergosterol: Terbinafina (TBF) e Itraconazol (ITZ). Análises funcionais individuais desses nove loci na presença de TBF e ITZ (ou do análogo Cetoconazol - CTZ) apresentaram níveis significantes de resistência após transfecção em células selvagens de L. (L.) major. Nesse trabalho apresentamos a metodologia de isolamento de um desses loci (cItz2), bem como a análise in silico das regiões cromossômicas correspondentes aos insertos dos nove cosmídios no genoma de L. (L.) major / Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated nine different loci of L. (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, Terbinafine (TBF) and Itraconazole (ITZ). Individual functional analysis of these nine loci in the presence of TBF and/or ITZ (or the ITZ analog Ketoconazole, CTZ), have showed significant levels of resistance after transfection into L. (L.) major wild-type cells, followed by over expression induction. In this work, we show the insert mapping and chromosomal identification of one of these loci (cItz2), as well as discuss the in silico chromosomal analysis of the nine correspondent inserts in the L. (L.) major genome

Orientador: Cláudia Herrera Tambeli / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T16:27:32Z (GMT). No. of bitstreams: 1 Favaro-Moreira_NadiaCristina_D.pdf: 14440949 bytes, checksum: 19ac7e9f7196c13dfd756bd4150b1456 (MD5) Previous issue date: 2014 / Resumo: Disfunção temporomandibular (DTM) é um termo coletivo que abrange uma série de problemas clínicos que envolvem os músculos mastigatórios e a articulação temporomandibular (ATM) e está comumente associada à inflamação. Apesar de medicamentos anti-inflamatórios não esteróides (AINEs) serem frequentemente utilizados no controle de dor inflamatória, sabe-se que a dor inflamatória possui um componente simpático que pode predominar em casos menos responsivos a tratamentos com AINEs. Portanto, os objetivos deste estudo foram: (i) avaliar se os hormônios gonadais modulam a resposta antinociceptiva ao bloqueio dos ?-adrenoreceptores (ARs) na ATM de ratos, (ii) avaliar se um e três dias de tratamento com o ?-bloqueador não seletivo para AR ?1 e ?2 nadolol reduzem a dor em pacientes com DTM significativamente mais do que o placebo, (iii) avaliar se as mulheres experimentam relativamente maior benefício com o tratamento com nadolol que os homens dependendo do seu estado hormonal, e (iv ) comparar o efeito do nadolol com o efeito do ibuprofeno. O primeiro objetivo foi desenvolvido em ratos machos e fêmeas, intactos ou gonadectomizados (com ou sem reposição hormonal) através da coadministração de formalina e antagonista específico para AR ?1 , ?2 e ?3 na região da ATM. O comportamento nocieptive foi quantificado e utilizado para a análise estatística. Para o segundo objetivo Nadolol (40 mg/dia), ibuprofeno (400 mg/dia) ou placebo foi administrado em 27 mulheres que não usam contraceptivo oral (CO), 28 mulheres usando CO e 29 homens, que estavam de acordo com os Critérios Diagnósticos para Pesquisa (Research Diagnostic Chriteria) para DTM. Eles completaram um estudo cruzado, aleatorizado, duplo-cego e com controle placebo. As mulheres participaram durante três meses (durante a fase menstrual e durante a fase peri-ovulatória em mulheres que não usam CO, e durante a fase menstrual e durante a fase de uso do CO em mulheres usando CO), em um total de 6 etapas de análise ( 2 por mês), e homens participaram durante um mês com três etapas de análise com um período de 6 dias entre cada etapa. Cada etapa consistiu de uma avaliação de basal e duas avaliações durante o tratamento, uma no primeiro e o outra no terceiro dia de tratamento. A dor foi avaliada por meio da utilização da escala visual analógica (VAS) e as comparações foram feitas através do pré-tratamento (basal), o primeiro e o terceiro dia de intervenção (pós-tratamento). O bloqueio dos ARs ? reduz significativamente a nocicepção da ATM em ratos machos e fêmeas, mas as fêmeas são mais sensíveis. Os hormônios gonadais reduziram a resposta ao bloqueio de ARs ? na ATM de machos e fêmeas. No estudo em humanos um e três dias de tratamento com nadolol ou ibuprofeno produz uma analgesia em mulheres e homens significativamente maior que placebo, mas as mulheres são mais sensíveis independente do estado hormonal. Em resumo, estes dados demonstram que os hormonios sexuais podem modular o efeito analgésico de bloqueadores de ARs ? dependendo dos níveis séricos dos hormonios gonadais, do subtipo de ARs ? ativado e da dose de droga administrada. A maior eficácia no tratamento da dor em mulheres é clinicamente relevante uma vez que a DTM é mais prevalente e mais severa em mulheres do que em homens / Abstract: Temporomandibular disorder (TMD) is a collective term embracing a number of clinical problems that involve the masticatory muscles, and the temporomandibular joint (TMJ), commonly associated with inflammation. Despite anti-inflammatory drugs (NSAIDs) are frequently used in the control of inflammatory pain, it is well known that inflammatory pain has a sympathetic component that might predominate in the cases less responsive to NSAIDs treatments. Therefore, the aims of this study were: (i) to evaluate whether gonadal hormones modulate the antinociceptive responsiveness to the blockade of ?-adrenoreceptors (ARs) in the TMJ of rats, (ii) to evaluate whether one and three days of treatment with the nonselective ?1 and ?2-AR antagonist nadolol would reduce clinical pain symptoms in TMD patients significantly more than placebo, (iii) to evaluate whether women would experience relatively greater benefit from nadolol than men depending on their hormonal status, and (iv) compared the effect of nadolol with the effect of ibuprofen. The first aim was developed in male and female rats, intact or gonadectomized (with or without hormone replacement), by coadministration of formalin and specific antagonist for ?1, ?2 and ?3-ARs in the TMJ region. The nocieptive behavior was quantified and used for estatistical analysis. For the second aim Nadolol (40 mg/day), ibuprofen (400 mg/day) or placebo was administrated in 27 women not using oral contraceptive (OC), 28 women using OC, and 29 men which met the Research Diagnostic Criteria for TMD. They completed a randomized, crossover, double¿blind, placebo controlled study. Women participated for three months (during menstrual phase and during peri-ovulatory phase in women not using OC, and during menstrual phase and during OC using phase in women using OC) in a total of 6 stages of analysis (2 per month), and men participated for one month whith three stages of analysis and 6 days of wash out. Each stage consisted of a baseline evaluation and two evaluations during treatment, one on the first and the other on the third day of treatment. Clinical pain ratings were obtained by Visual Analog Scale (VAS) and comparisons were made across the pre-treatment (baseline), the first and the third day of intervention (post-treatment). Blockade of ?-ARs significantly reduce the TMJ nociception in male and female rats, but females are more responsive. The gonadal hormones reduce the responsiveness to the blockade of ?-ARs in the TMJ of males and females rats. In the human study one and three days of treatment with nadolol or ibuprofen produces analgesia in TMD women and men significantly more than placebo, but women are more responsive independent of their hormonal status. In summary, these data demonstrate that gonadal hormones can modulate the analgesic effect of ?-ARs blockers depending on the gonadal hormones serum levels, on the ?-ARs activated subtype and on the dose of drug administered. The greater treatment efficacy in women is of clinical relevance since TMD is more prevalent and severe in women than in men / Doutorado / Fisiologia Oral / Doutora em Odontologia

Orientador: Ricardo de Lima Zollner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T18:58:24Z (GMT). No. of bitstreams: 1 ZanellatoFabbri_Natalia_D.pdf: 35087770 bytes, checksum: d1d79dc3e2bc0a5a2a05b4261a363a74 (MD5) Previous issue date: 2014 / Resumo: Apesar das diversas terapias disponíveis para o tratamento da rinite alérgica (RA), muitos pacientes não obtêm alívio dos sintomas com uso de um único fármaco e apresentam frequentemente queixa da manutenção dos sintomas mesmo sob tratamento. Estudos clínicos aleatorizados compararam a eficácia de anti-histamínicos e corticoides intranasais, isolados e associados e demonstraram que as terapias com drogas combinadas apresentam melhores resultados. A RA é um fator de risco para o desenvolvimento de obstrução de VAI e estudos clínicos com pacientes asmáticos demostraram redução da responsividade brônquica e sintomas de asma apenas com tratamento tópico nasal. O objetivo do presente estudo foi avaliar (1) o efeito do tratamento tópico nasal com azelastina (AZE), budesonida (BUD) e combinação AZE/BUD na obstrução nasal e sintomas de RA; (2) o efeito do estímulo nasal inespecífico com histamina na função pulmonar; e (3) o efeito dos tratamentos tópicos nasais na função pulmonar de pacientes com RA. O desenho do presente trabalho foi aleatorizado, cruzado e cego composto por 3 tratamentos. 28 pacientes participaram do estudo, com tratamento tópico nasal de 30 dias e intervalo de 7 dias entre os tratamentos. Os pacientes foram submetidos ao protocolo de TPN com histamina, avaliado por escore de sintomas, rinometria acústica e espirometria. Nossos resultados mostraram que a terapia com AZE/BUD é mais efetiva na prevenção da obstrução nasal e sintomas da RA comparada ao tratamento com as drogas isoladas. Além disso, encontramos indivíduos com alterações na função pulmonar após estímulo nasal inespecífico e controle destas alterações após tratamento tópico nasal, sugerindo influência positiva do tratamento nasal na função pulmonar de indivíduos com hiperreatividade brônquica / Abstract: Despite the several therapies available for the treatment of allergic rhinitis (AR) many patients do not get relief of symptoms using a single drug and often have the maintenance of symptoms even under treatment. Randomized clinical trials comparing the efficacy of antihistamines and intranasal corticosteroids, isolated and associates, show that the combination drug therapies have better outcomes. The AR is a risk factor for the development of obstruction in lower airways and clinical studies with asthmatic patients demonstrated reduction of bronchial hyperresponsiveness and asthma symptoms after just nasal topical treatment. The aim of this study was to evaluate (1) the effect of treatment with topical nasal azelastine (AZE), budesonide (BUD) and combined drugs (AZE/BUD) in nasal obstruction and symptoms of RA; (2) the effect of non-specific nasal challenge with histamine in lung function; and (3) the effect of topical nasal treatment on lung function in patients with RA. The design of this study was randomized, crossover and blind consisting of 3 periods of treatment with nasal sprays. 28 patients participated in the study, composed for 3 periods of treatment (30 days) and 7-day interval between treatments. Patients underwent protocol nasal provocation test with histamine assessed by symptom scores, acoustic rhinometry and spirometry. Our results showed that therapy with AZE/BUD is more effective in preventing nasal obstruction and symptoms of RA compared to treatment with drugs isolated. Furthermore, we find individuals with changes in pulmonary function after nonspecific nasal stimulation and control of these changes after nasal topical treatment, suggesting a positive influence of nasal treatment on lung function in subjects with bronchial hyperreactivity / Doutorado / Clinica Medica / Doutora em Clínica Médica