Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Subbaratnam Muthukrishnan / In this study, I have used dsRNA-mediated down-regulation of transcripts/proteins of several potential targets in the model beetle, Tribolium castaneum to identify a molecular target of the “chitin inhibitor”, diflubenzuron (DFB). The elytron of the red flour beetle, T. castaneum, was chosen as the model tissue for studying the mode of action of DFB and its molecular target(s). We have standardized the protocol for topical administration of DFB on precisely aged prepupae to achieve the desired level of mortality (90-95%) on day 5 of the pharate adult stage. Exposure of prepupae to DFB at 1000 ppm results in a near complete loss of chitin in the newly forming adult procuticle of the elytron and the body wall. Global analysis of transcripts by RNA Seq was carried out to look for differential expression of several critical genes of cuticle assembly in these insects compared to mock-treated controls. Interestingly, genes directly involved in the biosynthetic pathway of chitin were not among those affected by DFB. However, immunolocalization studies have shown that several proteins of chitin metabolism including chitin synthase A, which is involved in the synthesis of cuticular chitin, are present in near normal amounts but are mislocalized in DFB-treated insects. Assays for chitin synthase using elytral extracts have indicated that the enzyme preparations from DFB-treated insects are catalytically inactive. By using RNA interference and competition studies with fluorescently-tagged glibenclamide (a sulfonylurea compound) or DFB, we have identified a long-sought molecular receptor of DFB as an ABCC class transporter. DFB-treatment and RNAi of a specific ABCC-type transporter gene lead to identical phenotypes including loss of chitin, loss of laminar architecture of the cuticle, and mislocalization of CHS from its normal plasma membrane location to intracellular locations presumably by affecting vesicular transport. Further, using an in vitro chitin synthesizing system consisting of microsomes prepared from elytral tissue, which exhibits all of the hallmarks of cuticular chitin-synthesizing epidermal cells including sensitivity to DFB, further insights into the mechanistic details of how this class of insecticides inhibits chitin synthesis have been obtained.

Polímeros heterocíclicos abrangem uma grande variedade de materiais, desde simples polímeros lineares sintetizados a partir de monômeros do tipo heterocíclicos vinílicos até polímeros altamente funcionalizados e reticulados. Neste trabalho realizou-se a modificação química da poliacrilonitrila com a incorporação de grupos tetrazol em diferentes teores (1%, 2,5%, 5% e 10%). Os copolímeros de acrilonitrila e vinil-tetrazol obtidos foram caracterizados por FTIR e o seu comportamento térmico analisado por DSC e TGA. Os polímeros heterocíclicos foram avaliados como inibidores de corrosão para aço-carbono em meio ácido obtendo-se bons resultados e alcançando, em alguns casos, uma eficiência de inibição média superior a 70% / Heterocyclic polymers enclose a great variety of materials, since simple linear polymers synthesized from monomers of vinyl heterocyclics to polymers highly functionalized and crosslinked. In this work was carried out the chemical modified of polyacrilonitrile with incorporation of tetrazole groups in different quantities (1%, 2,5%, 5% and 10%). The acrilonitrile and vinyl-tetrazole copolymers were characterized by FTIR and its thermal behavior analyzed by DSC and TGA. The heterocyclic polymers were evaluated as corrosion inhibitor to carbon steel in acidic medium. It was obtained good results and in some cases inhibitor efficient average higher than 70% were reached

Aims Protein Phosphatase Inhibitor 1 (I-1) functions as an amplifier of the β-adrenergic cascade in cardiomyocytes. Once activated via PKA, I-1 specifically blocks PP-1-mediated dephosphorylation of phospholamban and the ryanodine receptor-1. In heart failure I-1 activity as well as its expression is significantly reduced. It is still unclear whether this adaptation is protective or detrimental. This work aims at examining the impact of I-1 depletion on the course of pressure-induced heart failure, more precisely on acute and long-term mortality, on cardiac morphology and function and on expression levels of hypertrophy markers. Results may help evaluating the benefit of putative I-1 inhibiting substances in the therapy of heart failure. Methods and Results 25 I-1KO and 28 WT mice (C57Bl/6J, age- and sex-matched) underwent transverse aortic constriction (TAC). Cardiac function was assessed via transthoracic echocardiography prior to the intervention and weekly afterwards. Additionally, mice were exposed to β-adrenergic stimulation by injection of dobutamine once prior to TAC and two times afterwards, each controlled by echocardiography. For male mice acute survival was significantly increased in WT compared to I-1KO, whereas the mortality of surviving animals did not differ during the investigation period. For female mice no difference was seen in acute mortality after TAC, but during heart failure progression I-1KO revealed a significantly better survival. Prior to TAC contractility in I-1KO after application of dobutamine was significantly lower than in WT. This effect was mainly induced by female mice. Overall female mice of both WT and I-1KO showed smaller increases in heart rate (HR) and stroke volume (SV) when stimulated. In contrast, following TAC neither line- nor sex-dependent differences were found according to β-adrenergic stimulation. The comparison of hypertrophy markers in control groups revealed clearly decreased levels for I-1KO compared to WT. Conclusion In pressure-induced heart failure, I-1 knockout alters cardiac contractility and modulates mortality in a phase- and sex-dependent way. The depletion is detrimental for male mice in the acute phase of cardiac stress, whereas it is protective for female mice during heart failure progression. The increased mortality in the acute phase might result from the loss of I-1 as an amplifier of β-adrenergic signaling as this leads to a restriction of contractile adaptation. The increased survival in heart failure progression might be caused by a reduced transmission of pathologically increased sympathetic activity on the SR due to the depletion of I-1. Additionally, hypertrophy marker analyses point to differences in expression levels even under non-pathological conditions. / Ziel Der Proteinphosphatase-Inhibitor I-1 wirkt als ein Verstärker der β-adrenergen Kaskade in Kardiomyozyten. Nach PKA-abhängiger Phosphorylierung hemmt er spezifisch die Dephosphorylierung von PLB und RYR-2 durch die Proteinphosphatase-1. Im Rahmen einer Herzinsuffizienz sind sowohl Aktivität als auch Expression von I-1 deutlich reduziert. Hierbei ist unklar, ob dies eine protektive oder eine schädliche Adaption der β-adrenergen Kaskade darstellt. Diese Arbeit untersucht den Einfluss einer Depletion des I-1 (I-1KO) im Rahmen der druckinduzierten Herzinsuffizienz auf die akute bzw. auf die langfristige Mortalität, auf die kardiale Morphologie und Funktion sowie auf die Expression typischer Hypertrophiemarker. Hieraus sollen Erkenntnisse über den Nutzen der Verwendung putativ I-1 inhibierender Substanzen in der Behandlung der Herzinsuffizienz gewonnen werden. Methoden und Resultate 25 I-1KO- sowie 28 WT-Mäuse (C57Bl/6J, age and sex matched) erhielten eine Transverse Aortic Constriction (TAC). Die kardiale Funktion wurde einmalig vor der Intervention sowie danach wöchentlich mittels TTE untersucht. Zusätzlich wurden die Tiere einmalig vor TAC und zweimalig danach unter echokardiographischer Kontrolle mittels Dobutamin β-adrenerg stimuliert. Für die männlichen Tiere zeigte sich in den ersten Tagen nach TAC eine signifikant erhöhte Überlebensrate des WT gegenüber I-1KO. Die Mortalität der überlebenden männlichen Tiere unterschied sich hingegen nicht über den Versuchszeitraum. Für die weiblichen Tiere bestand kein Unterschied in der akuten Sterblichkeit nach TAC, während sich im Verlauf eine signifikant bessere Überlebensrate der weiblichen I-1KO gegenüber WT zeigte. Vor TAC wurde eine signifikant herabgesetzte Kontraktilität (FAS) des I-1KO unter Dobutamin festgestellt, der im Wesentlichen durch die weiblichen Tiere bewirkt wird. Insgesamt zeigten die weiblichen Tiere beider Linien unter β-adrenerger Stimulation eine geringere Zunahme von Herzfrequenz (HR) und Schlagvolumen (SV). Hingegen waren nach TAC keine linien- oder geschlechtsabhängigen Unterschiede unter Dobutamingabe feststellbar. Ein Vergleich der Hypertrophiemarker in der Kontrollgruppe zeigte für I-1KO ein deutlich vermindertes Niveau der Marker gegenüber WT. Ergebnis Der I-1-Knockout verändert die kardiale Kontraktilität und wirkt sowohl in phasen- als auch in geschlechtsabhängiger Weise auf die Mortalität infolge druckinduzierter Herzinsuffizienz. Er ist nachteilig für männliche Tiere in der akuten Phase kardialer Belastung, während er für weibliche Tiere im weiteren Verlauf protektive Wirkung entfaltet. Eine erhöhte Mortalität in der akuten Phase kann durch den Ausfall der Verstärkerfunktion des I-1 erklärt werden, da hiermit eine Einschränkung der akut notwendigen kontraktilen Adaptionsfähigkeit einhergeht. Ein Überlebensvorteil bei chronischer kardialer Belastung könnte darauf zurückzuführen sein, dass die pathologisch erhöhte sympathische Aktivierung der β-adrenergen Kaskade infolge der I-1-Depletion eine geringere Auswirkung auf die Zielstrukturen des aktivierten I-1 am Sarkoplasmatischen Retikulum hat. Darüber hinaus lassen die Analysen der Hypertrophiemarker eine veränderte Genexpression zwischen I-1KO und WT auch unter nicht-pathologischen Bedingungen vermuten.

A malária é a doença tropical com maior taxa de mortalidade global. O surgimento de resistência às terapias de primeira linha reforça a necessidade do desenvolvimento de novos candidatos a fármacos. O objetivo deste trabalho foi a descoberta de inibidores e otimização da atividade destas moléculas como candidatos a compostos líderes para o desenvolvimento de novos agentes antimaláricos. Nesse sentido, realizamos a triagem da coleção Malaria Box e identificamos 11 moléculas de diferentes classes químicas como candidatos a inibidores da enzima enolase de Plasmodium falciparum (Pfeno). Em seguida, determinamos a potência inibitória contra a enzima alvo (IC50 entre 11 – >1.000 μM), atividade antiplasmodial in vitro contra a forma eritrocítica do parasito (EC503D7 entre 5,6–1.600 nM) e citotoxicidade (MCL50 > 19 μM) do subconjunto de 11 compostos do Malaria Box (1–11). Ensaios de combinação com o antimalárico artesunato e estágio de ação foram conduzidos para avaliar o potencial de associação e qual fase do ciclo eritrocítico seria mais suscetível as moléculas desse subconjunto. Os resultados obtidos indicam que alguns compostos apresentam caráter aditivo (2 e 9) ou antagônico (1, 3–8, 10 e 11) em relação ao artesunato e ação rápida (1–3, 5, 7, 9–11) ou lenta (4, 6 e 8) no desenvolvimento do parasita. Diante desses resultados, um conjunto de critérios estruturais e de atividade biológica foi estabelecido para a seleção de um candidato para estudos de relação estrutura-atividade (SAR). O derivado de hidrazinobenzimidazol (4) foi selecionado como hit inicial e 24 derivados (12–35) foram planejados, sintetizados e tiveram a atividade de inibição enzimática (IC50 entre 44 – >200 μM), atividade antiplasmodial contra cepas sensível (EC503D7 entre 0,19–14 μM) e resistente (EC50K1 entre 0,15–2,4 μM) e citotoxicidade (MCL50 > 3,7 μM) determinadas no primeiro ciclo de SAR. Os dados obtidos sugerem que a enzima Pfeno não é o alvo principal de ação dos derivados hidrazinobenzimidazol, contudo, a atividade antiplasmodial da série indicou razoável distribuição em termos de potência e variabilidade estrutural que permitiram a construção de um modelo HQSAR (q2= 0,64 e r2 = 0,93) adequado para o planejamento e predição da atividade inibitória de oito novos derivados hidrazinobenzimidazol (36–43). Os novos derivados foram sintetizados e tiveram sua atividade antiplasmodial (EC503D7 entre 0,10–2,3μM), citotoxicidade (MCL50 > 3 μM) e seletividade (SI > 5) determinadas. Os dados de validação prospectiva do modelo indicaram boa correlação entre a atividade real e predita para os novos derivados. Além disso, neste segundo ciclo de SAR foi descoberto o composto 41 como o mais potente (EC50 = 0,1 μM) e seletivo (SI > 2.000) da série investigada neste trabalho. Nossos resultados indicam que os derivados hidrazinobenzimidazol são moléculas atrativas para a descoberta de compostos líderes para o desenvolvimento de candidatos a fármacos antimaláricos. / Malaria is the tropical disease with the highest overall mortality rate. The emergence of resistance to first-line therapies reinforces the need for the development of new drug candidates. The main goal of this work was the discovery and optimization of these molecules as lead candidates for the development of new antimalarial agents. In this sense, we screened Malaria Box collection and identified 11 molecules from different chemical classes as inhibitor candidates of Plasmodium falciparum enolase enzyme (Pfeno). Then, we determined the inhibitory potency against the target enzyme (IC50 between 11 – >1.000 μM), in vitro antiplasmodial activity against the erythrocytic form of the parasite (EC503D7 between 5,6–1.600 nM) and cytotoxicity (MCL50 > 19 μM) of the 11 compounds subset from Malaria Box (1–11). Combination with artesunate and stage of action assays were conducted to evaluate the potential of association and which erythrocytic stage would be more susceptible to the molecules of this subset. The results obtained indicate that some compounds showed additive (2 and 9) or antagonistic (1, 3–8, 10 and 11) effect with artesunate and fast (1–3, 5, 7, 9–11) or slow (4, 6 and 8) acting on parasite development. In view of these, a set of structural and biological activity criteria was established for the selection of a candidate for structure-activity relationship (SAR) studies. The hydrazinobenzimidazole derivative (4) was selected as hit and 24 derivatives (12–35) were designed, synthesized and had the enzyme inhibitory activity (IC50 between 44 – >200 μM), antiplasmodial activity against sensitive strains (EC503D7 between 0,19–14 μM) and resistant (EC50K1 between 0,15–2,4 μM) and cytotoxicity (MCL50 > 3,7 μM) determined in the first round of SAR. The collected data suggest that Pfeno is not the main target of the hydrazinobenzimidazole derivatives. However, the antiplasmodial activity of the series indicated a reasonable distribution in terms of potency and structural variability which allowed us the development of a HQSAR model (q2 = 0,64 and r2 = 0,93) suitable for the design and prediction of the inhibitory activity of eight new hydrazinobenzimidazole derivatives (36-43). The new derivatives were synthesized and had the antiplasmodial activity (EC503D7 between 0,10–2,3μM), cytotoxicity (MCL50 > 3 μM) and selectivity (SI > 5) evaluated. The prospective validation of the HQSAR model indicated good correlation between the actual and predicted activity for the new derivatives. Moreover, in the second round of SAR, compound 41 was discovered as the most potent (EC50 = 0,1 μM) and selective (SI > 2,000) in these series. Our results indicate that the hydrazinobenzimidazole derivatives are attractive molecules for the discovery of new lead compounds for the development of antimalarial drug candidates.

nÃo hà / O aumento nos Ãltimos anos de indivÃduos imunocomprometidos, como portadores da SÃndrome da ImunodeficiÃncia Adquirida, de doenÃas malignas, transplantados e outros usuÃrios de terapias imunossupressoras, favorece o surgimento de infecÃÃes oportunistas, principalmente as de teor fÃngico, como a candidÃase e a criptococose. Apesar de a terapia antifÃngica atual ser eficiente na maioria dos casos, algumas vezes fazem-se necessÃrias novas drogas que atuem como alternativa ou como coadjuvantes no tratamento para potencializar o efeito dos antifÃngicos utilizados. As estatinas sÃo fÃrmacos hipolipemiantes mais prescritos mundialmente para doenÃas cardiovasculares. Entretanto, recentemente, tem sido descritos outros efeitos benÃficos destas drogas, como, por exemplo, o controle de infecÃÃes. Este trabalho teve como objetivo determinar a atividade antifÃngica in vitro das estatinas ante 51 cepas de Candida, sendo 16 de C. albicans, 11 de C. krusei, 12 de C. tropicalis e 12 de C. parapsilosis, e 25 cepas de Cryptococcus, sendo 12 de C. gattii e 13 de C. neoformans, por meio de testes de microdiluiÃÃo em caldo, segundo documento M27-A3 padronizado pelo CLSI. O intervalo de concentraÃÃo testado para pravastatina foi de 50 a 0,0977 mg/mL, para sinvastatina, 1 a 0,0020 mg/mL e para atorvastatina, 10 a 0,0200 mg/mL. Pravastatina inibiu 37 leveduras do gÃnero Candida apresentando concentraÃÃo inibitÃria mÃnima (CIM) na faixa de 1,56 a 6,25 mg /mL e as cepas restantes nÃo foram inibidas mesmo na maior concentraÃÃo testada (50 mg /mL), enquanto que sinvastatina e atorvastatina apresentaram atividade antifÃngica sobre todas as 51 cepas avaliadas, apresentando CIMs de 0,02 a 1 mg / mL e 0,04 a 5,00 mg / mL, respectivamente. Para o gÃnero Cryptococcus, apenas 4 cepas foram inibidas ante a pravastatina (CIM = 25 mg / mL), por outro lado, sinvastatina inibiu todas as 25 cepas (CIM = 0,06 a 1 mg / mL), e atorvastatina apenas 8 cepas (CIM = 0,62 a 2,5 mg / mL), sendo que as 17 restantes nÃo foram inibidas mesmo na maior concentraÃÃo testada ( ≥ 10 mg / mL). Foi determinada concentraÃÃo fungicida mÃnima (CFM) de pravastatina sobre 15 cepas do gÃnero Candida (CFM = 3,12 a 25 mg / mL), de sinvastatina sobre 34 cepas (CFM = 0,03 a 1 mg / mL), e de atorvastatina sobre 16 cepas (CFM = 0,04 a 0,31 mg / mL). Para o gÃnero Cryptococcus, das 25 cepas testadas, pravastatina exibiu CFM sobre apenas 3 cepas (CFM = 50 mg / mL), sinvastatina sobre 21 cepas (CFM = 0,12 a 1 mg / mL), e atorvastatina sobre 1 cepa (CFM = 1 mg / mL). Esta atividade inibitÃria in vitro de estatinas sobre espÃcies de Candida e Cryptococcus, abre uma perspectiva importante para a investigaÃÃo do possÃvel uso destas drogas com finalidade antifÃngica in vivo. / In the past years, fungal opportunistic infections, especially, candidiasis and cryptococcosis, have become more frequent because of the increase in the number of immunocompromised individuals, such as AIDS, transplant and cancer patients and those that are on immunosuppressive therapy. In spite of being effective, sometimes it is necessary to use new drugs as alternatives or as adjuvants in order to potentiate the effect of the classical antifungal therapy. Statins are the most prescribed hypolipemiant drugs worldwide for preventing cardiovascular diseases. However, other benefic effects for these drugs have been described, such as the control of infections. This work aimed at determining the antifungal activity of statins against Candida spp. and Cryptococcus spp. The minimum inhibitory concentrations (MICs) for three different statins (pravastatin, simvastatin and atorvastatin) were determined against 51 strains of Candida spp. (16 C. albicans, 11 C.krusei, 12 C. tropicalis and 12 C. parapsilosis) and 25 strains of Cryptococcus spp. (12 C. gattii and 13 C. neoformans), through broth microdilution assay, according to the Clinical Laboratory Standards Institute (CLSI - Document M27-A3). The concentration tested for pravastatin ranged from 50 to 0.0977 mg/mL, for simvastatin, it ranged from 1 to 0.0020 mg/mL and, for atorvastatin, it varied from 10 to 0.0200 mg/mL. Pravastatin inhibited 37 Candida strains, with MICs varying from 1.56 to 6.25 mg/mL and the remaining strains were not inhibited, even at the highest concentration tested (50 mg/mL). Simvastatin and atorvastatin, on the other hand, inhibited all 51 Candida strains evaluated, presenting MICs ranging from 0.02 to 1 mg/mL and from 0.04 to 5 mg/mL, respectively. Concerning Cryptococcus spp., only four strains were inhibited by pravastatin (MIC=25 mg/mL), while all 25 strains were inhibited by simvastatin (0.06≤MIC≤1 mg/mL) and eight were inhibited by atorvastatin (0.62≤MIC≤2.5 mg/mL) and the remaining 17 were not susceptible to the highest atorvastatin concentration tested (10 mg/mL). The minimum fungicidal concentrations (MFCs) for the tested statins were also determined. The MFC for pravastatin against Candida spp. was determined against 15 strains (3.12≤MIC≤25 mg/mL). The MFC values for simvastatin were determined for 34 strains of Candida spp. (0.03≤MFC≤1 mg/mL), while those for atorvastatin were determined against 16 strains (0.04≤MFC≤0,31 mg/mL). Concerning Cryptococcus spp., the 25 strains tested, MFC values for pravastatin were found against three strains (MFC=50 mg/mL), while those for simvastatin were determined against 21 strains (0.12≤MFC≤1 mg/mL) and those for atorvastatin were determined against one single strain (MFC=1 mg/mL). This in vitro inhibitory activity of statins against Candida spp. and Cryptococcus spp. creates an important perspective for the use of these drugs in vivo in order to control fungal infections.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Protease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 ÂC â EcCI; from 37 to 60 ÂC â EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM â EcCI; from 1 to 100 mM â EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents. / Inibidores de proteases sÃo proteÃnas que inibem a atividade catalÃtica de enzimas, sendo bastante comuns em sementes de plantas. As proteases desempenham papÃis centrais no desenvolvimento de muitas doenÃas. O controle de sua atividade realizado por inibidores de proteases despertou o interesse sobre estas molÃculas como agentes quimiopreventivos, especialmente sobre o cÃncer. Os efeitos anticarcinogÃnicos das sementes de leguminosas presentes comumente na dieta, bem como de espÃcies vegetais subexploradas, tÃm sido extensivamente investigados. O objetivo deste trabalho foi purificar, caracterizar bioquimicamente e avaliar o potencial quimiopreventivo in vitro de inibidores de proteases de sementes de Enterolobium contortisiliquum, utilizando como modelo cÃlulas de adenocarcinoma colorretal humano. Foram purificados dois inibidores de proteases denominados EcCI e EcTI. AtravÃs da sequÃncia N-terminal, EcCI foi identificado como um inibidor da famÃlia Kunitz. EcTI foi identificado, por meio de peptide mass fingerprinting e por anÃlise da sequÃncia N-terminal, como aquele descrito previamente na literatura (NCBI Protein BLAST; nÃmero de acesso: sp|P86451.1|ITRY_ENTCO). Suas massas moleculares determinadas por SDS-PAGE e espectrometria de massas sÃo, respectivamente, 18,5 kDa e 19.710,4 Da (EcCI); 20,2 kDa e 19.813,22 Da (EcTI). Ambos os inibidores sÃo formados de duas subunidades proteicas e apresentam isoformas cujos pIâs sÃo Ãcidos, entre 5 e 6. Frente a quimotripsina, EcCI à um inibidor nÃo competitivo e apresenta ki de 8 x 10-8 M, enquanto EcTI à inibidor competitivo com ki de 48 x 10-8 M. Frente à tripsina, EcTI apresenta inibiÃÃo nÃo competitiva e ki de 2,8 x 10-8 M. EcCI e EcTI apresentam atividade inibitÃria de quimotripsina alta (93%) e moderada (38%), respectivamente. Apenas EcTI foi capaz de inibir a tripsina (100%). EcCI e EcTI apresentam alta atividade inibitÃria de elastase neutrofÃlica (85 e 75%, respectivamente). Os dois inibidores inibiram sutilmente a elastase pancreÃtica (ca. de 10%) e nenhum foi capaz de inibir papaÃna e bromelaÃna. Os dois inibidores apresentam alta estabilidade à variaÃÃo de temperatura (de 37 a 70 ÂC para EcCI e de 37 a 60 ÂC para EcTI), pH (2 a 12) e concentraÃÃo de DTT (de 1 a 10 mM para EcCI e de 1 a 100 mM para EcTI). EcCI e EcTI inibiram a proliferaÃÃo de cÃlulas de adenocarcinoma colorretal humano da linhagem HT29 com CI50 de 35,5 e 20,4 x 10-6 M, respectivamente, indicando que esses inibidores apresentam bom potencial quimiopreventivo e, portanto, sÃo molÃculas bastante interessantes do ponto de vista biotecnolÃgico.

A angiogênese é um processo fundamental e fisiológico de organismos vertebrados, sendo responsável pela formação de novos vasos sanguíneos a partir dos já existentes. Entretanto, a angiogênese pode ocorrer também em condições patológicas, como causa ou consequência de doenças. Um exemplo disso está nos tumores, que para crescer além de alguns milímetros cúbicos, necessitam de um suprimento adequado de oxigênio e nutrientes, e, portanto, dependem da angiogênese. Por isso, compostos que inibem a angiogênese já estão em uso na clínica, não só para o tratamento de tumores, mas também de outras doenças dependentes da angiogênese, as retinopatias. Neste projeto, daremos continuidade à linha de pesquisa do nosso grupo, que procura identificar e validar peptídeos com potencial translacional (pré-fármacos), por apresentarem atividade anti-angiogênica. Utilizando a metodologia do Phage Display, nosso grupo identificou e caracterizou um hexapeptídeo, que foi selecionado por interagir com os receptores do principal fator iniciador da angiogênese, o VEGF (fator de crescimento endotelial vascular). Os receptores de VEGF (ou VEGFR) são proteínas do tipo receptor tirosina quinase, expressos em células endoteliais e essenciais para a iniciação e progressão da neovascularização. O hexapeptídeo identificado em nosso laboratório liga-se ao VEGFRs e inibe a formação de vasos sanguíneos in vivo em modelos animais de angiogênese. Neste trabalho, procuramos estender os estudos com este hexapeptídeo para identificar o sítio de ligação do mesmo no VEGFR e avançar em modelos que permitam a determinação dos requisitos estruturais de interação peptídeoreceptor. Com estes conhecimentos, poderemos num futuro próximo, caminhar para o desenvolvimento racional de moléculas peptideomiméticas com propriedades anti-angiogênicas. / Angiogenesis is a fundamental and physiological process for vertebrate organisms, being responsible for the formation of new blood vessels, sprouting from the existent ones. However, angiogenesis may occur in pathological conditions, being cause or consequence of diseases. One example is tumor development. To grow beyond a few cubic millimeters, tumors need a suitable supply of oxygen and nutrients, and, therefore, they are dependent of angiogenesis. In fact, anti-angiogenic compounds are already in therapeutic use, targeting not only tumors but other angiogenesis dependent diseases, like retinopathies. In this project, we expand research from our own group to identify and develop anti-angiogenic peptides with translational potential (pre-drugs). Using Phage Display methodology, our group identified and characterized a hexapeptide, that was selected based on its capacity to interact with the receptors for the main initiator factor of angiogenesis, the VEGF (Vascular Endothelial Growth Factor). VEGF receptors (VEGFR) are tyrosine kinase proteins, expressed by endothelial cells and essential for neovascularization initiation and progress. The hexapeptide identified in our lab binds to VEGFRs and inhibit blood vessel formation in vivo when tested in an angiogenesis animal model. In this study, we seek to further understand the interaction of this hexapeptide with its receptor by identifying its binding domain on VEGFR and develop models that will allow the determination of the structural requirements for interaction of this receptor ligand pair. With this knowledge, we can in a near future progress to a rational development of novel peptidemimetic molecules with angiogenic properties similar to this hexapeptide.

Com a introdução de uma nova classe de antiretrovirais integrantes da terapia anti-retroviral altamente ativa (HAART) para o tratamento das infecções pelo vírus da imunodeficiência humana, começaram a ser descritos inúmeros efeitos secundários.Na tentativa de se estabelecer um modelo animal para o estudo destes efeitos buscou-se uma espécie com similaridade no perfil e metabolismo lipídico. Iniciou-se estudo em Mesocricetus auratus. Foram avaliados o perfil lipídico e glicêmico,função hepática e renal, níveis de auto-anticorpos anti ox-LDL, perfil eletrocardiográfico, alterações histopatológicas renais e cardíacas nos animais sob dieta hiperlipídica e normal,tratados com Indinavir, inibidor de protease utilizado na HAART. Observou-se uma diminuição da sobrevida nos animais tratados com indinavir, aumento do nível sérico de triglicérides e glicose, redução de auto-anticorpos anti ox-LDL,aumento do segmento QRS no eletrocardiograma, presença de fibrose renal e cardíaca, hipercelularidade glomerular nos animais tratados com a droga com ou sem dieta hiperlipídica quando comparados com os controles. Concluimos que Mesocricetus auratus se apresenta como um bom modelo para o desvendamento dos mecanismos patológicos observados na HAART. / With the introduction of a new antiretroviral class use, integrants of highly active anti-retroviral therapy (HAART) for the treatment of infections by human immunodeficiency virus, several side effects started to be described.To establish an animal model for the study of these side effects, was chosen specie that have similarities in the lipidic profile and metabolism. A study in Mesocricetus auratus was started. It was evaluated the lipidic and glicemic profile ,hepatic and renal function, the levels of auto-antibodies against ox-LDL, electrocardiographic profile and renal and cardiac histopathological alterations in these animals under hyperlipidic and normal diets,treated with Indinavir, a protease inhibitor used in HAART.It was observed a decrease in the survival rate in the animals treated with Indinavir; an increase of the triglycerides and glucose serum level; reduction of anti ox-LDL auto-antibodies; increased QRS segment in the electrocardiogram; presence of renal and cardiac fibrosis; glomerular hypercellularity in the animals treated with the drug, with or without hyperlipidic diet when compared with the controls. We conclude that the Mesocricetus auratus is a good model for disclosure of the pathological mechanisms generated by HAART.

Class of 2012 Abstract / Specific Aims: To evaluate the quality of evidence in the literature substantiating major drug-drug interactions of the HMG-CoA reductase inhibitors (statins) atorvastatin, lovastatin, and simvastatin with the azole anti-fungals fluconazole, itraconazole, and ketoconazole. Methods: In this descriptive retrospective analysis, a list of articles reporting on each drug-drug interaction was compiled from the online databases Medline and International Pharmaceutical Abstracts, and the drug compendia Micromedex and Facts & Comparisons. The studies included in this analysis were primary literature reports, written in English, and consisted of human subjects. All studies included were evaluated using a 5-point quality of evidence scale developed to assess drug-drug interactions (van Roon scale). This scale rates the study type from lowest to highest quality, from zero to four. Case reports were additionally analyzed using the Drug Interaction Probability Scale (DIPS). The DIPS tool uses 10 questions to evaluate the probability that an adverse event is caused by a drug-drug interaction. Main Results: Twenty-one studies met the selection criteria. There were three studies involving atorvastatin, four studies involving lovastatin, and fourteen studies involving simvastatin. The mean quality of evidence score on the van Roon scale was 2.0 + 0.77, where atorvastatin studies had a score of 2.3 + 1.15, lovastatin had a score of 2.25 + 0.95 and simvastatin had a score of 1.86 + 0.66. Seventy-one percent of the studies reviewed were case reports. Conclusions: The reports substantiating some drug-drug interactions may be of little and low quality evidence.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in travellers and young children in developing countries. We previously characterized three vaccine candidates (MipA, Skp, and ETEC_2479) which effectively protected mice in an intranasal ETEC challenge model after immunization. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. In this study, we examined the potential of these antigens to affect the host intestinal microbiota and subsequently found no significant impact on healthy of host after vaccination. In addition, we also optimized the types of adjuvants and forms of antigens and evaluated the efficacy in a mouse intranasal challenge model. Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that cause global public health threads. EHEC possesses the potential to cause gastroenteritis, hemorrhagic colitis and hemolytic uremic syndrome (HUS), which may lead to renal failure. Type III secretion system (T3SS) is a hallmark of EHEC, characterized by the needle-like structure and a variety of effectors injected into host cells. NleB, one of T3SS effectors, is a glycosyltransferase with the ability to catalyze the transfer of N-acetyl-D-glucosamine (N-GlcNAc) to host proteins to suppress the activation of NF-kB signaling pathway. In this study, we employed luminescence-based glycosyltransferase assay and high-throughput screening using a chemical library of various compounds. A total of 128 chemicals was selected with significant inhibition on NleB glycosyltransferase activity for further pharmaceutical study as novel therapy against EHEC infection.