Islet transplantation is an attractive method to achieve blood glucose homeostasis. However, β-cell function declines over time. Therefore, it is necessary to explore strategies to enhance the β-cell mass and function. Also, because there is a severe shortage of human cadaver tissue, alternative sources of insulin secreting tissue need to be examined. Neonatal porcine islet (NPI) tissue has emerged as an attractive alternative source of β-cells. The aim of this thesis was to optimize the culturing conditions of NPIs pre-transplantation so that the available tissue can be used as efficiently and economically as possible. The results from this study indicate that the treatment of NPI cultures with z-VAD-FMK, a pan caspase inhibitor and general protease inhibitor significantly enhances β-cell survival. Additionally, the optimum length of culturing NPIs pre-transplantation appears to be 3-5 days. Since widespread cell death stimulates immunogenic response, this treatment also has the potential benefit of reducing immunosuppression needs in the recipient. / Experimental Surgery

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / This systematic review and meta-analysis tries to determine whether there is an association between the use of protease inhibitors (PIs) and the incidence of diabetes mellitus (DM) and/or metabolic syndrome (MS) in HIV-infected patients. A systematic literature search was performed using MEDLINE/PubMed, CENTRAL, LILACS, and EMBASE. Included articles were observational studies published on or prior to November 2015 that met specific inclusion criteria. Pooled relative risks (RRs) and hazard ratios (HRs) were calculated. Nine articles met the inclusion criteria, describing 13,742 HIV patients. Use of PIs was associated with the development of MS (RR: 2.11; 95% CI 1.28–3.48; p-value 0.003). No association between the use of PIs and development of DM was found: the HR for the incidence of DM among patients using PIs was 1.23 (95% CI 0.66–2.30; p-value: 0.51) and the RR was 1.25 (95% CI 0.99–1.58; p-value 0.06). Use of PIs in HIV-infected patients is associated with an increased risk of MS. No evidence of an increased risk of DM was found. However, because MS is a precursor to DM, it is possible that studies with a longer follow-up duration are needed in order to detect an association between PI use and onset of DM. / First, we would like to thank our families for all their support. Second, we would like to thank the Universidad Peruana de Ciencias Aplicadas, the Health Sciences Department, and the School of Medicine for their support and for all the tools they have provided throughout this process. Finally, we want to thanks to Dr Gwenyth O. Lee and Dr Daniela E. Kirwan for their comments. / Revisión por pares

BEZERRA, Lady Clarissa Brito da Rocha. Prospecção de moléculas com potencial nutracêutico em sementes de enterolobium contortisiliquum (VELL.) morong.: purificação e caracterização parcial de três inibidores de quimotripsina. 2010. 97 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2010. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-01T12:44:59Z No. of bitstreams: 1 2010_dis_lcbrbezerra.pdf: 1826588 bytes, checksum: df35a29a0078f414afc02a541cfda647 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-12T23:30:59Z (GMT) No. of bitstreams: 1 2010_dis_lcbrbezerra.pdf: 1826588 bytes, checksum: df35a29a0078f414afc02a541cfda647 (MD5) / Made available in DSpace on 2016-07-12T23:30:59Z (GMT). No. of bitstreams: 1 2010_dis_lcbrbezerra.pdf: 1826588 bytes, checksum: df35a29a0078f414afc02a541cfda647 (MD5) Previous issue date: 2010 / Leguminous seeds are main sources of food proteins for humans and animals. Other interests in these plant proteins concerning their nutraceutical properties have been raised, since many bioactive proteins, previously referred only as anti-nutritional factors, have exerted beneficial effects on health, acting as chemopreventive agents against several diseases. Among these are protease inhibitors, which play important roles in plant defense and have biological properties of interest for biotechnological applications in pharmaceutical and medical areas. This study aimed to evaluate the nutritional and nutraceutical potential of Enterolobium contortisiliquum seeds, focusing on the purification and partial characterization of chymotrypsin inhibitors and assess their antiproliferative effect against human tumor cells. E. contortisiliquum seeds are an excellent protein source, with about 50% of this macronutrient on a dry basis. The presence of protease inhibitors in high quantities suggests that these molecules may exert a nutraceutical application. Three chymotrypsin inhibitors, denominated EcCI1, EcCI2 and EcCTI were purified and partially characterized. EcCI1, EcCI2 and EcCTI show molecular weights of 17, 17 and 19 kDa, respectively. EcCI1 and EcCI2 are non-competitive inhibitors with ki of 4 x 10-8 and 2.5 x 10-8 M, respectively, while EcCTI inhibits chymotrypsin competitively with ki of 5 x 10-8 M. Only EcCTI was able to inhibit trypsin (98%). EcCI1 and EcCI2 successfully inhibited leukocyte elastase (about 70%), but EcCTI (20%). The three inhibitors slightly inhibited pancreatic elastase (about 20%) and none was able to inhibit papain. The three inhibitors have a high thermal stability (37 to 90 °C for EcCI2 and 37 to 100 °C for EcCI1 and EcCTI), pH (2 to 12) and DTT concentrations ranging from 1 to 100 mM for EcCI1 and EcCTI and from 1 to 10 mM for EcCI2. Further studies are needed to better characterize these inhibitors. None of the inhibitors promoted antiproliferative effect against four tumorigenic cell lines tested. / Sementes de leguminosas constituem uma das principais fontes de proteína na alimentação humana e animal. Outros interesses têm sido despertados para o potencial nutracêutico inerente a essas proteínas vegetais, uma vez que muitas proteínas bioativas, até então referidas apenas como fatores antinutricionais, têm exercido efeitos benéficos no organismo, atuando na cura e/ou prevenção de várias doenças. Dentre esses, destacam-se os inibidores de proteases, os quais desempenham importantes funções na defesa de plantas e apresentam propriedades biológicas de interesse para aplicações biotecnológicas nas áreas farmacológica e médica. O objetivo deste trabalho foi avaliar o potencial nutricional e nutracêutico de sementes de Enterolobium contortisiliquum, enfocando a purificação e caracterização parcial de inibidores de quimotripsina e avaliar seu efeito antiproliferativo contra células tumorigênicas humanas. As sementes de E. contortisiliquum são uma excelente fonte de proteínas, apresentando cerca de 50% desse macronutriente em base seca. A presença de inibidores de proteases em quantidades elevadas sugere que essas moléculas possam vir a ter uma aplicação nutracêutica. Dessa forma, foram purificados três inibidores de quimotripsina, denominados EcCI1, EcCI2 e EcCTI, os quais foram parcialmente caracterizados. As massas moleculares aparentes determinada para os três inibidores é de 17, 17 e 19 kDa, respectivamente. EcCI1 e EcCI2 são inibidores do tipo não competitivo e apresentam ki de 4 x 10-8 e 2,5 x 10-8 M, respectivamente. Em contrapartida, EcCTI inibe a quimotripsina de forma competitiva e apresenta ki de 5 x 10-8 M. Apenas EcCTI foi capaz de inibir a tripsina (98%). EcCI1 e EcCI2 inibiram satisfatoriamente a elastase neutrofílica (ca. de 70%), mas não EcCTI (20%). Os três inibidores inibiram sutilmente a elastase pancreática (ca. de 20%) e nenhum foi capaz de inibir a papaína. Os três inibidores são altamente estáveis às condições extremas de temperatura (de 37 a 90 °C para EcCI2 e de 37 a 100 °C para EcCI1 e EcCTI ), pH (2 a 12) e DTT nas concentrações de 1 a 100 mM para EcCI1 e EcCTI e de 1 a 10 mM para EcCI2. Outros estudos são necessários para melhor caracterizar esses inibidores. Nenhum dos inibidores promoveu efeito antiproliferativo contra as quatro linhagens de células tumorigênicas testadas.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Yunjeong Kim / Coronaviruses are pathogens that cause diverse diseases in humans and animals. The studies in this dissertation are focused on feline coronavirus (FCoV), ferret coronavirus (FRCoV) and mink coronavirus (MCoV). FCoV and FRCoV infections typically cause enteritis in cats and ferrets, respectively. However, a 100% fatal systemic disease called feline infectious peritonitis (FIP) can develop in some FCoV infected cats and a fatal systemic disease resembling FIP can develop in some FRCoV infected ferrets. MCoV causes enteritis which results in significant economic loss to mink farmers. No effective vaccine or treatment is available despite the increasing importance of these viral diseases. We have previously reported the synthesis of inhibitors against 3C-like protease (3CLpro) of FCoV and demonstrated the antiviral efficacy of a 3CLpro inhibitor for treating FIP. FRCoV and MCoV 3CLpro are closely related to FCoV 3CLpro. Therefore, we investigated the structure-function relationships of our 3CLpro inhibitors to identify the struc-tural requirements of inhibitors for FRCoV and MCoV. This is the first report of antiviral com-pounds against FRCoV and MCoV. We have previously conducted a field trial with a potent 3CLpro inhibitor, GC376, in cats with naturally occurring FIP. Comparison of the FCoV 3CLpro amino acid sequences from the pre- and post-treatment samples in one cat showed amino acid changes in 3CLpro. Hence, we generated recombinant 3CLpros carrying the amino acid changes and characterized the effects of these amino acid changes in FCoV 3CLpro on its susceptibility to GC376. We observed that these amino acid changes did not markedly affect the activity of GC376 in fluorescence resonance energy transfer (FRET) assay, explaining the absence of clinical drug resistance in this cat during the field trial.

Os inibidores de proteases desempenham nas plantas funções como: defesa contra ataque de predadores de sementes, regulação de enzimas endógenas e fontes de proteínas e aminoácidos. Muitos destes inibidores são utilizados em estudos bioquímicos, bem como no tratamento de patologias humanas como inflamação e câncer. Neste trabalho, um inibidor de serinoprotease, presente na semente de Stryphnodendron barbatinan (barbatimão), foi purificado, caracterizado e denominado SbKI. Sementes de barbatimão maduras foram trituradas, até a obtenção de uma farinha, e esta foi suspensa em PBS, pH 7,4 (1 :5 m/v), sob agitação por 14 horas a 4°C. O extrato foi centrifugado, filtrado e tratado com PVPP, sendo denominado EB, o qual apresentou inibição da coagulação sanguínea e da atividade de algumas serinoproteases. O inibidor SbKI foi purificado utilizando-se três procedimentos cromatográficos: cromatografia de exclusão molecular (Superdex-75, 10/30), troca iônica (Mono-S HR, 5/5), ambas acopladas em um sistema &TA Purifier e fase reversa (C-18, Waters 250 x 4,6mm) acoplada a um sistema HPLC. Em cada etapa de purificação a presença do inibidor foi monitorada pelos testes de atividade inibitória da tripsina e da coagulação, ambos in vitro. SDS-PAGE, sob condições redutoras, mostrou que o inibidor é formado por duas cadeias polipeptídicas (cadeia pesada e leve) unida por ligação dissulfeto. As cadeias foram separadas pela cromatografia de &se reversa após serem reduzidas e alquiladas. Suas seqüências N-terminais foram determinadas pela degradação de Edman, em seqüenciador automatizado, apresentando alta identidade seqüencial com inibidores do tipo Kunitz de outras leguminosas. A determinação da massa/molecular do inibidor e de suas cadeias isoladas, foram determinadas por espectroscopia de massa (LCtESI-MS system) mostrando massas moleculares de 19.570Da7 15530Da e 4040Da, respectivamente. A espectroscopia de dicroísmo circular (CD) revelou que o inibidor é formado predominantemente por elementos beta e estruturas desordenadas. SbKI foi estável a variações de pHs (2-12) e temperaturas extremas e a temperatura de transição foi calculada em 73,3\" C. A determinação das constantes de inibição (KI) foi realizada para as serinoproteases tripsina (KI = 5,5 nM) e calicreína plasmática (KI = 1,l nM). / Proteinase inhibitors perform many beneficia1 roles in plants such as defense against the attack of seed predators, regulation of endogenous enzymes and sources of proteins and amino acids. Many inhibitors are used in biochemistry research, as well as human pathology treatment such as inflammation and cancer. In this work, a serino proteinase inhibitor found in Stryphnodendron barbatiman seeds (barbatimão) was purified, characterized and denoted SbKI. Mature barbatimão seeds were ground and suspended in PBS pH 7.4 (15 wlv) and stirred for 14 hours at 4OC. The suspension was centrifuged, filtered and treated with PVPP and denoted EB. This EB inhibited blood coagulation and some serine proteinases activities. The inhibitor SbKI was purified by three chromatography step: molecular exclusion (on Supredex-75, 10/30), ion exchange (on Mono-S, 5/5), both connected to AKTA Purifíer System and reversed phase (on C-18, Waters 250 x 4.6 mm) connected to HPLC System. In each purification step the presence of inhibitor was monitored, in vitro, by trypsin and coagulation inhibitory activity. SDS-PAGE, reduced conditions, showed two polypeptide chains (heavy and light chains) linked by one disulphide bridge. The chains were separated by reversed phase chromatography aíter reduced and alquilated. The N-terminal sequence were performed on automated protein sequencer by Edman degradation and showed homology with Kunitz type inhibitors from Leguminosae. Molecular weight of inhibitor and its chains were determined by mass spectrometry (LC/ESI-MS System) and showed molecular weight of 19.570Da, 15.530Da and 4040Da, respectively. Circular dichroism spectroscopy showed SbKI is constituted predominantly by P elements and unordered structures. SbKI was stable over extreme ranges of pH (2-12) and temperature and the transition temperature 73.3\"C investigated by CD and fluorescence emission spectroscopies. Inhibition constants (Ki) were determined by typsin (Ki = 5.5 nM) and human plasmatic kallikrein (Ki = 1.1 mM)

Mesmo com o grande número de estudos relacionados à proteases do subtipo B e de como suas mutações podem interferir na estrutura, na resistência a inibidores e na eficiência catalítica da enzima, existe ainda uma lacuna de como as mudanças polimórficas de proteases de HIV de outros subtipos de HIV-1 interferem nesses fatores. Nesse contexto insere-se esse trabalho, que utilizou proteases de HIV-1 isoladas de pacientes brasileiros HIV-1 infectados com o subtipo F, e outros dois mutantes, sendo que um do subtipo F e outro do subtipo B para ensaios frente a seis inibidores comercialmente disponíveis: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir e saquinavir. Nossos resultados experimentais revelam que os seis inibidores comerciais estudados são significantemente menos ativos para o subtipo F e para as mutantes quando comparados ao subtipo B. Além disso, os valores de vitalidade dessas proteases também são considerados maiores que os obtidos para a proteína selvagem do subtipo B. O acúmulo de mutações comumente detectadas e o polimorfismo natural tornam a protease selvagem do subtipo F cataliticamente suficiente para manter a viabilidade do vírus e garantir alto grau de resistência cruzada frente a todos os inibidores estudados. / Despite years of intense research around the world, HIV continues to represent considerable therapeutical challenge. In order to gain more insights into resistance of polymorphic mutations of existing HIV subtypes toward commercially available pharmaceutics, we studied inhibition of subtypes B and F HIV proteases (PRs) [native and two mutant enzymes clinically identified in Brazilian patients] by six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir). Our results show that all these inhibitors have significantly higher Ki values for the subtype F HIV PR (Fwt) and both mutant enzymes than that for the B subtype HIV PR (Bwt). Furthermore, the biochemical fitnesses of these proteases, or their vitalities, are also considerably higher than that of Bwt. The accumulation of commonly detected resistant mutations in HIV PRs with natural polymorphisms turns Fwt sufficiently catalytically active to guarantee the virus viability and confers it a large degree of cross resistance against all studied inhibitors.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Sementes de leguminosas constituem uma das principais fontes de proteÃna na alimentaÃÃo humana e animal. Outros interesses tÃm sido despertados para o potencial nutracÃutico inerente a essas proteÃnas vegetais, uma vez que muitas proteÃnas bioativas, atà entÃo referidas apenas como fatores antinutricionais, tÃm exercido efeitos benÃficos no organismo, atuando na cura e/ou prevenÃÃo de vÃrias doenÃas. Dentre esses, destacam-se os inibidores de proteases, os quais desempenham importantes funÃÃes na defesa de plantas e apresentam propriedades biolÃgicas de interesse para aplicaÃÃes biotecnolÃgicas nas Ãreas farmacolÃgica e mÃdica. O objetivo deste trabalho foi avaliar o potencial nutricional e nutracÃutico de sementes de Enterolobium contortisiliquum, enfocando a purificaÃÃo e caracterizaÃÃo parcial de inibidores de quimotripsina e avaliar seu efeito antiproliferativo contra cÃlulas tumorigÃnicas humanas. As sementes de E. contortisiliquum sÃo uma excelente fonte de proteÃnas, apresentando cerca de 50% desse macronutriente em base seca. A presenÃa de inibidores de proteases em quantidades elevadas sugere que essas molÃculas possam vir a ter uma aplicaÃÃo nutracÃutica. Dessa forma, foram purificados trÃs inibidores de quimotripsina, denominados EcCI1, EcCI2 e EcCTI, os quais foram parcialmente caracterizados. As massas moleculares aparentes determinada para os trÃs inibidores à de 17, 17 e 19 kDa, respectivamente. EcCI1 e EcCI2 sÃo inibidores do tipo nÃo competitivo e apresentam ki de 4 x 10-8 e 2,5 x 10-8 M, respectivamente. Em contrapartida, EcCTI inibe a quimotripsina de forma competitiva e apresenta ki de 5 x 10-8 M. Apenas EcCTI foi capaz de inibir a tripsina (98%). EcCI1 e EcCI2 inibiram satisfatoriamente a elastase neutrofÃlica (ca. de 70%), mas nÃo EcCTI (20%). Os trÃs inibidores inibiram sutilmente a elastase pancreÃtica (ca. de 20%) e nenhum foi capaz de inibir a papaÃna. Os trÃs inibidores sÃo altamente estÃveis Ãs condiÃÃes extremas de temperatura (de 37 a 90 ÂC para EcCI2 e de 37 a 100 ÂC para EcCI1 e EcCTI ), pH (2 a 12) e DTT nas concentraÃÃes de 1 a 100 mM para EcCI1 e EcCTI e de 1 a 10 mM para EcCI2. Outros estudos sÃo necessÃrios para melhor caracterizar esses inibidores. Nenhum dos inibidores promoveu efeito antiproliferativo contra as quatro linhagens de cÃlulas tumorigÃnicas testadas / Leguminous seeds are main sources of food proteins for humans and animals. Other interests in these plant proteins concerning their nutraceutical properties have been raised, since many bioactive proteins, previously referred only as anti-nutritional factors, have exerted beneficial effects on health, acting as chemopreventive agents against several diseases. Among these are protease inhibitors, which play important roles in plant defense and have biological properties of interest for biotechnological applications in pharmaceutical and medical areas. This study aimed to evaluate the nutritional and nutraceutical potential of Enterolobium contortisiliquum seeds, focusing on the purification and partial characterization of chymotrypsin inhibitors and assess their antiproliferative effect against human tumor cells. E. contortisiliquum seeds are an excellent protein source, with about 50% of this macronutrient on a dry basis. The presence of protease inhibitors in high quantities suggests that these molecules may exert a nutraceutical application. Three chymotrypsin inhibitors, denominated EcCI1, EcCI2 and EcCTI were purified and partially characterized. EcCI1, EcCI2 and EcCTI show molecular weights of 17, 17 and 19 kDa, respectively. EcCI1 and EcCI2 are non-competitive inhibitors with ki of 4 x 10-8 and 2.5 x 10-8 M, respectively, while EcCTI inhibits chymotrypsin competitively with ki of 5 x 10-8 M. Only EcCTI was able to inhibit trypsin (98%). EcCI1 and EcCI2 successfully inhibited leukocyte elastase (about 70%), but EcCTI (20%). The three inhibitors slightly inhibited pancreatic elastase (about 20%) and none was able to inhibit papain. The three inhibitors have a high thermal stability (37 to 90 ÂC for EcCI2 and 37 to 100 ÂC for EcCI1 and EcCTI), pH (2 to 12) and DTT concentrations ranging from 1 to 100 mM for EcCI1 and EcCTI and from 1 to 10 mM for EcCI2. Further studies are needed to better characterize these inhibitors. None of the inhibitors promoted antiproliferative effect against four tumorigenic cell lines tested

Novel conserved amino acid variations of papaya cystatin (PC) were investigated by amino acid substitutions using oryzacystatin-I (OCI) as a model plant cystatin for comparison. These amino acid residues in the conserved motifs are involved in binding with cysteine proteases, these include the GG (Gly-Gly) in the N-terminal region for both OCI and PC, the (Q)QVVAG (Gln-Val-Val-Ala-Gly) motif for OCI and (Q)AVVEG (Ala-Val-Val-Glu-Gly) motif for PC in the first inhibitory loop, and the PW (Pro-Trp) motif for OCI and LW (Leu-Trp) motif for PC in the second inhibitory loop. Recombinant OCI and PC mutant proteins were expressed in Escherichia coli and were tested for altered inhibitory activity against commercial cysteine proteases (papain and cathepsin L) and extracts from Colorado potato beetle (Leptinotarsa decemlineata) larvae, from banana weevil larvae (Cosmopolites sordidus) and tobacco leaf extracts (Nicotiana benthamiana). In all tests higher amounts of PC had to be used to obtain similar inhibition levels as OCI. Changing the amino acid Q at position 52 to E in OCI in the first inhibitory loop, had lowered the Ki value of the mutant against the commercial proteases. Concurrently the same amino acid string (EQ) in PC had resulted in a significantly decreased Ki value compared to PC wild-type and other mutants. All other OCI mutants were less efficient than the wild-type OCI, whereas all PC first inhibitory loop mutants had improved inhibitory activity against protease activity with the highest improvement against the protease extracts was found for the substitution of E with A at position 55. This study has shown the importance of the three conserved motifs and that it is possible to improve the binding capacity of a plant cystatins to cysteine protease activity by amino acid substitution using site-directed mutagenesis. By mutating individual amino acid residues in the first binding loop of the relatively “weak” papaya cystatin to amino acid residues found in OCI caused a significant improvement in inhibitory potency of PC. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13363号 / 論医博第2205号 / 新制||医||1045(附属図書館) / (主査)教授 妹尾 浩, 教授 今中 雄一, 教授 川上 浩司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Since 1981, HIV/AIDS has affected over 70 million individuals worldwide. Due to the incorporation of Combination Antiretroviral Therapy (cART), this deadly virus has now become a manageable chronic illness with a reduction in mortality and morbidity rates. Combination therapy targets multiple stages of the HIV replication cycle including fusion, entry, reverse transcription, integration, and maturation. The HIV-1 protease enzyme is responsible for cleavage and processing of viral polyproteins into mature enzymes and is a common therapeutic target for inhibition of HIV. To date, there have been many protease inhibitors approved by the FDA and introduced into the market. However, mutations within the protease enzyme has rendered some of these inhibitors ineffective. This has led to an ever-growing need to develop novel protease inhibitors to combat drug resistance through mutations. Described herein is the design, synthesis, and biological evaluation of HIV-1 protease inhibitors featuring a novel hexahydropyrrolofuran (HPF) bicyclic scaffold as a P₂ ligand to target binding interactions with Asp29 and Asp30. The HPF ligand provides a molecular handle that allows for further structure-activity discoveries within the enzyme. The HIV-1 protease inhibitors discussed feature carbamate, carboxamide, and sulfonamide derivatives which displayed good to excellent activity.</p>