Iminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy.

Na última década, a análise da estrutura proteica baseada em teoria de redes/grafos tem emergido. A abstração da estrutura tridimensional proteica em forma de uma rede, leva em consideração os resíduos de aminoácidos e suas interações através do espaço, e apresenta um conjunto de conexões e propriedades mais complexas do que aquelas visualizadas apenas com a estrutura covalente. A análise da estrutura proteica identificou que as proteínas pertencem às redes de classes de \"mundo pequeno\" (small-world) e \"sem escala\" (scale-free), o que significa que seus resíduos de aminoácidos são altamente agregados e que existem poucas conexões entre 2 resíduos quaisquer da proteína. A identificação dos resíduos com alto grau de conexão, chamados centrais (\"resíduos hubs\"), é feita pela determinação do caminho mais curto que conecta um dado resíduo aos demais compreendidos nesta rede. A remoção destes resíduos centrais (hubs) afeta a integridade da rede de forma mais contundente diferentemente da remoção de resíduos que não são centrais. Até o momento estes \"resíduos hubs\" ainda não foram experimentalmente correlacionados com as propriedades enzimáticas de proteínas. Para tal finalidade, a estrutura terciária de uma β-glicosidase de Spodoptera frugiperda (Sfβgli) foi analisada como uma rede. Após calcular-se os caminhos médios entre todos os pares de aminoácidos da β-glicosidase, encontrou-se 11 resíduos centrais (\"resíduos hubs\"). Alinhamento de sequências e comparações estruturais indicaram alta conservação destes \"resíduos hubs\". Nosso objetivo foi produzir esta β-glicosidase mutando-se a maioria dos \"resíduos hubs\" e 3 aminoácidos não centrais (\"não hubs\"), expressar estes mutantes em E. coli, determinar suas propriedades enzimáticas como atividade catalítica e preferência pelo substrato e verificar a estabilidade destes mutantes em experimentos de inativação térmica. Os resultados obtidos sugerem que mutações nos \"resíduos hubs\" não afetam as propriedades catalíticas, contudo as enzimas com mutações nos \"resíduos hubs\" apresentaram uma menor estabilidade térmica. Estes resultados sugeriram que os \"resíduos hubs\" são relevantes na difusão da energia cinética (vibração) introduzida na estrutura desta β-glicosidase pelo seu aquecimento / In recent years, graph-theoretic approaches have established that protein structures can be modeled as complex networks of interacting residues. Proteins structures can be represented as small-world and scale-free networks that are usually highly clustered with few links connecting any pair of nodes. The identification of nodes with high connection degrees, called hubs, is made by determining the shortest path linking one amino acid to the further nodes comprising the network. Targeted removal of the hubs has greater affect on the integrity of the network structure in contrast to a random removal of amino acid residues comprising the network. Nevertheless these hubs had not previously been correlated with enzymatic properties. The tertiary structure of β-glycosidase from S. furgiperda (Sfβgly) was analyzed as a network. After calculating the averaged paths between all pairs of amino acid residues of Sfgly, we defined 11 hubs, which have the highest centrality on the network. Sequence alignment and structural comparison showed that these hubs residue are conserved among β-glycosidases. Our goal was to mutate most hubs and 3 ´non-hubs´ residues from Sfβgly, express these mutant enzymes in E. coli, test their enzymatic properties as catalytic efficiency and substrate preference, and verify the thermal stability of these mutants. The results implied that mutations in these hubs do not cause changes in catalytic properties although enzymes containing mutations in hubs showed lower thermal stability. Based on that, it was suggested that hub residues are important in the diffusion of kinetic energy (vibrations) introduced in the Sfβgly structure by heating

Introdução e objetivos: β-glicosidases da família GH1 das glicosil-hidrolases possuem um dobramento do tipo barril (β/α)8. Propõe-se que proteínas com este dobramento, que usualmente classificam-se como tendo um único domínio, na verdade são compostas por dois domínios, cada um deles correspondendo a um \"meio barril\" (β/α)4. Assim, as proteínas com dobramento barril (β/α)8 seriam provenientes de uma duplicação e fusão gênica de um ancestral \"meio barril\" (β/α)4. O objetivo geral deste projeto é investigar a existência de dois domínios (β/α)4, as metades N- e C-terminal, na estrutura (β/α)8 barril da β-glicosidase A de Thermotoga maritima (bglTm) e β-glicosidase B de Paenibacillus polymyxa (bglB) por meio de análise da desnaturação térmica e química dessas enzimas. Resultados: Para atingir esse objetivo foram introduzidas mutações que rompem contatos não covalentes entre os supostos domínios destas β-glicosidases. Os segmentos de DNA que codificam as enzimas selvagem e mutantes foram clonados em plasmídeo de expressão pLATE51 e as enzimas recombinantes expressas em Escherichia coli BL21(DE3). Foram purificadas com sucesso as enzimas selvagens e duas mutantes de bglTm, denominadas T1 e T2, que possuem 2 e 4 mutações respectivamente em resíduos na interface inter-metades. Para confirmar o enovelamento destas proteínas recombinantes foi empregada a análise de estruturas secundárias por dicroísmo circular, também o espectro de fluorescência intrínseca de triptofano e sua supressão por acrilamida e finalmente foram determinados parâmetros cinéticos. Observou-se que não houve mudanças significativas na exposição dos triptofanos das proteínas recombinantes, sugerindo que se encontram enoveladas, o que está de acordo com a observação de que as proteínas recombinantes mantêm sua atividade catalítica. Já pela análise de dicroísmo circular concluiu-se que a mutante T1 possui dobramento semelhante à selvagem bglTm e que T2 apresenta diferenças significativas, sendo as porcentagens de α-hélice de 21%, 22% e 10% para bglTm, T1 e T2, respectivamente. Em seguida demonstrou-se que T1 mantém a termo estabilidade semelhante à selvagem, enquanto que T2 tem termo estabilidade reduzida, apresentando kobs de 0,3 min-1 em 80°C e de 0,06 min-1 em 75 °C.. O cálculo de Tm através de Differential Scanning Fluorimetry foi feito para bglB e T2 (42 e 81.7 °C respectivamente), enquanto que bglTm e T1 mantiveram-se estáveis na faixa de temperatura analisada (até 95°C). Na análise do efeito da temperatura sobre a estrutura das mutantes T1 e T2 não se observou nenhuma evidência da presença dos dois supostos domínios (β/α)4. A análise da desnaturação por cloreto de guanidina mostrou que o c50 diminuiu para T2 (2,4 M), mas não mostrou alteração para T1 (4,5 M) em comparação à bglTm (4,3 M). Coerentemente, a estabilidade da enzima selvagem e de T1 na ausência de desnaturante é a mesma (ΔGH2O = 5,2 kcal/mol), mas se reduziu para a T2 (ΔGH2O = 3,5 kcal/mol). Os cálculos do parâmetro m mostraram uma cooperatividade semelhante na desnaturação de bglTm, T1 e T2, não evidenciando independência entre os dois supostos domínios (β/α)4. Em conclusão, a análise estabilidade da β-glicosidase bglTm frente à temperatura e ao cloreto de guanidina não revelaram a presença dos domínios (β/α)4 que correspondem às metades N- e C-terminal desta β-glicosidase. / Introduction and Aims: β-glucosidases from the family GH1 of the glycosil-hidrolases presents a (β/α)8 barrel folding. These proteins are usually classified as single domain, however it has been alternatively proposed that they actually are formed by two \"half barrel\" (β/α)4. Thus, (β/α)8 barrel proteins had evolved from an \"half barrel\" ancestor that underwent a duplication-fusion event. The general goal of this project is the search for the two putative (β/α)4 domains, which form the N- and C-terminal ends of the β-glucosidase A from Thermotoga maritima (bglTm) and β-glucosidase B from Paenibacillus polymyxa (bglB), detecting their presence through the thermal and chemical stability of these (β/α)8 barrel proteins. Results: Site-directed mutagenesis was employed to replace residues forming non-covalent interaction between the putative (β/α)4 domains. DNA segments coding for bglB, bglTm and mutant bglTm were cloned into the pLATE51 expression vector and produced as recombinant proteins in E. coli BL21(DE3). The bglB, bglTm and two mutant bglTm, hereafter called T1 and T2, with 2 and 4 mutations respectively on residues in the interface between the protein halves, were purified. They were stable folded as shown by detecting their catalytic activity upon two different substrates and also by circular dichroism (CD) and tryptophan fluorescence analysis. Nevertheless, T2 showed a decrease in the α-helix content (10 %) in the CD analysis, whereas bglTm and T1 are similar (22 %). The wild-type bglTm and T1 are thermostable, whereas T2 was inactivated after pre-incubation at high temperature (kobs = 0,3 min-1 at 80 °C and 0,06 min-1 at 75 °C). The Differential Scanning Fluorimetry experiments revealed Tm of 42 e 81.7 °C for bglB and T2, respectively, whereas wild-type bglTm and mutant T1 did not showed any thermal transition up to 95 °C. Indeed, the analysis of the thermal stability of T1 and T2 did not reveal any evidence of the putative (β/α)4 domains. Following that, the analysis of the protein denaturation by guanidine hydrochloride showed that the c50 for T2 was reduced (2.4 M), whereas no modification was observed for bglTm and T1 (4,3 and 4,5 M, respectively). In agreement the stability of bglTm and T1 (ΔGH2O = 5,2 kcal/mol) is similar, but it was reduced for T2 (ΔGH2O = 3,5 kcal/mol). The m parameter showed a similar cooperative denaturation for wild-type bglTm and mutants T1 and T2, but no evidence of the independent unfolding of the putative (β/α)4 domains was found. Conclusion: In conclusion, the analysis of the thermal and chemical stability of the bglTm did not reveal the presence of the putative (β/α)4 domains that form the N- and C-terminal end of these β-glucosidase.

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2013 / Diabetes mellitus is one of the fast growing chronic metabolic disorders throughout the world. It has become a life threatening disease and health burden. So far it can only be managed with commercial therapeutic agents, proper diet and exercise. People particularly from developing countries use medicinal plants to treat this condition. According to WHO, about 80% of the population in developing countries are dependable on medicinal plants. This prompted many researchers to explore the effectiveness and safety of these plants. In the current study ten medicinal plants were randomly chosen, screened for antidiabetic activity by testing their ability to inhibit α-amylase and α-glucosidase enzymes. The plants were tested using in vitro assays. The finely powdered leaves of each plant were extracted with hexane, chloroform, acetone and ethyl acetate. Phytoconstituents of each plant extracts were analyzed using both qualitative and quantitative methods. All plant extracts tested positive for phenols, flavonoids and all negative for starch. Their compounds were better separated in the TEA mobile system on the TLC plates. All plant extracts had more of total phenolics ranging between 0.1-400 GAE/mg than total flavonoids and condensed tannins. Antioxidant activity of the plant extracts was tested quantitatively at various concentrations using DPPH. Most plant extracts were able to scavenge the radicals produced by DPPH at highest concentration of 2.5 mg/mℓ. Not all plant extracts with the highest number of total phenolics had the highest antioxidant activity. For antidiabetic in vitro assays, plant extracts inhibited various percentages of both α-amylase and α-glucosidase activity at concentrations ranging between 0.019- 2.5 mg/mℓ. The best overall activity against both enzymes was observed in acetone and ethyl acetate plant extracts. Cassia abbreviata and Helinus integrifolius were even more active than acarbose which was used as positive control. These plant extracts inhibited both the enzymes in a dose dependent and non-competitive manner. Seeing that both extracts of C. abbreviata and H. integrifolius were consistent when inhibiting both enzymes, they were further evaluated for their effect on glucose uptake by the C2C12 muscle and H-II-4-E liver cells. All the plant extracts tested were able to increase glucose uptake in the muscle cells. However optimal increase was seen in the liver cells when treated with 250 µg/mℓ of acetone and ethyl acetate extracts of C. abbreviata. The cytotoxicity effects of both acetone and ethyl acetate of C. abbreviata and H. integrifolius was tested using the xCelligence system on RAW 264.7 cells. Different cell indexes were obtained after treating the cells with different concentrations (0.05,0.1 and 0.25 mg/mℓ) of each plant extracts respectively. The system was run for three days but the toxic effects of plant extracts were analyzed for the first ten hours. The results obtained shows that cell index decreased as the concentration of the plant extracts was increased. All the plant extracts were less toxic as compared to positive control, Actinomycin D. The leaves of H. integrifolius were further exhaustively extracted with hexane, dichloromethane, acetone, ethyl acetate and methanol respectively. Since the DCM extracts yielded the highest mass in quantity, it was further used for isolation of active compounds. Column chromatography and bioassay guided fraction led to isolation of a mixture of triterpenes identified as α and β-amyrin. The structure was elucidated using nuclear magnetic resonance technique. The inhibitory capability of the isolated compound against α-amylase enzyme was less than the crude extract which inhibited more than 50% of the activity at a concentration of 1 mg/mℓ.Based on the enzymes assays and cell culture work it can be concluded that C. abbreviata and H. integrifolius species are the best inhibitors of carbohydrate digesting enzymes, and therefore be used to manage postprandial hyperglycemia in the people with type 2 diabetes. However more work still need to be conducted for further isolation of more active compounds.

In this thesis, carbohydrate mimicry is investigated through the syntheses of carbohydrate analogues and evaluation of their inhibitory effects on carbohydrate-processing enzymes. Galactofuranosides are interesting structures because they are common motifs in pathogenic microorganisms but not found in mammals. M.tuberculosis, responsible for the disease tuberculosis, has a cell wall containing a repeating unit of alternating (1→5)- and (1→6)-linked β-D-galactofuranosyl residues. Synthetic inhibitors of the enzymes involved in the biosynthesis of the cell wall could find great therapeutic use. The first part of this thesis describes the first synthesis of the hydrolytically stable carbasugar analogue of galactofuranose, 4a-carba-β-D-Galf, and the synthetic work of synthesising β-linked pseudodisaccharides containing carba-Galf, which were tested for glycosyltransferease inhibitory activity. The pseudodisaccharide carba-Galf-(β1→5)-carba-Galf was found to be a moderate inhibitor of the glycosyltransferase GlfT2 of M.tuberculosis. The thesis also describes how a general method towards biologically relevant α-linked carba-Galf ethers was developed. The final part of this thesis is focussed on the formation of nitrogen-linked monosaccharides without the participation of the anomeric centre. Such a mode of coupling is called tail-to-tail neodisaccharide formation. The couplings of carbohydrate derivatives via the Mitsunobu reaction are successfully reported herein. The method describes the key introduction of an allylic alcohol in the electrophile and the subsequent functionalisation of the alkene to obtain the neodisaccharide. Two synthesised neodisaccharides presented in this thesis have been sent to be tested for glycosidase inhibitory activity. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript. Paper 6: Manuscript.

Made available in DSpace on 2016-06-02T19:56:45Z (GMT). No. of bitstreams: 1 3875.pdf: 1789055 bytes, checksum: a2495b6571816afdda74f70d8d9b300d (MD5) Previous issue date: 2011-02-25 / Universidade Federal de Sao Carlos / B-glucosidase (BG) is an important enzyme for several biotechnological applications. This enzyme plays an important role in hydrolyses of lignocellulosic biomass in order to produce second generation ethanol (2G ethanol). The enzimatic hydrolysis of cellulose requires the sinergystic action of endoglucanases, exoglucanases and β-glucosidases. Endo e exoglucanases are strongly inhibited by cellobiose and its accumulation into reaction medium decreases the hydrolysis rate. The supplementation of the reaction medium with BG can reduce the inhibition effect, leading to higher conversions of cellulose to glucose. In this work, BG was immobilized on different solid supports in order to obtain an active and stable derivative to be used in hydrolyses of sugarcane bagasse. BG was immobilized on glyoxyl-agarose (GA) and polyacrylic matrix (MP) at 25oC and pH 9.0 and 4.8, respectively. To improve the immobilization yield on glyoxyl-agarose at pH 9.0, a chemical amination of the enzyme surface was required. However, BG was inactivated during the immobilization reaction due to alkaline conditions that are required to immobilize enzymes on glyoxylagarose support. Nevertheless, the presence of a competitive inhibitor (glucose) during immobilization of BG preserved about 70% of the initial activity. However, the reduction step with sodium borohydride (end point of the reaction) drastically reduced the derivative activity even in the presence of glucose. The BG immobilization in presence of competitive inhibitor allowed the preparation of a derivative approximately 4 times more active than one prepared in inhibitor absence. On the other hand, the best derivative was prepared adsorbing the enzyme on polyacrylic resin covered with carboxylic groups. After four hours of reaction, the immobilization yield and the recovered activity were ca. 71% and 97%, respectively. Pretreated sugarcane bagasse (10% w/v, dry basis) was hydrolyzed at 50oC, pH 4.8 (50 mM sodium citrate buffer), for 24 h, using soluble cellulase (Acellerase 1500) in the enzyme/substrate ratio of 20 FPU/gcellulose. Hydrolyses under same conditions were performed by supplementing the reaction medium with BG immobilized on glyoxyl-agarose (BG-GA) or BG immobilized on polyacrylic resin (GA-MP) in the enzyme/substrate ratio of 120 U/gcellulose. Five batches were performed under - xi - conditions described above by reusing the immobilized BG and non-converted cellulose after thoroughly washing with distilled water. The supplementation of the reaction medium with immobilized BG enhanced the cellulose conversions in all batches. This behavior is due to the fact that BG removes cellobiose from the reaction medium, avoiding its accumulation, which could inhibit the endoglucanases and exoglucanases. However, a decrease of the cellulose conversion after the second batch was observed (cellulose conversion decreased from ca. 50% to 15-25%). Anyway, this work shows that supplementation of the commercial enzymatic complexes with immobilized BG is advantagous. However, the stabilization of the immobilized BG is still required. / B-Glicosidase (BG) é uma enzima de grande importância em inúmeras aplicações biotecnológicas. Essa enzima desempenha um papel muito importante na hidrólise enzimática da biomassa lignocelulósica visando a produção de etanol de segunda geração (etanol 2G). A hidrólise enzimática da celulose requer a ação sinergística de endoglicanases, exoglicanases e β-glicosidases. Endo e exoglicanases são fortemente inibidas por celobiose e seu acúmulo no meio reacional reduz a taxa de hidrólise. A suplementação do meio reacional com BG pode reduzir o efeito inibitório, levando a conversões maiores de celulose a glicose. Neste trabalho BG foi imobilizada em diferentes suportes sólidos visando a obtenção de um derivado ativo e estável para uso em reações de hidrólise de bagaço de cana-de-açúcar. BG foi imobilizada em glioxil-agarose (GA) e resina poliacrílica catiônica (MP) a 25ºC e pH 9,0 e 4,8, respectivamente. Para melhorar o rendimento de imobilização de BG em glioxilagarose a pH 9,0 foi necessária uma aminação química da superfície da enzima. Entretanto, BG era inativada durante a imobilização, devido às condições alcalinas requeridas para imobilização de enzimas em glioxilagarose. Contudo, a presença de um inibidor competitivo (glicose) durante a imobilização de BG preservou aproximadamente 70% da atividade inicial. Mesmo na presença de glicose, a etapa de redução com borohidreto de sódio (finalização da reação de imobilização) reduziu drasticamente a atividade da enzima imobilizada. A imobilização de BG na presença de inibidor competitivo permitiu a preparação de um derivado cerca de 4 vezes mais ativo que aquele preparado na ausência do inibidor. Por outro lado, o melhor derivado foi preparado adsorvendo BG em resina poliacrílica funcionalizada com grupos carboxílicos. Após quatro horas de reação, o rendimento de imobilização e a recuperação de atividade foram aproximadamente 71% e 97%, respectivamente. Bagaço de cana pré-tratado (10% m/v, base seca) foi hidrolisado a 50°C, pH 4,8 (tampão citrato de sódio 50 mM), por 24h, utilizando celulase solúvel (Acellerase 1500) na relação enzima/substrato de 20 FPU/gcelulose. Hidrólises nas mesmas condições foram realizadas - ix - suplementando o meio reacional com BG imobilizada em glioxil-agarose (BGGA) ou BG imobilizada em matriz poliacrílica (BG-MP) na relação enzima/substrato de 120 U/gcelulose. Cinco bateladas foram realizadas nas condições descritas acima, reutilizando a BG imobilizada e a celulose não convertida, após lavagem abundante com água destilada. A suplementação do meio reacional com BG imobilizada contribuiu para a obtenção de maiores conversões de celulose em todas as bateladas, devido ao fato da BG remover celobiose do meio reacional, evitando seu acúmulo, o qual poderia inibir a ação das endoglucanases e exoglucanases. Entretanto, observou-se uma redução da conversão de celulose após a segunda batelada (de 50% para 15-25%). De qualquer forma, esse trabalho mostra que a suplementação dos complexos enzimáticos comerciais com BG imobilizada é vantajosa, entretanto, a estabilidade da BG imobilizada ainda precisa ser melhorada.

Introdução e objetivos: β-glicosidases da família GH1 das glicosil-hidrolases possuem um dobramento do tipo barril (β/α)8. Propõe-se que proteínas com este dobramento, que usualmente classificam-se como tendo um único domínio, na verdade são compostas por dois domínios, cada um deles correspondendo a um \"meio barril\" (β/α)4. Assim, as proteínas com dobramento barril (β/α)8 seriam provenientes de uma duplicação e fusão gênica de um ancestral \"meio barril\" (β/α)4. O objetivo geral deste projeto é investigar a existência de dois domínios (β/α)4, as metades N- e C-terminal, na estrutura (β/α)8 barril da β-glicosidase A de Thermotoga maritima (bglTm) e β-glicosidase B de Paenibacillus polymyxa (bglB) por meio de análise da desnaturação térmica e química dessas enzimas. Resultados: Para atingir esse objetivo foram introduzidas mutações que rompem contatos não covalentes entre os supostos domínios destas β-glicosidases. Os segmentos de DNA que codificam as enzimas selvagem e mutantes foram clonados em plasmídeo de expressão pLATE51 e as enzimas recombinantes expressas em Escherichia coli BL21(DE3). Foram purificadas com sucesso as enzimas selvagens e duas mutantes de bglTm, denominadas T1 e T2, que possuem 2 e 4 mutações respectivamente em resíduos na interface inter-metades. Para confirmar o enovelamento destas proteínas recombinantes foi empregada a análise de estruturas secundárias por dicroísmo circular, também o espectro de fluorescência intrínseca de triptofano e sua supressão por acrilamida e finalmente foram determinados parâmetros cinéticos. Observou-se que não houve mudanças significativas na exposição dos triptofanos das proteínas recombinantes, sugerindo que se encontram enoveladas, o que está de acordo com a observação de que as proteínas recombinantes mantêm sua atividade catalítica. Já pela análise de dicroísmo circular concluiu-se que a mutante T1 possui dobramento semelhante à selvagem bglTm e que T2 apresenta diferenças significativas, sendo as porcentagens de α-hélice de 21%, 22% e 10% para bglTm, T1 e T2, respectivamente. Em seguida demonstrou-se que T1 mantém a termo estabilidade semelhante à selvagem, enquanto que T2 tem termo estabilidade reduzida, apresentando kobs de 0,3 min-1 em 80°C e de 0,06 min-1 em 75 °C.. O cálculo de Tm através de Differential Scanning Fluorimetry foi feito para bglB e T2 (42 e 81.7 °C respectivamente), enquanto que bglTm e T1 mantiveram-se estáveis na faixa de temperatura analisada (até 95°C). Na análise do efeito da temperatura sobre a estrutura das mutantes T1 e T2 não se observou nenhuma evidência da presença dos dois supostos domínios (β/α)4. A análise da desnaturação por cloreto de guanidina mostrou que o c50 diminuiu para T2 (2,4 M), mas não mostrou alteração para T1 (4,5 M) em comparação à bglTm (4,3 M). Coerentemente, a estabilidade da enzima selvagem e de T1 na ausência de desnaturante é a mesma (ΔGH2O = 5,2 kcal/mol), mas se reduziu para a T2 (ΔGH2O = 3,5 kcal/mol). Os cálculos do parâmetro m mostraram uma cooperatividade semelhante na desnaturação de bglTm, T1 e T2, não evidenciando independência entre os dois supostos domínios (β/α)4. Em conclusão, a análise estabilidade da β-glicosidase bglTm frente à temperatura e ao cloreto de guanidina não revelaram a presença dos domínios (β/α)4 que correspondem às metades N- e C-terminal desta β-glicosidase. / Introduction and Aims: β-glucosidases from the family GH1 of the glycosil-hidrolases presents a (β/α)8 barrel folding. These proteins are usually classified as single domain, however it has been alternatively proposed that they actually are formed by two \"half barrel\" (β/α)4. Thus, (β/α)8 barrel proteins had evolved from an \"half barrel\" ancestor that underwent a duplication-fusion event. The general goal of this project is the search for the two putative (β/α)4 domains, which form the N- and C-terminal ends of the β-glucosidase A from Thermotoga maritima (bglTm) and β-glucosidase B from Paenibacillus polymyxa (bglB), detecting their presence through the thermal and chemical stability of these (β/α)8 barrel proteins. Results: Site-directed mutagenesis was employed to replace residues forming non-covalent interaction between the putative (β/α)4 domains. DNA segments coding for bglB, bglTm and mutant bglTm were cloned into the pLATE51 expression vector and produced as recombinant proteins in E. coli BL21(DE3). The bglB, bglTm and two mutant bglTm, hereafter called T1 and T2, with 2 and 4 mutations respectively on residues in the interface between the protein halves, were purified. They were stable folded as shown by detecting their catalytic activity upon two different substrates and also by circular dichroism (CD) and tryptophan fluorescence analysis. Nevertheless, T2 showed a decrease in the α-helix content (10 %) in the CD analysis, whereas bglTm and T1 are similar (22 %). The wild-type bglTm and T1 are thermostable, whereas T2 was inactivated after pre-incubation at high temperature (kobs = 0,3 min-1 at 80 °C and 0,06 min-1 at 75 °C). The Differential Scanning Fluorimetry experiments revealed Tm of 42 e 81.7 °C for bglB and T2, respectively, whereas wild-type bglTm and mutant T1 did not showed any thermal transition up to 95 °C. Indeed, the analysis of the thermal stability of T1 and T2 did not reveal any evidence of the putative (β/α)4 domains. Following that, the analysis of the protein denaturation by guanidine hydrochloride showed that the c50 for T2 was reduced (2.4 M), whereas no modification was observed for bglTm and T1 (4,3 and 4,5 M, respectively). In agreement the stability of bglTm and T1 (ΔGH2O = 5,2 kcal/mol) is similar, but it was reduced for T2 (ΔGH2O = 3,5 kcal/mol). The m parameter showed a similar cooperative denaturation for wild-type bglTm and mutants T1 and T2, but no evidence of the independent unfolding of the putative (β/α)4 domains was found. Conclusion: In conclusion, the analysis of the thermal and chemical stability of the bglTm did not reveal the presence of the putative (β/α)4 domains that form the N- and C-terminal end of these β-glucosidase.

Made available in DSpace on 2016-08-16T18:18:42Z (GMT). No. of bitstreams: 1 TESE_TONICLEY ALEXANDRE DA SILVA.pdf: 1267921 bytes, checksum: 5198168103d56188dc3c8fa6fbffc2fb (MD5) Previous issue date: 2015-06-26 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / Diabetes mellitus is a chronic disorder affecting the metabolism of carbohydrates, fats and proteins, leading to hyperglycemia, due to reduced sensitivity, or insufficient insulin production. Plant species are often used by the population as a complementary treatment of diabetes among the most cited epecies is Anacardium occidentale L. This study evaluated the effect of hydroalcoholic extract of Anacardium occidentale L. and compounds on the α-amylase and α-glucosidase in vitro and the effects of A. occidentale on the production of insulin and blood glucose levels in murine diabetes. For this systematized up the information on scientific and ethnobotanical data on the anti-diabetes activity A. occidentale, considering the toxicity and pharmacological effects. We evaluated the effect of the aerial parts of A. occidentale and its major compounds (anacardic acid, shikimic, gallic and ellagic) on inhibition of α-glucosidase and α-amylase enzymes in vitro. Evaluated the effect of the extract of A. occidentale flowers in controlling type 2 diabetes, considering the production of glucose, insulin, and in vivo cytokines. To achieve the in vitro assays hydroalcoholic extracts of flowers were prepared (EFL), leaves (EF) and shell (EC) stem of A. occidentale, preparations containing compounds present in the extracts as well as mixtures of such compounds: anacardic acid (AA ), shikimic (AC), gallic (AG) and ellagic (AE) tested in various concentrations (0.5, 5, 50 and 500 mg/Kg). In vitro assays have evaluated the effect of extracts and compounds on the inhibition of α-amylase and human salivary α-glucosidase from Saccharomyces cerevisiae. To conduct in vivo testing diabetes was induced in male Swiss mice, three months old, with fructose and streptozotocin. The animals were divided into 6 groups of 5 animals each, treated with saline, metformin and EFL in concentrations of 0.5; 5; 50 and 500 mg/Kg evaluated by oral tolerance test glucose, glycemic variation, consumption of water and feed, changes in body weight, concentration of glycated hemoglobin, insulin, frutosaminas, triglycerides, total cholesterol and HDL and LDL fractions, production of cytokines IFN-, TNF-, IL-4 and IL-10. The in vitro results showed that the EFL showed the highest inhibitory effect on the activity of α-amylase and α-glucosidase than the other extracts enzymes even at low concentrations, possibly due to the presence of anacardic acid and ellagic acid, since these two compounds present in the crude extract, when evaluated alone, or in combination with shikimic acid showed a similar effect to the EFL. In vivo the EFL reduced the glycemic peak, consumption of water and feed, the concentration of glycated hemoglobin, frutosaminas, triglycerides, total cholesterol and LDL. On the other hand, occurred in the same group increase in the HDL fraction, and increase in the concentration of anti-inflammatory cytokines IL-10 and IL-4. Together, the results indicate that the EFL with proven effectiveness in controlling diabetes mellitus due to enzyme inhibition, and reducing glucose homeostasis in insulin production, possibly due to the increase in peripheral sensitivity. / A diabetes mellitus é um distúrbio crônico, que afeta o metabolismo de carboidratos, gorduras e proteínas, ocasionando hiperglicemia, em virtude de redução da sensibilidade ou da produção insuficiente de insulina. Espécies vegetais são frequentemente utilizadas pela população como tratamento complementar da diabetes entre as epecies mais citadas está Anacardium occidentale L. O presente estudo avaliou o efeito do extrato hidroalcoólico de Anacardium occidentale L. e de compostos sobre as enzimas α-amilase e α-glicosidase in vitro e os efeitos de A. occidentale sobre a produção de insulina e a glicemia sanguinea no diabetes murino. Para isso sistematizou-se as informações sobre dados etnobotânicos e científicos acerca da atividade anti-diabetes de A. occidentale, considerando a toxicidade e os efeitos farmacológicos. Avaliou-se o efeito das partes aéreas de A. occidentale e de seus compostos majoritários (ácidos anacárdico, chiquímico, gálico e elágico), sobre inibição das enzimas α-glicosidase e α-amilase in vitro. E avaliou-se o efeito do extrato das flores de A. occidentale no controle da diabetes tipo 2, considerando a produção de glicose, insulina e citocinas in vivo. Pra realização dos ensaios in vitro foram preparados os extratos hidroalcoólicos das flores (EFL), folhas (EF) e casca (EC) do caule de A. occidentale e preparações com compostos presentes nos extratos, bem como misturas desses compostos: ácido anacárdico (AA), chiquímico (AC), gálico (AG) e elágico (AE) testados em varias concentrações (0,5; 5; 50 e 500 mg/Kg). Os ensaios in vitro avaliaram a ação dos extratos e dos compostos sobre a inibição da α-amilase de saliva humana e a α-glicosidase de Saccharomyces cerevisiae. Para realização dos ensaios in vivo foi induzida diabetes em camundongos Swiss machos, três meses de idade, com frutose e estreptozotocina. Os animais foram divididos em 6 grupos com 5 animais cada, tratados com salina, metformina e EFL nas concentrações de 0,5; 5; 50 e 500 mg/Kg avaliados por do teste de tolerância oral a glicose, variação glicêmica, consumo de água e ração, variação ponderal, concentração de hemoglobina glicada, insulina, frutosaminas, triglicérides, colesterol total e frações HDL e LDL, produção das citocinas IFN-, TNF-, IL-4 e IL-10. Os resultados in vitro mostraram que o EFL apresentou o maior efeito inibitório da atividade das enzimas α-amilase e α-glicosidase do que os demais extratos, mesmo nas menores concentrações, possivelmente devido a presença do ácido anacárdico e elágico, já que estes dois compostos presentes no extrato bruto, quando avaliados isoladamente, ou em associação com o ácido chiquímico, apresentaram efeito similar ao EFL. Na avaliação in vivo o EFL reduziu o pico glicêmico, o consumo de água e de ração, a concentração de hemoglobina glicada, frutosaminas, triglicérides, colesterol total e fração LDL. Por outro lado, ocorreram nesse mesmo grupo aumento da fração HDL do colesterol e aumento na concentração de citocinas anti-inflamatórias IL-10 e IL-4. Em conjunto, os resultados indicam que o EFL apresenta ação efetiva no controle da diabetes mellitus, devido a inibição enzimática, regulação glicêmica e redução da produção de insulina, possivelmente em decorrência do aumento da sensibilidade periférica.

Na última década, a análise da estrutura proteica baseada em teoria de redes/grafos tem emergido. A abstração da estrutura tridimensional proteica em forma de uma rede, leva em consideração os resíduos de aminoácidos e suas interações através do espaço, e apresenta um conjunto de conexões e propriedades mais complexas do que aquelas visualizadas apenas com a estrutura covalente. A análise da estrutura proteica identificou que as proteínas pertencem às redes de classes de \"mundo pequeno\" (small-world) e \"sem escala\" (scale-free), o que significa que seus resíduos de aminoácidos são altamente agregados e que existem poucas conexões entre 2 resíduos quaisquer da proteína. A identificação dos resíduos com alto grau de conexão, chamados centrais (\"resíduos hubs\"), é feita pela determinação do caminho mais curto que conecta um dado resíduo aos demais compreendidos nesta rede. A remoção destes resíduos centrais (hubs) afeta a integridade da rede de forma mais contundente diferentemente da remoção de resíduos que não são centrais. Até o momento estes \"resíduos hubs\" ainda não foram experimentalmente correlacionados com as propriedades enzimáticas de proteínas. Para tal finalidade, a estrutura terciária de uma β-glicosidase de Spodoptera frugiperda (Sfβgli) foi analisada como uma rede. Após calcular-se os caminhos médios entre todos os pares de aminoácidos da β-glicosidase, encontrou-se 11 resíduos centrais (\"resíduos hubs\"). Alinhamento de sequências e comparações estruturais indicaram alta conservação destes \"resíduos hubs\". Nosso objetivo foi produzir esta β-glicosidase mutando-se a maioria dos \"resíduos hubs\" e 3 aminoácidos não centrais (\"não hubs\"), expressar estes mutantes em E. coli, determinar suas propriedades enzimáticas como atividade catalítica e preferência pelo substrato e verificar a estabilidade destes mutantes em experimentos de inativação térmica. Os resultados obtidos sugerem que mutações nos \"resíduos hubs\" não afetam as propriedades catalíticas, contudo as enzimas com mutações nos \"resíduos hubs\" apresentaram uma menor estabilidade térmica. Estes resultados sugeriram que os \"resíduos hubs\" são relevantes na difusão da energia cinética (vibração) introduzida na estrutura desta β-glicosidase pelo seu aquecimento / In recent years, graph-theoretic approaches have established that protein structures can be modeled as complex networks of interacting residues. Proteins structures can be represented as small-world and scale-free networks that are usually highly clustered with few links connecting any pair of nodes. The identification of nodes with high connection degrees, called hubs, is made by determining the shortest path linking one amino acid to the further nodes comprising the network. Targeted removal of the hubs has greater affect on the integrity of the network structure in contrast to a random removal of amino acid residues comprising the network. Nevertheless these hubs had not previously been correlated with enzymatic properties. The tertiary structure of β-glycosidase from S. furgiperda (Sfβgly) was analyzed as a network. After calculating the averaged paths between all pairs of amino acid residues of Sfgly, we defined 11 hubs, which have the highest centrality on the network. Sequence alignment and structural comparison showed that these hubs residue are conserved among β-glycosidases. Our goal was to mutate most hubs and 3 ´non-hubs´ residues from Sfβgly, express these mutant enzymes in E. coli, test their enzymatic properties as catalytic efficiency and substrate preference, and verify the thermal stability of these mutants. The results implied that mutations in these hubs do not cause changes in catalytic properties although enzymes containing mutations in hubs showed lower thermal stability. Based on that, it was suggested that hub residues are important in the diffusion of kinetic energy (vibrations) introduced in the Sfβgly structure by heating

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2016-09-26T14:16:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Simone_Pacheco_Frutos da família Myrtaceae_Caracterização físico-química e potencial .pdf: 1364587 bytes, checksum: 6df19769efc9216d28d9614070c8e6af (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-26T18:34:47Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Simone_Pacheco_Frutos da família Myrtaceae_Caracterização físico-química e potencial .pdf: 1364587 bytes, checksum: 6df19769efc9216d28d9614070c8e6af (MD5) / Made available in DSpace on 2016-09-26T18:34:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Simone_Pacheco_Frutos da família Myrtaceae_Caracterização físico-química e potencial .pdf: 1364587 bytes, checksum: 6df19769efc9216d28d9614070c8e6af (MD5) Previous issue date: 2015-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / As espécies vegetais Campomanesia xanthocarpa (guabiroba), Eugenia uniflora (pitanga), Eugenia pyriformis (uvaia), Psidium cattleianum (araçá) e Syzygium cumini (jambolão) estão presentes na Floresta Atlântica e são utilizadas pela população, para tratar diversas patologias, especialmente o diabetes melito tipo 2. Entretanto, a eficácia destes tratamentos e o mecanismo envolvido ainda não foram totalmente elucidados. Neste contexto, o presente estudo teve como objetivo principal avaliar o potencial dos compostos naturais destes frutos em inibir as enzimas α-amilase e α- glicosidase que estão envolvidas no metabolismo de carboidratos. Estes frutos também foram avaliados físico-quimicamente, realizando-se dentre outras análises a quantificação dos compostos fenólicos totais e a determinação da atividade antioxidante (métodos ABTS e DPPH). Os extratos metanólicos de P. cattleianum (acesso 44), S. cumini e E. pyriformis (acessos 11 e 15) inibiram de forma significativa a atividade da α-amilase. Os extratos metanólicos de P. cattleianum (acessos 44 e 87) também inibiram a atividade da α-glicosidase, utilizando-se os substratos maltose e sacarose. Em virtude da atividade antioxidante, da elevada quantidade de compostos fenólicos e da capacidade de inibição das enzimas digestivas do metabolismo de carboidratos, os frutos de P. cattleianum (acessos 44 e 87), S. cumini e E. pyriformis (acessos 11 e 15) podem apresentar potencial uso no manejo da hiperglicemia pós-prandial. / Campomanesia xanthocarpa (guabiroba), Eugenia uniflora (pitanga), Eugenia pyriformis (uvaia), Psidium cattleianum (araçá) and Syzygium cumini (jambolão) grow in the Brazilian Atlantic Forest and their fruits are commonly used as medicine to treat diseases related to carbohydrate metabolism, such as diabetes. The effectiveness of these treatments has not been demonstrated neither the biochemical mechanism involved. Therefore this study was devised to evaluate the potential of natural compounds of these fruits to inhibit key enzymes α-amylase and α- glucosidase involved in the carbohydrate metabolism. The fruits were also subjected to physicochemical characterization, quantification of phenolics compounds and antioxidant activity (ABTS and DPPH methods). The methanolic extracts of P. cattleianum (access 44), S. cumini, E. pyriformis (accesses 11 and 15) distinctively inhibited α-amylase activity. The methanolic extracts of P. cattleianum. (accesses 44 and 87) also inhibited α-glycosidase activity, with either maltose or sucrose as substrate. By having antioxidant activities, a fairly content of phenolic compounds, and capacity to inhibit carbohydrate digestive enzymes, P. cattleianum (access 44 and 87), S. cumini and E. pyriformis (accesses 11 and 15) could be good candidates to be used in the management of postprandial hyperglycemia.