Intracellular protein degradation is required for maintaining the cellular proteome and regulating cellular processes. This pathway involves proximal ATP-dependent proteases that unfold and translocate proteins targeted for degradation into catalytic chambers. The large peptides produced are further cleaved by ATP independent endopeptidases, aminopeptidases and carboxypeptidases to release free amino acids. Lon and Clp are the key ATP-dependent proteases in prokaryotes and 26S proteasomes in eukayotes. In general, enzymes involved in the distal processing of peptides are ATP-independent, display greater redundancy and their orthologs are present in most organisms. The aim of the present study was to generate biochemical and functional insights on the ATP-independent enzyme, Peptidase N (PepN), which belongs to the M1 family. Previous studies in our laboratory identified Escherichia Coli PepN, to harbor both amino and endopeptidase activitities. In addition, it is responsible for the cleavage of majority of aminopeptidase substrates in E. Coli and is known to be involved in Sodium salicylate(NaSal)-induced stress. The present study consists of four parts. First, intracellular proteolysis plays an important role for virulence in pathogens. Therefore, it becomes important to study the biochemical properties and roles of enzymes involved in protein degradation. In this direction, a study was initiated to characterize the biochemical properties of Peptidase N from Salmonella enterica serovar Typhimurium(S. typhimurium). To study the contribution of PepN to the overall cystosolic protein degradation in S.typhimurium, a targeted deletion in pepN was generated. Cystosolic lysates of S. typhimurium wild type(WT) and ΔpepN strains were examined for their ability to cleave a panel of aminopeptidase and endopeptidase substrates. The ΔpepN strain displayed greatly reduced cleavage of nine out of a total of thirteen exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. S. typhimurium PepN also cleaved the endopeptidase substrate Suc-LLVY-AMC, similar to E. Coli PepN. To understand the physiological role of PepN, WT and ΔpepN were subjected to different stress conditions. During nutritional downshift in combination with high temperature stress, the growth of ΔpepN was significantly reduced compared to WT. Importantly, the PepN overexpressing strains grew better than WT, demonstrating an enhanced ability to overcome this stress combination. The above study clearly underscores the importance of PepN, to play distinct roles during stress. The significance of this study lies in understanding the biochemical and functional properties of a M1 family member from a pathogenic organism. Second, peptidases belonging to the M1 family are widely distributed with orthologs found across different kingdoms. The key amino acids in the catalytic domain are conserved in this family. However, amino acids present in the C-termini are variable and the three available crystal structures of M1 family members display distint differences in organization of this domain. To investigate the functional role of C-termini, progressive deletions were generated in PepN from E.Coli and Tricorn interacting factor F2 from Thermoplasma acidophilum(F2). Catalytic activity was partially reduced inPepN lacking four aa from C-terminus (PepNΔC4) whereas it is greatly reduced in F2 lacking ten amino acids from C-terminus(F2ΔC10) or eleven amino acids from PepN (PepNΔC11). To understand the mechanistic reasons involved, biochemical and biophysical studies were performed on purified WT and C-termini deleted proteins. Increased binding to 8-amino- 1- naphthalene sulphonic acid (ANS) was observed for all C-termini deleted proteins revealing greater numbers of surface exposed hydrophobic amino acids. Further, trypsin sensitivity studies demonstrated that mutant proteins were more sensitive compared to WT. Notably, expression of PepNΔC4, but not PepNΔC11, in E ColiΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating a physiological role for the C-terminus. Together, these studies reveal involvement of distal amino acids in the C-termini of two distant M1 family members in repressing the exposure of apolar residues and enhancing enzyme function. Third, the crystal structure of E. coliPepN displayed the presence of Zn2+. To study the role of metal cofactor, apo-PepN was isolated by chelating the holoenzyme with 1,10-phenanthroline. Among different metals tested, only Zn2+ rescued the greatly reduced catalytic activity of the apo-PepN. Further confirmatory studies were performed using pepN mutants in the conserved GXMEN and HEXXH motifs. No major structural differences were observed in purified mutants(E264A, H297A, and E298A) using circular dichroism (CD) and intrinsic fluorescence studies; however, they lacked catalytic activity. These studies clearly demonstrate that Zn2+ was essential for catalysis but not for the overall structural integrity of PepN. Estimation of the Zn2+ content by atomic absorption spectrometry demonstrated that the WT contained one molecule of zinc per molecule of enzyme. Similar results were obtained in purified proteins of E264A and E298A. residues involved in catalysis. However the Zn2+ amount was greatly reduced in H297A, which is involved in Zn2+ binding. Further, the in vivo role of metal cofactor and catalyis were studied during two established stress conditions. Over expression of the mutants, unlike WT, was unable to rescue the growth of ΔpepN during nutritional down shift and high temperature stress. These results demonstrate that E264, H297 and E298 were required for PepN function during nutritional downshift and high temperature stress. However during NaSal-induced stress condition, overexpression of WT or mutants reduced growth of ΔpepN, demonstrating that PepN function was independent of catalytic activity or metal cofactor. Further studies identified the YL motif, which is conserved in all members of the M1 family, to play a role during NaSal-induced stress. Over expression of Y185F or L186Q did not modulate catalytic activity although growth reduction of ΔpepN in the presence of NaSal was compromised. To understand the mechanisms by which the YL motif plays a role during this condition, Y185F and L186Q mutant proteins were purified. In vitro, both mutant proteins were found to aggregate at a lower temperature and their catalytic activities were more sensitive to temperature, compared to WT. Steady state analysis of WT, Y185F and L186Q were performed to study the modulation of PepN amount during stress conditions. Steady state amounts of Y185F and L186Q mutant proteins were greatly decreased compared to WT, during NaSal-induced stress. Most likely, the lowered amounts of Y185F and L186Q mutant proteins contribute to growth advantage during NaSal-induced stress. Thus, the YL motif in E. Coli PepN reduces protein aggregation and enhances the structural integrity of PepN during selective stress conditions in vivo. In summary, this study clearly identifies metal cofactor and peptidase-dependent and –independent motifs to play distinct functional roles in PepN. Fourth, the crystal structures of known M1 family members have shown that the catalytic domain and mechanism of action are similar. To identify novel residues that may modulate the catalytic activity of PepN, multiple sequence alignment of important M1 family members were performed. The alignment identified a subset of M1 family members, including PepN, containing an aspargine residue which is present two amino acids before glycine in the GAMEN motif. A closer investigation of thecrystal structure of PepN revealed an interaction between N259(Catalytic domain) with Q821 (C-terminal domain). To understand the functional role of this interaction, site-specific mutants were generated: N259D, Q821E and a double mutant, N259D & Q821E. Spectroscopic studies did not reveal any significant differences with respect to global structure or protein stability between purified WT and mutant enzymes. Also, binding to substrates by mutant enzymes was not affected as judged by Km values. However, the Kcat of PepN containing N259D or Q821E was enhanced with respect to both aminopeptidase and endopeptidase substrates. On the other hand, there was significant decrease in the catalytic activity of the double mutant. Modeling studies demonstrate that the N259-Q821 interaction is located in the vicinity of residues important for catalysis in PepN and specific alterations in this interaction may affect the compactness of the catalytic domain. In summary, this study provides a functional role for the N259-Q821 interaction in modulating the catalytic activity of PepN. Mammalian orthologs of M1 family members play important roles in different physiological processes, e.g. angiogenesis, blood pressure, inflammation, MHC class I antigen presentation etc. PepN is a well characterized M1 family member of microbial origin. The present study on E. Coli PepN provides new knowledge on the roles of: a) distal C-terminal amino acids in repressing exposed hydrophobic amino acids; b) the conserved YL motif during NaSal-induced stress condition; c) the N259 and Q821 interaction in modulating enzymatic activity. The implications of these results on other members of the M1 family are discussed.

Analysis of the genomic sequences of Streptococcus mutans UA159 and Group B Streptococcus (GBS) strains Streptococcus agalactiae NEM316 and S. agalactiae 2603V/R indicated the presence of two putative collagenase genes in each organism. smcol1 from S. mutans was previously cloned and analyzed and the results indicated that the enzyme belonged to the U32 family of collagenases/peptidases. This enzyme shares homology with the prtC of Porphyromonas gingivalis, one of the principal examples of the U32 family of peptidases. Considering the potential role of these enzymes in the pathogenicity of P. gingivalis (periodontitis or gum disease), GBS (premature rupture of the amniochorionic membrane) and S. mutans (dental root decay), it is necessary to study these enzymes and establish their role in the virulence of these organisms. Toward this goal the present study has focused on cloning collagenase 2 (smcol2) from S. mutans and cloning collagenase 1 (gbscol1), and collagenase 2 (gbscol2), from GBS. The information obtained will contribute to a further understanding of the U32 peptidase family.

African animal trypanosomosis is a devastating disease affecting livestock mainly found in sub-Saharan Africa. This disease is known as nagana and is transmitted by the trypanosome parasite from the tsetse fly vector to a mammalian host. There are three African trypanosomes namely Trypanosoma vivax, T. congolense and T. brucei brucei that are the causative agents responsible for this disease in African cattle. This disease is serious since it not only affects livestock but also has a negative impact on the sub-Saharan African economy. There is, therefore, a great demand for better control methods of the disease and suitable diagnostic methods. Current control measures such as the use of trypanocidal drugs, tsetse fly eradication methods and trypanotolerant cattle have become inadequate. The defence mechanism of the trypanosome to continuously change its surface coat by a process of antigenic variation has made it impossible to produce a suitable vaccine. Therefore, chemotherapy is still one of the key approaches for control of this wasting disease. The long existence of the current trypanocidal drugs has allowed the development of drug resistance. The development of new chemotherapeutic drugs is focused on targeting the pathogenic factors such as parasite cysteine peptidases that contribute to the disease. Vivapain is the main cysteine peptidase of T. vivax and shares high sequence identity with congopain, the main cysteine peptidase of T. congolense, which was previously shown to be a pathogenic factor contributing to trypanosomosis. Vivapain, thus, has potential as a target for chemotherapeutic drug design. Hence, the first part of this study involved the recombinant expression and enzymatic characterisation of vivapain for future production of new synthetic inhibitors for the use in new trypanocidal drugs. The catalytic domain of vivapain (Vp) was recombinantly expressed in the Pichia pastoris yeast expression system and enzymatically characterised. The main finding from this study was that Vp was only able to hydrolyse a substrate if the P2 position was occupied by either a hydrophobic Phe or Leu residue. Vp was also found to be active close to physiological pH and was inhibited by the reversible cysteine peptidases, leupeptin, antipain and chymostatin and the irreversible cysteine peptidases L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64), iodoacetic acid (IAA) and iodoacetamide (IAN). A further important aspect of controlling trypanosomosis is the diagnosis of the disease. Clinical, parasitological, molecular and serological techniques have been applied and used to diagnose trypanosomosis. One of the most promising serological techniques has proven to be the enzyme-linked immunosorbent assay (ELISA), more specifically the antibody and antigen detection ELISAs. The main requirement for this technique is a readily available and reproducible antigen such as that produced by recombinant expression. While there are recombinant antigens that are available to be used to detect T. congolense, T. brucei brucei and even T. evansi infections, there are none available to detect T. vivax infections. In the second part of this study, a mutant inactive full length form of vivapain (FLVp) was expressed in a bacterial expression system for the detection of T. vivax infections. Antibodies against this antigen were produced in both chickens and mice. Both the chicken IgY and mice sera were able to detect the recombinant FLVp in western blots. The mice sera were also able to detect native vivapain in a T. vivax lysate, which is very promising for future use of the FLVp antigen and the corresponding antibodies in diagnosis of T. vivax infections in sera of infected animals. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Kyoto University (京都大学) / 0048 / 新制・論文博士 / 博士(工学) / 乙第12200号 / 論工博第3989号 / 新制||工||1438(附属図書館) / 26272 / UT51-2008-C970 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生 / 学位規則第4条第2項該当

Made available in DSpace on 2016-06-02T20:21:34Z (GMT). No. of bitstreams: 1 6066.pdf: 3990215 bytes, checksum: 05550a0cc5074637afaa52685749e202 (MD5) Previous issue date: 2014-02-25 / Financiadora de Estudos e Projetos / Brazil is a major exporter of oranges in the world, with the state of São Paulo, the largest citrus producer in the country. In the last years plants of the genus citrus have been strongly affected by various diseases and among them, the disease Huanglongbing (HLB) have occupied prominent role and importance due to the devastating damage caused to citrus production worldwide. Caused by Candidatus Liberibacter spp., a bacterium that colonizes and blocks the conductive vessels of elaborated sap (phloem) and transmitted by the vector, hemiptera, the psyllid Diaphorina citri. In this context, it is necessary to search for strategies to control the disease in citrus orchards. In order to identify a cysteine peptidase D. citri, in this work, the identification, recombinant expression and characterization of a cysteine peptidase D. citri, the type (cathepsin B) called DiaciCATHB was performed. The identification of an ORF for cysteine peptidase was performed using the database of the transcriptome of D. citri. Recombinant expression was performed in Pichia pastoris yeast cells. For activation of the recombinant enzyme important factors were observed, acidic conditions and incubation in suitable temperature and time. Kinetic characterization of the enzyme was verified by hydrolysis of the synthetic substrate Z-Phe-Arg-MCA, resulting in a (km) of 15.7 mM. Inhibition of the enzymatic activity tests were conducted using the recombinant inhibitor of cysteine peptidases CaneCPI-4, resulting in the inhibition constant (Ki) of 0.05 nM. The analysis of gene expression DiaciCATHB demonstrated that gene expression occurs in all stages of insect development, there is however a significant difference in expression level. According to the developmental stages of the insect D. citri, the expression level gradually increases DiaciCATHB, being higher in nymph and adult stages. The present results do not prove that the location and function performed by the enzyme DiaciCATHB, but regardless, the cysteine peptidase in this study has the potential to become an important target for use in future studies on insect control. / O Brasil é um dos maiores exportadores de laranjas do mundo, sendo o estado de São Paulo o maior produtor de citros no país. Nos últimos anos as plantas do gênero citrus têm sido fortemente afetadas por diversas patologias e dentre estas, a doença Huanglongbing (HLB) têm ocupado papel de destaque e importância devido aos danos devastadores causados a citricultura mundial. Causada pela Candidatus Liberibacter spp., uma bactéria que coloniza e obstrui os vasos condutores da seiva elaborada (floema) e transmitida pelo vetor, hemíptero, o psilídeo Diaphorina citri. Neste contexto, se faz necessário à busca por estratégias de controle para a doença nos pomares de citros. Com a finalidade de identificar uma cisteíno peptidase de D. citri, no presente trabalho, foi realizada a identificação, expressão recombinante e caracterização de uma cisteíno peptidase de D. citri, do tipo (catepsina B) denominada DiaciCATHB. A identificação de uma ORF para cisteíno peptidase de foi realizada através do banco de dados do transcriptoma de D. citri. A expressão recombinante foi realizada em células da levedura Pichia pastoris. Para a ativação da enzima recombinante fatores importantes foram observados, como condições ácidas e incubação em temperatura e tempo adequados. A caracterização cinética da enzima foi verificada pela hidrólise do substrato sintético Z-Phe-Arg-MCA, resultando em um (Km) de 15,7 μM. Testes de inibição da atividade enzimática foram realizados utilizando a o inibidor de cisteíno peptidases recombinante CaneCPI-4, resultando na constante de inibição (Ki) de 0,05 nM. A análise de expressão do gene DiaciCATHB demonstrou que a expressão do gene ocorre em todas as fases de desenvolvimento do inseto, havendo no entanto, uma diferença significativa no nível de expressão. De acordo com as fases de desenvolvimento do inseto D. citri, o nível de expressão de DiaciCATHB aumenta gradualmente, sendo maior nas fases de ninfa e adulto. Os resultados obtidos nesse trabalho não comprovam qual a localização e função exercida pela enzima DiaciCATHB, porém, independente disso, a cisteíno peptidase em estudo apresenta potencial para tornar-se um importante alvo para a utilização em futuros estudos no controle do inseto.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-22Bitstream added on 2014-06-13T20:35:41Z : No. of bitstreams: 1 goncalves_an_me_sjrp.pdf: 651485 bytes, checksum: f6bb7160fc2afa8aae9c65f75fff26da (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Há um grande interesse no mercado de enzimas para descobertas de novas fontes para prospecção, tendo em vista que as enzimas de origem animal ou vegetal não suprem totalmente a demanda mundial. Desta forma busca-se não apenas novas fontes, mas também materiais fermentescíveis de baixo custo, assim agregando valor ao produto final. Neste trabalho investigou-se a produção de peptidase utilizando o fungo Aspergillus flavus, através de dois processos biofermentativos, sólido e submerso, usando farelo de trigo e meio complexo, respectivamente. Alguns parâmetros foram analisados para a obtenção de uma melhor produção. Para o bioprocesso fermentativo sólido foi investigado a influência da concentração de esporos, suplementação do meio com fontes adicionais de nitrogênio, temperatura e tempo. Obtivemos para essas variações a melhores condições quando o fungo era fermentado somente em farelo de trigo, sem suplementação de fontes adicionais de nitrogênio, com a proporção de 2,5x 10 6 esporos/ 5g meio a 30°C e em 96 horas de fermentação. Para o bioprocesso fermentativo submerso avaliamos a influência da concentração de esporos, suplementação do meio com fontes de carbono e com caseína, temperatura, pH e tempo. Obtivemos como melhores condições quando o fungo era fermentado em meio sem suplementação de fonte de carbono adicional, com 0,5% de caseína, 5x10 5 esporos/mL de meio a 30°C em pH 8 por 168 horas. Os extratos enzimáticos de ambos bioprocessos foram caracterizados bioquimicamente avaliando desta forma: pH ótimo, temperatura ótima e classe da peptidase. A partir desses dados foram realizados métodos cromatográficos para obtenção da peptidase pura e com essa amostra realizou-se a caracterização bioquímica funcional e a determinação dos... / There is a great interest by enzyme’s market on discovery of new sources for prospecting, considering that the enzymes from animal or plant do not fully supply the global demand. Therefore, the research of new sources, is not only of new sources, but also fermentable material with low cost are also required, in order to add value to the final product. In this study it was investigated the production of peptidase using the fungus Aspergillus flavus, through two processes, solid and submerged fermentation, using wheat bran and complex media, respectively. Some parameters were analyzed to obtain the best production. In solid fermentation bioprocess were investigated the influence of spore concentration of supplementation of the medium with additional sources of nitrogen, temperature and time. We obtained for these variations the best conditions when the fungus was fermented wheat bran only, without supplementation of additional sources of nitrogen, with a density of 2.5 x 10 6 spores / 5 g medium at 30 ° C and 96 hours of fermentation. In the submerged fermentation bioprocess were evaluated the effect of spore concentration supplementation of the medium with carbon sources and with casein, temperature, pH and time. We obtained as best conditions when the fungus was fermented in medium without supplemental carbon source added with 0.5% casein, 5x10 5 spores / ml medium at 30°C at pH 8 for 168 hours. The enzymatic extracts obtained by these bioprocesses were biochemically characterized by testing optimum pH, optimum temperature and class of peptidase. From these data it was obtained a pure peptidase by using chromatographic methods and this sample was used for functional biochemical characterization and determination of kinetic parameters, using the peptide substrate of FRET. The fungus Aspergillus flavus... (Complete abstract click electronic access below)

Made available in DSpace on 2016-06-02T19:02:40Z (GMT). No. of bitstreams: 1 3427.pdf: 6177352 bytes, checksum: f9f6df521c16815add3a5b1dba3f6753 (MD5) Previous issue date: 2009-04-30 / Financiadora de Estudos e Projetos / Cystatins are proteins that inhibit specifically cysteine peptidases. The Canecystatin 1 gene which codifies a protein containing 106 amino acids residues was identified in sugarcane and possesses significant similarity with Oryzacystatin, a cystatin from rice. In order to obtain a cystatin with improved activity, direct evolution experiments were carried out. A DNA shuffling library was constructed using these two cystatins. One clone named A10PL3 obtained from these shuffled cystatins was selected, expressed in E.coli, purified and an analyzed by activity assays. These results showed that the activity of hybrid protein A10PL3 increased, in particular regarding its inhibitory activity on cathepsin B compared with its two precursors. The present study aimed to revert the changes of individual clone A10PL3 through site-directed mutations, generating three mutant cystatins: Mutant I (Thr17Ile), Mutant II (Gln 84Leu) and Mutant III (Thr17Ile); (Gln84Leu). Assays for inhibitory activity against the human cathepsins B and L were performed. Structural studies were also made by means of molecular modeling of proteins by homology that were enabled to understand the molecular mechanisms related to improvement of the inhibitory activity of these cystatins. These studies therefore corroborate with the observed data previously which demonstrated the improvement of specific protein A10PL3 in cathepsin B inhibition (Ki 16 nM) in relation to their parents. The mutants I, II and III, did not present improvement in inhibitory activity against cathepsin B. The structural studies revealed that the mutations performed on cystatin A10PL3 destabilized the hydrophobic core making it more flexible, thus increasing the inhibitory activity on cathepsin B. The absence of interactions underlying the hydrophobic core resulted in a trend of lower solubility, probably due to their inability to adopt a compact formation, which resulted in the exposure of some residues which are part of that core, which can lead to aggregation and also contribute to increasing the flexibility of cystatin, influencing their inhibitory activity. / Cistatinas são proteínas que inibem especificamente cisteíno peptidases. A canacistatina 1, uma proteína com 106 resíduos de aminoácidos, apresenta grande similaridade com a proteína Orizacistatina 1, uma cistatina de arroz. Com o objetivo de se obter uma cistatina com a atividade inibitória melhorada, experimentos de evolução molecular direta de proteínas foram realizados, através da construção de uma biblioteca de DNA shuffling usando estas duas cistatinas. Um clone denominado A10PL3 foi selecionado, expresso, purificado e submetido a ensaios de inibição de atividade enzimática. Foi demonstrado que a proteína A10PL3 exibiu aumento da atividade inibitória contra catepsina B humana em comparação com seus dois precursores. O presente estudo teve como objetivo reverter as alterações do clone A10PL3 através de mutações sítio-dirigidas, gerando mais três cistatinas mutantes: Mutante I (Thr17Ile), Mutante II (Gln 84 Leu) e Mutante III (Thr17Ile); (Gln84Leu). Ensaios de atividade inibitória dos mutantes contra as catepsinas humanas B e L foram realizados. Além disso, foram desenvolvidos estudos estruturais por meio de modelagem molecular de proteínas por homologia que permitiram a compreensão dos determinantes moleculares relacionados à melhoria da atividade inibitória destas cistatinas. Os resultados aqui apresentados são importantes, pois corroboram com os dados observados anteriormente, demonstrando a melhora na especificidade da atividade inibitória da proteína A10PL3 contra a catepsina B (Ki = 16 nM) em relação aos seus parentais. Os mutantes I, II e III não foram capazes de inibir a catepsina B. Os estudos estruturais revelaram que as mutações na cistatina A10PL3 desestabilizaram o núcleo hidrofóbico provavelmente tornando a região N-terminal da proteína mais flexível, influenciando a atividade inibitória contra a catepsina B. A desestabilização do núcleo hidrofóbico resultou na tendência de uma menor solubilidade, provavelmente devido à sua tendência de expor resíduos que fazem parte desse núcleo, o que pode levar à agregação e também contribuir para o aumento da flexibilidade da cistatina.

Orientador: Hamilton Cabral / Banca: Eleonora Cano Carmona / Banca: Maria de Lourdes Teixeira de Moraes Polizeli / Resumo: Há um grande interesse no mercado de enzimas para descobertas de novas fontes para prospecção, tendo em vista que as enzimas de origem animal ou vegetal não suprem totalmente a demanda mundial. Desta forma busca-se não apenas novas fontes, mas também materiais fermentescíveis de baixo custo, assim agregando valor ao produto final. Neste trabalho investigou-se a produção de peptidase utilizando o fungo Aspergillus flavus, através de dois processos biofermentativos, sólido e submerso, usando farelo de trigo e meio complexo, respectivamente. Alguns parâmetros foram analisados para a obtenção de uma melhor produção. Para o bioprocesso fermentativo sólido foi investigado a influência da concentração de esporos, suplementação do meio com fontes adicionais de nitrogênio, temperatura e tempo. Obtivemos para essas variações a melhores condições quando o fungo era fermentado somente em farelo de trigo, sem suplementação de fontes adicionais de nitrogênio, com a proporção de 2,5x 10 6 esporos/ 5g meio a 30°C e em 96 horas de fermentação. Para o bioprocesso fermentativo submerso avaliamos a influência da concentração de esporos, suplementação do meio com fontes de carbono e com caseína, temperatura, pH e tempo. Obtivemos como melhores condições quando o fungo era fermentado em meio sem suplementação de fonte de carbono adicional, com 0,5% de caseína, 5x10 5 esporos/mL de meio a 30°C em pH 8 por 168 horas. Os extratos enzimáticos de ambos bioprocessos foram caracterizados bioquimicamente avaliando desta forma: pH ótimo, temperatura ótima e classe da peptidase. A partir desses dados foram realizados métodos cromatográficos para obtenção da peptidase pura e com essa amostra realizou-se a caracterização bioquímica funcional e a determinação dos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: There is a great interest by enzyme's market on discovery of new sources for prospecting, considering that the enzymes from animal or plant do not fully supply the global demand. Therefore, the research of new sources, is not only of new sources, but also fermentable material with low cost are also required, in order to add value to the final product. In this study it was investigated the production of peptidase using the fungus Aspergillus flavus, through two processes, solid and submerged fermentation, using wheat bran and complex media, respectively. Some parameters were analyzed to obtain the best production. In solid fermentation bioprocess were investigated the influence of spore concentration of supplementation of the medium with additional sources of nitrogen, temperature and time. We obtained for these variations the best conditions when the fungus was fermented wheat bran only, without supplementation of additional sources of nitrogen, with a density of 2.5 x 10 6 spores / 5 g medium at 30 ° C and 96 hours of fermentation. In the submerged fermentation bioprocess were evaluated the effect of spore concentration supplementation of the medium with carbon sources and with casein, temperature, pH and time. We obtained as best conditions when the fungus was fermented in medium without supplemental carbon source added with 0.5% casein, 5x10 5 spores / ml medium at 30°C at pH 8 for 168 hours. The enzymatic extracts obtained by these bioprocesses were biochemically characterized by testing optimum pH, optimum temperature and class of peptidase. From these data it was obtained a pure peptidase by using chromatographic methods and this sample was used for functional biochemical characterization and determination of kinetic parameters, using the peptide substrate of FRET. The fungus Aspergillus flavus... (Complete abstract click electronic access below) / Mestre

Dipeptidyl peptidase-IV (DPP-IV) is a multifunctional transmembrane glycoprotein removing X-Pro dipeptide from the amino-terminus of the peptide chain. This evolutionary conserved sequence protects a number of biologically active peptides against the unspecific proteolytic cleavage. DPP-IV belongs into the group of "Dipeptidyl peptidase-IV Activity and/or Structure Homologues" (DASH), which, except the canonical DPP-IV, comprises fibroblast activation protein-α/seprase (FAP), and several other molecules. However several of DASH molecules are the enzymes, they execute at least some of their biological functions by non-proteolytic protein-protein interactions. DASH molecules, their substrates and binding partners are parts of "DASH system" which is affected in several pathological process including a cancer. Specifically DPP-IV and its closest structural relative FAP are among others expected to be involved in the development and progression of malignant glioma. In this study we showed the expression and colocalization of DPP-IV and FAP in glioma cells in vitro and in human high grade gliomas. In addition to the DPP-IV/FAP double positive transformed glial cells, we also identified a subpopulation of FAP positive mesenchymal cells located in the perivascular compartment. Moreover we described the...

Malignant gliomas belong to a highly aggressive class of tumours. Average patient survival time generally does not exceed 15 months. Despite intensive research, no therapeutic strategies capable of significantly extending the lives of those affected by the disease have been established to date. One potentially viable area of research into possible therapeutic targets in cancer therapy focuses on cell surface proteases. This group of proteins includes dipeptidyl peptidase IV (DPP-IV). Changes to DPP-IV expression have been established in the case of various cancer types including malignant gliomas. Understanding the role of DPP-IV in the biological processes of this malignant disease may thus contribute to the development of new therapeutic modalities. This thesis is therefore dedicated to establishing an orthotopic xenograft model as well as a genetically engineered model (GEM) of the glioma. The effects of DPP-IV on the growth of an experimental glioma were subsequently examined, as was the ratio of catalytic and non- catalytic mechanisms in this process. The GEM model was used for monitoring enzymatic activity and DPP-IV distribution. Non-invasive fluorescence imaging was employed in order to monitor the intraexperimental dynamics of experimental gliomas. The results indicated that DPP-IV...