A citocina IL-33 apresenta papel dual e está envolvida com a resolução ou progressão de inúmeras doenças, além disso, acredita-se que a via IL-33/ST2 esteja envolvida no equilíbrio entre a atividade de osteoclastos e osteoblastos. O objetivo deste estudo foi avaliar o papel do receptor ST2 no desenvolvimento e progressão de lesões periapicais experimentalmente induzidas em camundongos. Lesões periapicais foram induzidas em primeiros molares inferiores de camundongos WT e ST2 knockout (KO). Decorridos 7 e 14 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos e do volume por microtomografia computadorizada (&mu;CT); contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); expressão gênica de marcadores osteogênicos e osteoclastogênicos por q-PCR; quantificação de neutrófilos por ensaio de mieloperoxidases. Os linfonodos foram submetidos à análise da expressão dos fatores transcricionais T-bet, GATA-3, RORc e Foxp-3 por q-PCR. Análise estatística utilizada foi One-way ANOVA, seguido de pós-teste de Bonferroni. Aos 14 dias, observou-se maior extensão da lesão periapical em animais WT que em ST2KO (p<0,05). O tamanho da lesão nos animais ST2KO permaneceu igual em função do tempo. Foi observada maior quantidade de neutrófilos na lesão do grupo WT aos 7 dias, em comparação aos animais ST2KO (p<0,05). Na expressão de T-bet, GATA-3, RORc e Foxp-3 não foram observadas diferenças estatisticamente significantes. O número de osteoclastos contados nos animais ST2KO foi maior que o observado em WT aos 7dias e aos 14 dias (p<0,05). A expressão de Runx2 foi maior no grupo lesão dos animais ST2KO quando comparado a seu respectivo controle. Os outros marcadores relacionados com a formação óssea não apresentaram diferenças estatisticamente significantes. Dentre os marcadores relacionados com a reabsorção óssea, a catepsina K e o MMP-9 apresentaram maior expressão aos 14 dias, na lesão dos animais WT quando comparada à expressão na lesão dos animais ST2KO (p<0,05). Com base nos resultados obtidos no presente estudo, pode-se concluir que na ausência do receptor ST2 as lesões periapicais são menos extensas e embora em maior quantidade, os osteoclastos são menos ativos. Nossos resultados sugerem um importante papel da via IL-33/ST2 na ativação dos osteoclastos e desenvolvimento da lesão periapical. / The IL -33 cytokine presents a dual role and is involved either in the resolution and progression of many diseases. Furthermore, it is believed that this pathway is involved between osteoclast and osteoblast activity balance. The aim of this study was to evaluate the role of ST2 receptor in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and ST2 knockout (KO) mice. After 7 and 14 days, jaw samples were subjected to various analysis: determination of periapical lesions area by histology and volume by computed microtomography (&mu;CT); osteoclasts number by TRAP histoenzymology; osteogenic and osteoclastogenic markers expression by q-PCR; neutrophil quantification by myeloperoxidase activity. The expression of transcription factors T-bet, GATA-3, RORC and Foxp-3 in lymph nodes were analysed by q-PCR. Statistical analysis was done by One-way ANOVA and Bonferroni post-test. It was observed a greater extent in periapical lesions of WT compared to ST2KO animals at 14 days (p<0.05). There is no progression in the lesion of ST2KO mice with the time. A larger number of neutrophils in WT group was observed, compared to ST2KO mice evaluated at 7 days (p<0.05). The expression of T-bet, GATA-3, RORc and Foxp-3 were not statistically significant different among the groups. The number of osteoclasts in lesions of ST2KO animals were greater than the observed in WT, at 7 and 14 days (p<0.05). Although, other osteogenic markers showed no statistically significant difference, Runx2 expression in ST2KO was higher in lesion side compared to control side at 14 days. The markers related to bone resorption, cathepsin K and MMP-9, were significantly abrogated in the lesion side of ST2KO mice, at 14 days (p<0.05). Based on the results, it can be concluded that although larger amounts of osteoclast were counted in ST2KO, the lesion was less extensive and osteoclasts less active. It all suggests that the IL-33/ST2 pathway play an important role in osteoclasts activation and periapical lesion development.

La cathepsine D est une aspartyl protéase lysosomale surexprimée et hypersécrétée par les cellules épithéliales cancéreuses mammaires. C'est un marqueur de mauvais pronostic du cancer du sein. Elle stimule la prolifération des cellules cancéreuses, la croissance invasive des fibroblastes et la formation des métastases. Les travaux de l'équipe ont montré qu'elle peut agir indépendamment de son activité catalytique par interaction protéique. Le répresseur transcriptionnel Tricho-Rhino-Phalangeal Syndrome type 1, TRPS1, a été identifié comme un partenaire potentiel de la cathepsine D. Différentes études indiquent que des cystéines cathepsines peuvent être localisées au noyau et être protéolytiquement actives. Par exemple, la cystéine cathepsine L agit par protéolyse limitée sur le facteur de transcription CDP/Cux et sur l'histone H3 lorsqu'elle est localisée au noyau.Dans cette thèse nous avons étudié le rôle de la cathepsine D nucléaire dans des cellules cancéreuses mammaires. Nos résultats indiquent que la cathepsine D, comme TRPS1, est localisée au noyau et est associée à la chromatine dans les cellules positives aux récepteurs aux œstrogènes. De plus elle interagit de manière directe et endogène avec TRPS1 et participe à la régulation transcriptionnelle de PTHrP (parathyroïd hormone-related protein) un gène cible de TRPS1. Finalement nous avons identifié de nouveaux gènes co-régulés par TRPS1 et la cathepsine D dans le cancer du sein montrant que leur action n'est pas limitée à PTHrP. L'ensemble de ces résultats suggère que la cathepsine D est la première cathepsine identifiée comme un co-facteur transcriptionnel et que son rôle dans le cancer pourrait impliquer, en plus de ses activités extracellulaires, ses activités nucléaires. / Cathepsin D is a lysosomal aspartyl protease which is overexpressed and hyper-secreted by epithelial breast cancer cells. This is a poor prognosis factor in breast cancer. It stimulates cancer cell proliferation and metastasis formation. Team works have shown it can acts in an independent manner of its catalytic activity by protein interactions. The transcriptional repressor trichorhinophalangeal syndrome type 1 protein, TRPS1, has been identified as a new potential partner of Cathepsin D. Several studies indicate that cystein cathepsins can be localized in nucleus and are proteolytically actives. For example, the cystein Cathepsin L acts by limited proteolysis of the CDP/Cux transcription factor and histone H3 when located to the nucleus.During this thesis, we studied the role of nuclear Cathepsin D in breast cancer cells. Our results indicate that Cathepsin D, as TRPS1, is localized in nucleus and is associated with chromatin in estrogen-receptor positive breast cancer cells. Furthermore it interacts in a direct and endogenous manner with TRPS1 and participates to the transcriptional repression of PTHrP, parathyroïd hormone-related protein, a TRPS1 target gene. Finally, we identified new co-regulated genes by TRPS1 and Cathepsin D in breast cancer showing their action is not limited to PTHrP.Together, our results suggest that Cathepsin D is the first cathepsin identified as a transcriptional co-repressor and that its role in cancer may involve, in addition to its extracellular activities, its nuclear activities.

A partir da identificação de catepsinas L em ensaios in vitro e em zimogramas partimos para purificação desta enzima no inseto. A região V2 foi selecionada como fonte de obtenção da cisteína proteinase já que dentre os três ventrículos o segundo apresentou maior atividade específica. Após diversas tentativas de isolar esta proteinase, foi estabelecida uma marcha de purificação que envolvia em todas as etapas a participação de metil metanosulfonato (MMTS), o que inativa a proteinase evitando assim autólise ao longo do processo de purificação. A marcha consistiu de três passos cromatográficos (troca-iônica, filtração em gel e afinidade, nesta ordem) onde foi observada a presença de duas cisteína proteinases, cada uma apresentando respectivamente as seguintes massas moleculares 32 e 45 kDa (SDSPAGE). As duas cisteína proteinases possuem o mesmo pH ótimo igual a 6,3. Além disso, estas enzimas foram termicamente inativadas a 40 ºC segundo uma cinética de primeira ordem aparente, sugerindo a existência de apenas uma espécie molecular de cada enzima na preparação com meia vida de 5 minutos para cis 1 e 4,8 minutos para cis 2. Foi determinada a constante de dissociação entre enzimainibidor, onde foi observado os valores de 17,3 nM para cis 1 e 7,11 nM para cis 2 através da titulação por E-64. A eficiência de catálise cis 1 e cis 2 é maior para o substrato sintético Z-FR-MCA do que para Z-RR-MCA, indicando que tratava-se de catepsinas L. Com o intuito de descrever os mecanismos moleculares por trás dos fenômenos fisiológicos no intestino médio do Hemiptera Dysdercus peruvianus foi construída uma biblioteca de cDNA a partir de mRNA deste tecido. Utilizamos ESTs provenientes desta biblioteca com o objetivo de identificar genes transcritos relacionados com proteínas de transporte de glicose além de enzimas digestivas. Após o processamento das leituras, surgiram 1053 ESTs úteis. Montando estes ESTs por alinhamento de bases, foram produzidos 62 contíguos e 841 singletos, o que totaliza 903 seqüências únicas. Entre as seqüências homólogas encontradas as mais relevantes para o nosso estudo foram: &#946;-glicosidase (marcadora de membranas microvilares), &#945;-glicosidase (marcadora de membranas 8 perimicrovilares), aminopeptidase (espaço perimicrovilar), catepsina L (conteúdo de vesículas secretoras) e proteína transportadora de açúcar do tipo GLUT. Estas seqüências encontradas tiveram a sua transcrição específica (ou preferencial) averiguada por RT-PCR semiquantitativo nos diferentes tecidos do inseto estudado (intestino médio, túbulo de Malpighi, corpo gorduroso, glândula salivar, ventrículo 1, ventrículo 2 e ventrículo 3 do intestino médio). O transporte de glicose e água in vivo foi estudado. Para isso, os insetos foram alimentados com uma solução contendo glicose e corante não absorvível seguido de dissecação e análise do conteúdo luminal. O transporte de água e glicose foi inibido por floretina 0,2 mM (inibidor do uniportador GLUT) e por florizina 0,1 mM (inibidor do simportador SGLT) e ativado por K2SO4 50 mM. Isto sugere a presença do transportador do tipo uniportador (GLUT), e do simportador K+-glicose (SGLT), ambos co-transportando água. O transcriptoma revelou proteína homóloga a transportador GLUT cuja seqüência está parcialmente completa e foi analisada com ferramentas de bioinformática / After identification of cathepsins L in vitro assays and in zimograms we began to purify this enzyme in insect midgut. The region V2 was selected as a source of material for purifying a cysteine proteinase because it contains most of the activity of that proteinase. After several attempts to purify this proteinase, an effective process was developed that avoid autolysis with methyl methanethiosulfonate (MMTS), a sulfhydryl-reactive and reversible sulfonating reagent for thiol-containing molecules. The purify process was made by three chromatographic steps (anion-exchange column, gel filtration column and affinity column in this order), where two cysteine proteinase were purified, cys 1 and cys 2 with 32 and 45 kDa (SDS-PAGE). The two cysteine proteinases have the same pH optimum of 6.3. Besides that, these enzymes were thermicaly inactivated following apparent first-order kinetics with a half-life of 5 min (cys1) and 4.8 min (cys2) at 40 ºC. Both Cys are inhibited by E-64 with a KD of 17.3 nM (Cys 1) and 7.11 nM (Cys2). Both Cys are more active on Z-FR-MCA than on Z-RR-MCA, suggesting they are cathepsins-L. With purpose of describe the molecular mechanisms underlying physiological phenomena in midgut of the Hemiptera Dysdercus peruvianus a cDNA library was prepared from midgut mRNA. We used ESTs from this library to identify transcripts genes related with glucose transport proteins besides digestive enzymes. Analysis of 1053 high-quality expressed sequence tags (ESTs) yielded 903 unique sequences comprised of 62 contigs and 841 singlets. Among the homologous sequences found the following are more relevant to our aim: &#946;-glucosidase (microvillar membrane marker), &#945;-glucosidase (perimicrovillar membrane marker), aminopeptidase (perimicrovillar space marker), cathepsin L (vesicles content) and sugar transporter protein, GLUT. These sequences had its specific transcription (or preferential) verified by semi-quantitative RT-PCR on different insect tissues (malpighian tubules, salivary gland, fat body, midgut, midgut first ventriculus, second ventriculus and third ventriculus). The glucose and water absorption across the first ventriculus of the midgut of the Hemiptera Dysdercus peruvianus were determined. The insects were fed with a 10 glucose-non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventriculus contents. The transport of water and glucose can be inhibited by 0.2 mM phloretin (GLUT inhibitor) and by 0.1 mM phlorizin (SGLT inhibitor) and is activated by 50 mM K2SO4 The results suggest that D. peruvianus has a transporter uniporter like (GLUT) and K+-glucose symporter like SGLT, both co-transporting water. The transcriptome showed a GLUT homologous protein which sequence is almost complete and was analyzed by bioinformatics tools

A mosca domestica (Musca domestica) é um dos insetos largamente distribuído e conhecido pelo homem. A larva de M. domestica possui no conteúdo luminal do ventrículo anterior e médio uma atividade proteolítica com pH ótimo entre 3,0 3,5 e propriedades cinéticas similares a catepsina-D. Três cDNAs codificantes para preprocatepsina-D (ppCAD1, ppCAD2 e ppCAD3) foram clonados a partir de uma biblioteca de cDNA ventricular de larvas de M. domestica. As sequências possuem o peptídeo sinal, o propeptídeo e a enzima madura contendo os resíduos catalíticos e todos os resíduos de ligação ao substrato conservados, achados em uma catepsina-D lisossomal bovina. Um cladograma de sequências de aminoácidos de catepsinas-D de insetos e vertebrados depositados no GENBANK formou um grande grupo dividido em duas ramificações monofiléticas: Uma com sequências de vertebrados e a outra com sequências de catepsinas-D lisossomais de insetos incluindo a ppCAD1. A sequência do pepsinogênio humana, ppCAD2, ppCAD3 e uma sequência de D. melanogaster são excluídas desse grande grupo indicando uma função não lisossomal para essas sequências. ppCAD3 deve corresponder a uma catepsina-D digestiva encontrada no conteúdo luminal em larvas do inseto devido: (1) Análise por RT-PCR indicam que os transcritos codificantes para a CAD3 são expressos no ventrículo anterior e porção proximal do ventrículo médio. (2) pCAD3 recombinante após autoativação sob condições ácidas possui um pH ótimo entre 2,5 3,0 que é próximo ao pH luminal do ventrículo médio onde essa enzima atua. (3) Imunoblots das proteínas de diferentes tecidos e marcadas com o anticorpo preparado contra a pCAD3 foi positiva apenas nos tecidos e conteúdo do ventrículo anterior e médio. (4) CAD3 é localizada pela técnica de imuno-ouro no interior de vesículas de secreção e próxima a microvilosidades nas células do ventrículo anterior e médio. Esses dados suportam a idéia que ao se adaptar a um hábito detritivo, a CAD digestiva da mosca (e de outros Diptera Cyclorrhapha) resultou do mesmo gene ancestral da catepsina-D lisossomal intracelular, da mesma forma que se acredita que ocorreu com a pepsina em vertebrados. Uma limitada quantidade de informações de sequências de DNA e aspectos moleculares de M. domestica é disponível até o presente momento. Nós então propusemos gerar sequências de ESTs a partir de uma bibilioteca de cDNA ventricular da larva desse inseto. Um total de 826 ESTs randomicamente selecionados presentes no ventrículos de larvas de M. domestica foram seqüenciados e analisados com programas de bioinformática e separado em 323 clusters. As sequências foram manualmente anotadas e separadas em 3 categorias: (S) produtos provavelmente secretados, (H) produtos de metabolismo em geral (housekeeping) e (U) produtos sem função conhecida. Cento e sessenta clusters (423 ESTs) codificavam para proteínas secretadas tais como: lisozimas, lipases, tripsinas, quimotripsinas, dipeptidades, carboxipeptidase e &#945;-amilases. Cento e trinta e dois clusters (190 ESTs) codificavam para sequências de metabolismo em energético, síntese de proteínas, transdução de sinal e outras funções celulares. Noventa e cinco clusters (231 ESTs) codificaram para proteínas sem similaridades com proteínas conhecidas no banco de dados. Estudos de expressão, localização e imunocitoquímica de sequências alvos deverão no futuro nos fornecer um melhor entendimento da fisiologia digestiva da larva de Musca domestica em detalhes moleculares. Uma enzima lipolítica (LipMD) foi identificada no item anterior. A sequência de aminoácidos obtidas é homóloga a lipases e contém os três bem conservados resíduos de aminoácidos que compõe a tríade catalítica (Ser183, His273 e Asp208) para esse grupo de enzimas. O fragmento de cDNA codificante para a LipMD foi clonado em vetor pAE (Ramos et al., 2004) e expresso em E. coli produzindo uma enzima com massa molecular de 37,3 kDa. A LipMD recombinante foi purificada e é capaz de hidrolizar tributirina e um grande número de substratos (pNP-acetato a pNP-esterato) e possui um pH ótimo próximo de 7,5. Analise por RT-PCR mostrou que os transcritos codificantes para a LipMD são expressos apenas no ventrículo anterior. Western-blots após SDS-PAGE das proteínas de vários tecidos e marcados com o anticorpo produzido contra a LipMD revelou a ocorrência da enzima principalmente no conteúdo luminal do ventrículo anterior. A LipMD é localizada pela técnica de imuno-ouro no interior de vesículas de secreção próximas a microvilosidades no interior das células do ventrículo anterior. Esta enzima pode atuar como uma enzima lipolítica digestiva secretada nessa região do ventrículo de larvas de M. domestica / The house fly, Musca domestica is one the best known and most widely distributed insects known to humans. M. domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetical properties like cathepsin-D. Three cDNAs coding for preprocatepsin D-like proteinases (ppCAD1, ppCAD2, ppCAD3) were cloned from a M. domestica midgut cDNA library. The sequences encoding the signal peptide, propeptide and mature enzyme having all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin-D. A cladogram of sequences of insect and vertebrate cathepsin-D-like proteinases deposited on GENBANK form a large grouping divided into two monophyletic branches: one with vertebrate and the other with insect lysosomal sequences including ppCAD1. Human pepsinogen, ppCAD2, ppCAD3, and a sequence from Drosophila melanogaster are excluded indicating a nonlysosomal function for them. CAD3 should correspond to the digestive CAD found in enzyme assays because: (1) The mRNA for CAD3 is expressed (RT-PCR) only in the anterior and proximal middle midgut. (2) Recombinant pCAD3, after auto activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (3) Immunoblots of proteins from different tissues and stained with antiserum prepared against recombinant pCAD3 were positive only for the anterior and middle midgut tissue and contents. (4) CAD3 is localized with immunogold labeling inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to a detritivorous habit M. domestica digestive CAD (and of other Diptera Cyclorrhapha) resulted from the same archetypical gene as the intracellular cathepsin-D, paralleling what happened with vertebrates. A limited amount of data regarding DNA sequences and molecular aspects of Musca domestica species is avaliable. We proposed to generate ESTs sequences from a cDNA library constructed from larval midguts. A total of 826 randomly selected midgut derived cDNAs were sequenced and assembled based on their similarities into 323 clusters. The sequences were classified into three categories: (S) probably secretory products, (H) housekeeping products and (U) products with unknown cell localization and function. One hundred and sixty clusters (423 ESTs) encode putative secreted proteins such as lysozymes, lipases, trypsins, chymotrypsins, dipeptidases, carboxypeptidases A and &#945;-amylases. One hundred and thirty two clusters (190 ESTs) encode housekeeping sequences associated with energy metabolism, protein syntesis, signal transduction and other cellular functions. Ninety five clusters (213 ESTs) encode proteins with no similarity with known proteins. Expression and high-throughput bioassay screening of target sequences must provide us with a better understanding of the digestive physiology of Musca domestica midgut larvae, in molecular detail. A lipolytic enzyme (LipMD) was identified from expressed sequence tags (EST) constructed from midgut larvae cDNA library. The deduced amino acid sequence is homologous to lipases and contained the three well-conserved amino acid residues, Ser183, His273, and Asp208, which form the catalytic triad of the enzyme. The cDNA fragment encoding for LipMD was cloned into a pAE vector (Ramos et al., 2004) and expressed in E. coli producing an enzyme with a molecular mass of 37.3 kDa. The recombinant LipMD was purified and was able to hydrolyse tributyrin and a broad range of substrates, from C2 to C18 p-nitrophenyl-esters and displayed an optimal pH of approximately 7.5. RT-PCR analysis in tissue homogenates (anterior, middle and posterior midguts, hemolymph, fat body and Malpighian tubules) showed that LipMD mRNA transcripts were expressed only in anterior midgut. Western-blots after SDS PAGE of proteins from different tissues and stained with anti-LipMD serum revealed that the enzyme occurs mainly in the anterior midgut lumen. LipMD is localized with immunogold labeling inside secretory vesicles and around microvilli in anterior midgut cells. LipMD is a candidate to be the digestive lipolytic enzyme found in that midgut region

A partir da identificação de catepsinas L em ensaios in vitro e em zimogramas partimos para purificação desta enzima no inseto. A região V2 foi selecionada como fonte de obtenção da cisteína proteinase já que dentre os três ventrículos o segundo apresentou maior atividade específica. Após diversas tentativas de isolar esta proteinase, foi estabelecida uma marcha de purificação que envolvia em todas as etapas a participação de metil metanosulfonato (MMTS), o que inativa a proteinase evitando assim autólise ao longo do processo de purificação. A marcha consistiu de três passos cromatográficos (troca-iônica, filtração em gel e afinidade, nesta ordem) onde foi observada a presença de duas cisteína proteinases, cada uma apresentando respectivamente as seguintes massas moleculares 32 e 45 kDa (SDSPAGE). As duas cisteína proteinases possuem o mesmo pH ótimo igual a 6,3. Além disso, estas enzimas foram termicamente inativadas a 40 ºC segundo uma cinética de primeira ordem aparente, sugerindo a existência de apenas uma espécie molecular de cada enzima na preparação com meia vida de 5 minutos para cis 1 e 4,8 minutos para cis 2. Foi determinada a constante de dissociação entre enzimainibidor, onde foi observado os valores de 17,3 nM para cis 1 e 7,11 nM para cis 2 através da titulação por E-64. A eficiência de catálise cis 1 e cis 2 é maior para o substrato sintético Z-FR-MCA do que para Z-RR-MCA, indicando que tratava-se de catepsinas L. Com o intuito de descrever os mecanismos moleculares por trás dos fenômenos fisiológicos no intestino médio do Hemiptera Dysdercus peruvianus foi construída uma biblioteca de cDNA a partir de mRNA deste tecido. Utilizamos ESTs provenientes desta biblioteca com o objetivo de identificar genes transcritos relacionados com proteínas de transporte de glicose além de enzimas digestivas. Após o processamento das leituras, surgiram 1053 ESTs úteis. Montando estes ESTs por alinhamento de bases, foram produzidos 62 contíguos e 841 singletos, o que totaliza 903 seqüências únicas. Entre as seqüências homólogas encontradas as mais relevantes para o nosso estudo foram: &#946;-glicosidase (marcadora de membranas microvilares), &#945;-glicosidase (marcadora de membranas 8 perimicrovilares), aminopeptidase (espaço perimicrovilar), catepsina L (conteúdo de vesículas secretoras) e proteína transportadora de açúcar do tipo GLUT. Estas seqüências encontradas tiveram a sua transcrição específica (ou preferencial) averiguada por RT-PCR semiquantitativo nos diferentes tecidos do inseto estudado (intestino médio, túbulo de Malpighi, corpo gorduroso, glândula salivar, ventrículo 1, ventrículo 2 e ventrículo 3 do intestino médio). O transporte de glicose e água in vivo foi estudado. Para isso, os insetos foram alimentados com uma solução contendo glicose e corante não absorvível seguido de dissecação e análise do conteúdo luminal. O transporte de água e glicose foi inibido por floretina 0,2 mM (inibidor do uniportador GLUT) e por florizina 0,1 mM (inibidor do simportador SGLT) e ativado por K2SO4 50 mM. Isto sugere a presença do transportador do tipo uniportador (GLUT), e do simportador K+-glicose (SGLT), ambos co-transportando água. O transcriptoma revelou proteína homóloga a transportador GLUT cuja seqüência está parcialmente completa e foi analisada com ferramentas de bioinformática / After identification of cathepsins L in vitro assays and in zimograms we began to purify this enzyme in insect midgut. The region V2 was selected as a source of material for purifying a cysteine proteinase because it contains most of the activity of that proteinase. After several attempts to purify this proteinase, an effective process was developed that avoid autolysis with methyl methanethiosulfonate (MMTS), a sulfhydryl-reactive and reversible sulfonating reagent for thiol-containing molecules. The purify process was made by three chromatographic steps (anion-exchange column, gel filtration column and affinity column in this order), where two cysteine proteinase were purified, cys 1 and cys 2 with 32 and 45 kDa (SDS-PAGE). The two cysteine proteinases have the same pH optimum of 6.3. Besides that, these enzymes were thermicaly inactivated following apparent first-order kinetics with a half-life of 5 min (cys1) and 4.8 min (cys2) at 40 ºC. Both Cys are inhibited by E-64 with a KD of 17.3 nM (Cys 1) and 7.11 nM (Cys2). Both Cys are more active on Z-FR-MCA than on Z-RR-MCA, suggesting they are cathepsins-L. With purpose of describe the molecular mechanisms underlying physiological phenomena in midgut of the Hemiptera Dysdercus peruvianus a cDNA library was prepared from midgut mRNA. We used ESTs from this library to identify transcripts genes related with glucose transport proteins besides digestive enzymes. Analysis of 1053 high-quality expressed sequence tags (ESTs) yielded 903 unique sequences comprised of 62 contigs and 841 singlets. Among the homologous sequences found the following are more relevant to our aim: &#946;-glucosidase (microvillar membrane marker), &#945;-glucosidase (perimicrovillar membrane marker), aminopeptidase (perimicrovillar space marker), cathepsin L (vesicles content) and sugar transporter protein, GLUT. These sequences had its specific transcription (or preferential) verified by semi-quantitative RT-PCR on different insect tissues (malpighian tubules, salivary gland, fat body, midgut, midgut first ventriculus, second ventriculus and third ventriculus). The glucose and water absorption across the first ventriculus of the midgut of the Hemiptera Dysdercus peruvianus were determined. The insects were fed with a 10 glucose-non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventriculus contents. The transport of water and glucose can be inhibited by 0.2 mM phloretin (GLUT inhibitor) and by 0.1 mM phlorizin (SGLT inhibitor) and is activated by 50 mM K2SO4 The results suggest that D. peruvianus has a transporter uniporter like (GLUT) and K+-glucose symporter like SGLT, both co-transporting water. The transcriptome showed a GLUT homologous protein which sequence is almost complete and was analyzed by bioinformatics tools

This study examined if multi-joint resistance exercises could elicit expression of biomarkers associated with neuroprotection. Thirteen well-trained males performed 4 sets to failure at 80% of a one-repetition maximum (1RM) on the back squat, bench press, and deadlift. The biomarkers measured immediately pre- and post-exercise were brain derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), cathepsin B (CatB), and interleukin 6 (IL-6). There was a main time effect (p<0.01) for BDNF with significant increases in the deadlift (p=0.01) and bench press (p=0.01) conditions, but not the squat (p=0.21). There was a main time effect (p<0.01) for IL-6 with a significant increase in the squat (p<0.01). There was no significant increase in CatB or IGF-1 (p>0.05). Additionally, there was no significant relationship between BDNF and IL-6 response. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

La Cathepsine K est parmi la plus efficace des collagénases de mammifère pour cliver la triple hélice de collagène de type-1. Nous avons développé une série d'azanitriles, (CKI-8 and CKI-13) inhibiteurs de cathepsine K. CKI-8 (un isomère de CKI-13) et CKI-13 ne sont pas toxiques sur les osteoblastes Saos-2 et les cellules RAW 264.7 jusqu' à une concentration de 1000 nM, tandis qu'ils ne le sont pas jusqu'à une concentration de 100 nM sur les osteoclastes. CKI-8 n'affecte pas l'activité de la phosphatase alkaline ainsi que la minéralisation induite par les Saos-2 et par les osteoblastes primaires. CKI-13 diminue de 35 % la minéralisation induite par les Saos-2 tandis qu'il n'affecte pas la minéralisation induite par les osteoblastes primaires. L'addition de CKI-13 diminue l'activité de la phosphatase alkaline d'environ 20% (Saos-2) et de 40 % (osteoblastes primaires). La résorption osseuse sur des tranches d'os d'origine bovine est diminuée avec 10 nM de CKI-13, 100 nM de CKI- 8 et 100 nM d'inhibiteur commercial E64. CKI-8 et CKI-13 diminuent la mobilité des osteoclastes. Nous avons développé un dosage d'hydrolyse de PPi par la phosphatase alkaline au moyen de l'IR, ayant l'avantage de fonctionner sur des vésicules matricielles et des cellules avec des substrats naturels à un pH physiologique. La bande de PPi localisée à 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) et celles de Pi localisées à 1076 cm-1 (∑= 1346 ± 116 M-1.cm- 1) et à 991 cm-1 (∑= 493 ± 49 M-1.cm-1) ont servis à mesurer les concentrations du substrat et du produit / Cathepsin K is among the most potent mammalian collagenase, capable of cleaving the triple helix in type-I collagen. We developed a series of azanitriles (CKI-8 and CKI-13) which are inhibitors of cathepsin K. CKI-8 (an isomer of CKI-13) and CKI-13 did not induce significant toxicity on osteoblasts Saos-2 and RAW 264.7 cells up to 1000 nM, while they were not toxic on mature osteoclasts up to 100 nM. Commercial E64 inhibitor was not toxic in primary osteoclast cells up to 1000 nM. CKI-8 did not affect alkaline phosphatase activity as well the mineralization induced by Saos-2 cells and by primary osteoblasts. CKI-13 decreased by 35% the mineralization induced by Saos-2 cells while it did not on mineralization induced by primary osteoblasts. Addition of CKI-13 decreased alkaline phosphatase activity by around 20% (Saos-2 cells) and 45% (primary osteoblasts). Bone resorption on bovine slices decreased significantly with 10 nM of CKI-13, with 100 nM of CKI-8 and commercial inhibitor E64. Our findings indicated that CKI-8 and CKI-13 inhibited bone resorption and affected the mobility of osteoclast. To monitor directly the PPi hydrolytic activity by alkaline phosphatase, we developed an infrared (IR) assay taking the advantage to use natural substrate under physiological pH in matrix vesicles and in living cells. PPi band located at 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) and Pi bands located at 1076 cm-1 (∑= 1346 ± 116 M-1.cm-1) and at 991 cm-1 (∑= 493 ± 49 M-1.cm-1) served to measure the substrate and the product concentrations

Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.

It is suggested that Alzheimer’s disease (AD) is caused by an imbalance between production, degradation and clearance of the amyloid-β (Aβ) protein. This imbalance leads to aggregation of Aβ and tau proteins and neurodegeneration in the brain. Today there is increasing evidence that the balance between the protease cathepsin B and the protease inhibitor cystatin C affects the tendency for Aβ to aggregate. The primary aim of this thesis was to investigate Aβ, cystatin C and cathepsin B levels in blood and cerebro-spinal fluid (CSF) in relation to the risk of AD. Studies I &amp; II were based on the re-examinations of participants, at ages 70 and 77, in the Uppsala Longitudinal Study of Adult Men (ULSAM), a community-based prospective study initiated in 1970 (participants then being 50 years of age). In ULSAM, low plasma Aβ1-40 (Study I) and low serum cystatin C levels (Study II) were associated with a higher risk of AD. Studies III &amp; IV were based on a cross-sectional sample of people with AD, mild cognitive impairment and healthy controls, recruited at three Swedish Memory Disorder units: Uppsala University Hospital, Uppsala, Skåne University Hospital, Malmö, and Karolinska University Hospital, Huddinge, Stockholm. In Study III, CSF cystatin C levels were positively correlated with both Aβ1-42 and tau levels. In Study IV, individuals with AD had higher mean plasma cathepsin B levels than healthy controls. In conclusion, low plasma Aβ1-40 and low serum cystatin C levels may precede clinically manifest AD in elderly men, cystatin C levels are positively correlated with Aβ1-42 and tau levels in CSF, and mean plasma cathepsin B levels are higher in people with AD compared to healthy controls. In addition to Aβ1-42 and tau levels in CSF, Aβ1-40, cystatin C and cathepsin B levels in blood may reflect the risk of AD.

Das Glioblastoma multiforme gehört zu den ZNS-Tumoren neuroepithelialen Ursprungs. Es zeichnet sich durch ein multiformes Zellbild, einen geringen Differenzierungsgrad und eine schnelle Krankheitsprogression aus. Trotz mikrochirurgischer Entfernung und anschließender Radiochemotherapie entwickeln die Patienten im Durchschnitt nach 7 Monaten einen Rezidivtumor und haben eine mittlere Überlebenszeit von 14,6 Monaten. Die Rezidivneigung stellt somit ein großes Problem in der Behandlung von Glioblastompatienten dar. In früheren Arbeiten konnte nachgewiesen werden, dass die Rezidivtumore eine andere Zellzusammensetzung und auch ein aggressiveres Wachstumsverhalten als deren Primärformen aufweisen. Ziel dieser Arbeit war es, zu prüfen ob mittels 2D-Gelelektrophorese und anschließender MALDI-TOF-Massenspektrometrie Unterschiede im Proteinexpressionsmuster zwischen Gewebeproben vom Primärtumor eines Glioblastoms WHO Grad IV und dem korrespondierendem Rezidivtumor eines Patienten detektierbar sind. Hierbei wurden 43 Proteine als differentiell exprimiert erkannt, von denen mit Hilfe der MALDI-TOF-Massenspektrometrie sechs genauer charakterisiert wurden. Vier der sechs Proteine waren im Rezidivtumor erhöht: EnoylCoA-Hydratase, ATP-Synthase Untereinheit d, Tropomyosin alpha-3-Kette Isoform 2 und Cathepsin D. Die anderen zwei waren im Rezidivtumor niedriger ausgeprägt: Nukleosid-Diphosphatkinase A und L-3-Phosphoserin-Phosphatase. Eine weitere Untersuchung mittels Western-Blot-Analyse bestätigte, dass Cathepsin D (als eines der sechs charakterisierten Proteine) tatsächlich auch in den Rezidivtumoren dreier weiterer Patienten stärker exprimiert war als in den korrespondierenden primären Glioblastomen.