Estudamos a relação entre fenótipos do diabetes e obesidade com o genótipo da ECA, em humanos e camundongos. Analisamos fenótipos relacionados com obesidade em 1600 indivíduos da população de Vitória (genotipados para o polimorfismo I/D da ECA) e em camundongos com 1 a 3 cópias do gene da ECA, submetidos ou não à dieta rica em gordura ou diabetes. Camundongos com 3 cópias e alimentados com dieta rica em gordura apresentaram menor peso, gordura periepididimal e insulinemia. Já no grupo diabético os camundongos com 3 cópias apresentaram maior mortalidade. A relação fenótipo e genótipo encontrada nos camundongos repetiu-se no estudo populacional. Estes resultados dão suporte à idéia de que o gene da ECA é um importante fator determinante nos fenótipos relacionados a doenças complexas como a síndrome X. / We studied the relationship between obesity and diabetes phenotypes with ACE genotypes using a human population (1600 individuals where ACE I/D polymorphism was determined) and engineered ACE mice models (nondiabetic or diabetic under high fat diet or control diet). Body weight, periepididymal adipose tissue weight and insulin levels were lower in 3 copies under high fat diet. Diabetic 3 copies mice had higher mortality. Presence of D allele was significantly associated with decreased body fat percentage in individuals who were at increases risk of being hyperinsulinemic. Taken together these results provide evidence for a direct role of ACE in obesity phenotypes, which may be important to unravel the complexities of metabolic syndrome.

O sistema renina-angiotensina (SRA) circulante é um sistema endócrino que promove a produção de angiotensina (Ang) II, a qual exerce seus efeitos pela interação com receptores específicos. O conceito clássico do SRA circulante está sendo modificado, pois tem sido demonstrada a existência de sistemas locais capazes de gerar angiotensinas de forma independente do SRA circulante em vários tecidos e órgãos. Trabalhos recentes sugerem a existência de alguns componentes do SRA em tecido gengival e fibroblastos gengivais de diferentes espécies. Porém, não são encontrados na literatura achados inequívocos sobre a presença de importantes componentes do SRA, tais como renina e angiotensinogênio, no tecido gengival de rato. Portanto, os objetivos do presente trabalho foram: 1) estudar a expressão e localização de componentes do SRA no tecido gengival de rato e 2) estudar in vitro a funcionalidade do SRA local em homogenato de tecido gengival de rato quanto à formação de Ang II e outros peptídeos vasoativos a partir de precursores de Ang II. Transcrição reversa seguida de reação em cadeia da polimerase (RTPCR) foi utilizada para avaliar a expressão de RNAm. Análise imunohistoquímica foi utilizada para detecção e localização de renina no tecido gengival de rato. Um método fluorimétrico padronizado com o tripeptídeo Hipuril-Histidina-Leucina (Hip-His-Leu) foi usado para medir a atividade da ECA em homogenatos de tecido gengival de rato. A técnica de cromatografia líqüida de alto desempenho (HPLC) foi usada para analisar os produtos formados após a incubação de homogenatos de tecido gengival de rato com Ang I ou tetradecapeptídeo substrato de renina (TDP). RT-PCR revelou a expressão de RNAm para renina, angiotensinogênio, ECA e receptores de Ang II (AT1a, AT1b e AT2) em tecido gengival; em fibroblastos cultivados de tecido gengival foi observada expressão de RNAm para renina, angiotensinogênio e receptor AT1a. A técnica de imunohistoquímica demonstrou a existência de renina em vasos de tecido gengival de rato. Atividade da ECA foi detectada por meio do ensaio fluorimétrico (4,95±0,89 nmol His-Leu/g.min). Quando Ang I foi usada como substrato, análises de HPLC mostraram a formação de Ang 1-9 (0,576±0,128 nmol/mg.min), Ang II (0,066±0,008 nmol/mg.min) e Ang 1-7 (0,111±0,017 nmol/mg.min), enquanto que os mesmos peptídeos (0,139±0,031; 0,206±0,046 e 0,039±0,007 nmol/mg.min, respectivamente) e Ang I (0,973±0,139 nmol/mg.min) foram formados quando TDP foi usado como substrato. Adicionalmente, análises de HPLC revelaram a ausência de enzimas que degradam Ang II em homogenatos de tecido gengival de rato. Em conclusão, os resultados apresentados neste trabalho mostram claramente a existência de um SRA local em tecido gengival de rato, que é capaz de gerar Ang II e outros peptídeos vasoativos in vitro. Estudos adicionais são necessários para elucidar o papel deste sistema local no tecido gengival de rato. / Systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through the interaction with specific receptors. The concept of this classic circulating RAS has been modified since there is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of the circulating RAS. Recent works suggest the existence of some RAS components in the gingival tissue and cultured gingival fibroblasts of different species, but there is paucity of data in the literature regarding the unequivocal existence of crucial RAS components, such as renin and angiotensinogen, in the rat gingival tissue. Therefore, the aims of the present work were to: 1) study the expression and localization of RAS components in the rat gingival tissue and 2) evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different precursors of Ang II. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess mRNA expression. Immunohistochemical (IHC) analysis aimed to detect and localize renin in the rat gingival tissue. A standardized fluorimetric method with the tripeptide Hippuryl-Histidyl-Leucine (Hip-His-Leu) was used to measure tissue ACE activity in rat gingival tissue homogenates. High performance liquid chromatography (HLPC) was used to analyze the products formed after the incubation of rat gingival tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). RT-PCR revealed the mRNA expression for renin, angiotensinogen, ACE and Ang II receptors (AT1a, AT1b and AT2) in the rat gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen and AT1a receptor. IHC demonstrated the existence of renin in vessels of the rat gingival tissue. ACE activity was detected by the fluorimetric assay (4.95±0.89 nmol His-Leu/g.min). When Ang I was used as the substrate, HPLC analyses showed the formation of Ang 1-9 (0.576±0.128 nmol/mg.min), Ang II (0.066±0.008 nmol/mg.min) and Ang 1-7 (0.111±0.017 nmol/mg.min) whereas these same peptides (0.139±0.031; 0.206±0.046 and 0.039±0.007 nmol/mg.min, respectively) and Ang I (0.973±0.139 nmol/mg.min) were formed when TDP was the substrate. Additionally, HPLC revealed absence of Ang II degrading enzymes in rat gingival tissue homogenates. In conclusion, the results presented here clearly show the existence of a local RAS in the rat gingival tissue, which is capable of generating Ang II and other vasoactive peptides in vitro. Further studies are required to elucidate the role of this system in the rat gingival tissue.

O arroz (Oryza sativa L.), principal alimento para cerca de metade da população mundial, é consumido principalmente na forma polida. Contudo, o arroz integral vem se destacando, devido principalmente aos compostos bioativos presentes nas camadas mais externas do grão. Os benefícios à saúde são atribuídos, em parte, à sua capacidade de combater radicais livres e exercer atividades biológicas, tais como a inibição de determinadas enzimas. Neste trabalho foram analisados os teores de compostos fenólicos totais (FT), solúveis (FS) e insolúveis (FI) e avaliado o efeito do cozimento de 17 genótipos de arroz integral, sete com pericarpo pigmentado e dez genótipos não-pigmentados. Ainda foi avaliada a inibição da enzima conversora de angiotensina I (ECA) e da -amilase por esses compostos, no arroz cru e cozido. O arroz pigmentado se mostrou rico em compostos fenólicos, em média da ordem de 4200 µg eq. ácido ferúlico/g, devido aos seus altos teores de FS, constituídos principalmente por antocianinas e proantocianidinas. Os FI, representados principalmente pelos ácidos fenólicos contribuíram com apenas 20% dos compostos fenólicos totais. O arroz não-pigmentado contém, em média, ao redor de 1000 µg eq. ácido ferúlico/g, distribuídos quase equitativamente entre a fração solúvel e insolúvel. O cozimento do arroz provocou redução nos teores de FT e alteração na proporção entre FS e FI. Essas alterações foram mais pronunciadas no arroz com pericarpo vermelho, afetando principalmente a fração solúvel. O arroz preto, contudo, manteve a proporção entre FS e FI após o cozimento. A -amilase não foi inibida de forma significativa pelos fenólicos das amostras de arroz cozido. O arroz pigmentado inibiu mais fortemente a ECA do que o arroz não-pigmentado, levando a crer que a pigmentação seja um fator importante. No entanto, entre os diferentes genótipos pigmentados, o perfil de fenólicos parece ser o fator determinante para a maior ou menor atividade inibitória. O cozimento do arroz reduziu significativamente a inibição da ECA pelos fenólicos, fato observado principalmente nos genótipos vermelhos, devido à diminuição dos teores de FS e da capacidade inibitória dos fenólicos presentes. O arroz preto se destacou por ter o maior teor de fenólicos solúveis e a maior ação inibidora da ECA, após o cozimento. / Rice (Oryza sativa L.) sustains at present about half of the world´s population. Consumption is mainly in its milled form, but brown rice has prompted further research due to bioactive compounds present in the pericarp of the grain. Some of the positive health effects have been attributed to radical scavenging activity and other biological effects such as inhibition of certain enzymes. In this study it was analyzed the contents of total, soluble and insoluble phenolic compounds of 17 different genotypes of brown rice as well as the effect of cooking. Seven genotypes had pigmented pericarp and ten were non-pigmented. In addition, the extracts from crude and cooked rice were tested for their capacity to inhibit the angiotensin I (ACE) converting enzyme and -amylase activities. Pigmented rice genotypes were highest in phenolic compounds, with an average of about 4200 µg ferulic acid eq./g, due to their high contents of soluble phenolics, mostly represented by anthocyanins and proanthocyanidins. Insoluble phenolics, represented mainly by phenolic acids, contributed with only 20% of total phenolics. Non-pigmented rice showed overall lower levels of phenolics. The mean content was about 1000 µg ferulic acid eq./g, almost equally distributed between the soluble and insoluble fractions. Levels of total phenolics were significantly reduced by rice cooking and proportions between soluble and insoluble fractions were altered. These alterations were more pronounced for pigmented rice, and soluble phenolics were the most affected. However, after cooking, black rice was the only that maintained the original proportion between soluble and insoluble phenolics. Alpha-amylase was not significantly inhibited by phenolics after cooking. Pigmented rice showed a potent inhibition of ACE, much higher than of non-pigmented rice, which seems to indicate that color of the pericarp is an important factor. Nevertheless, different profiles of phenolic compounds may explain why individual pigmented genotypes with similar phenolic levels can have different ACE inhibiting capacities. Rice cooking reduced significantly the inhibition of ACE by phenolics, which feature was more pronounced in pigmented rice due to the reduction of soluble phenolics and the activity of individual phenolics. Among pigmented rice, the highest soluble phenolic content and the most potent ACE inhibition after cooking was observed for black rice turning it the most distinguished notable.

Evidências sobre a presença do sistema renina-angiotensina (SRA) na medula óssea e a possível participação da enzima conversora de angiotensina-I (ECA) na regulação hematopoética tem despertado o interesse da comunidade científica. Como a ECA também é um componente chave do sistema calicreína-cininas (SCC), é possível que elementos deste sistema, possam estar envolvidos no controle hematopoético. Assim, avaliamos a participação da ECA na regulação hematopoética de animais não modificados (WT) e nocautes dos receptores B1 de cininas (KOB1). Para isso, utilizamos técnicas de cultura de células de medula óssea, a saber: os ensaios clonogênicos em soft-ágar para granulócitos e macrófagos (CFU-GM) e o sistema de cultura líquida de longa duração (CLLD). Os resultados mostraram a presença da ECA em células das CLLD e indicaram a participação da enzima na proliferação de progenitores hematopoéticos possivelmente através do controle dos níveis de AcSDKP, pois o tratamento com o tetrapeptídeo e com captopril, reduziu significativamente o número CFU-GM in vitro e in vivo. Quando adicionado às CLLD, o AcSDKP foi capaz de aumentar significativamente a expressão do mRNA da ECA, sugerindo que seus níveis possam controlar a expressão gênica desta enzima. Em relação aos animais KOB1, os resultados mostraram maior atividade da ECA, acompanhado de aumento não significativo da expressão gênica e protéica da enzima. O tratamento das CLLD de animais WT com agonistas de receptores de cininas, não alterou a expressão gênica e a atividade da ECA. Assim, nossos dados sugerem que a ECA participa da regulação hematopoética neste modelo. No entanto, novos estudos serão necessários para a elucidação dos mecanismos envolvidos na expressão e/ou controle da atividade da ECA pelos receptores de cininas. / Evidences on the presence of the renin angiotensin system in the bone marrow and the possible participation of the angiotensin-I converting enzyme (ACE) in the hematopoietic regulation have aroused interest of the scientific community. As the ACE also is a common element of the kallikrein-kinin system (KKS), it is possible that elements of KKS, can be involved in the hematopoietic control. Thus, we evaluated the participation of the ACE on the hematopoietic regulation of wild-type (WT) and kinin receptor B1 knockout mice (KOB1). For this, we use techniques of bone marrow cell culture, to know the clonogenic assays for granulocyte-macrophage (GM-CFU) and the long term bone marrow cultures (LTBMC). The results shown the presence of the ACE in cells from LTBMC and its possible participation on hematopoietic proliferation through the control of AcSDKP levels, therefore the treatment with AcSDKP and captopril, decreased significantly the GM-CFU number in vitro and in vivo. When added to the LTBMC, the AcSDKP increase significantly the expression of ACE mRNA, suggesting that its levels could control the gene expression of this enzyme. In relation to KOB1 mice, the results shown increase of the ACE activity and not significant increase of the gene and protein expression of the enzyme. The treatment of the LTBMC of WT mice with kinins receptors agonists, did not modify the gene expression and the ACE activity. Thus, our data suggesting that ACE participate of the hematopoietic regulation in this model. However, new studies will be necessary to understand the involved mechanisms in the expression and/or control of ACE activity by kinins receptors.

Submitted by Cristiane Chim (cristiane.chim@ucpel.edu.br) on 2018-05-07T11:51:08Z No. of bitstreams: 1 LARISSA RIBAS RIBEIRO.pdf: 853387 bytes, checksum: b07d078ec1ece03014fcfd9275152a65 (MD5) / Made available in DSpace on 2018-05-07T11:51:08Z (GMT). No. of bitstreams: 1 LARISSA RIBAS RIBEIRO.pdf: 853387 bytes, checksum: b07d078ec1ece03014fcfd9275152a65 (MD5) Previous issue date: 2017-11-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES# / #2075167498588264571# / #600 / Introduction: Chronic kidney disease (CKD) is characterized by progressive decrease in glomerular filtration rate (GFR), eventually reaching the end-stage renal disease (ESRD) requiring renal replacement therapy (RRT). The decrease in GFR is associated with a gradual and linear increase in cardiovascular mortality. Dysfunction of the autonomic nervous system (ANS) with sympathetic overactivity has been well documented in patients with CKD, especially in people with ESRD. The renal ischemia causes both the excessive activation of the renin-angiotensin-aldosterone system (RAAS) by increasing renin release, as sympathetic ANS, through the afferent sympathetic nerves. The overactivated RAAS and sympathetic SNA feedback each other, which contributes to cardiovascular disease (CVD) in CKD. Despite the clear involvement of these systems in the pathogenesis of CVD in CKD, randomized clinical trials using drugs that block the RAAS or sympathetic SNA have not shown significant effects in the frequency of cardiovascular events in this population. A possible explanation for these negative findings is the genetic heterogeneity, such as the polymorphism in the gene for angiotensin converting enzyme (ACE). Objectives: To investigate the correlation between polymorphisms in the ACE gene and heart rate variability (HRV), a noninvasive tool used to assess ANS activity, in patients with CKD on hemodialysis treatment. Hypotheses: The D allele of the ACE gene, associated with increased activity of the RAAS, would be associated with increased sympathetic activity, which is reflected in lower HRV. If confirmed, the finding could point the subgroup of patients that may get additional benefit of drugs that act on the sympathetic and RAAS systems. Methodology: quantitative and cross-sectional study. The sample will consist of at least 129 adult patients with CKD on hemodialysis treatment (HD) for more than 90 days. It wil be obtained sociodemographic data, previous medical history and medications used. HRV analysis will be conducted through Micromed® ECG machine with registration pulse interval variance, root mean square of squared differences between consecutive intervals (RMSSD) and the percentage of intervals greater than 50 milliseconds (pNN50), a low frequency band (LF) and high frequency (HF), at a time of normovolemia after a midweek HD session. Analysis of hydration status will be made through multi-Frequency bioimpedance device. Polymorphism of the ACE gene will be assessed by polymerase chain reaction method in peripheral blood sample. Data analysis will be performed by ANOVA with comparison of the LF / HF ratio, a component of HRV analysis, between polymorphisms II, ID and DD of the ACE gene. Multivariate analysis with linear regression will be applied using as dependent variables the HRV parameters, and as independent variables the polymorphisms of ACE gene and all variables known to influence HRV, for adjustment purposes. The data will be analyzed using the statistical package STATA 14.0. / Introdução: A doença renal crônica (DRC) caracteriza-se pela redução progressiva da taxa de filtração glomerular (TFG), eventualmente alcançando o estágio de doença renal crônica terminal (DRCT) com necessidade de terapia renal substitutiva (TRS). A diminuição da TFG está associada a um aumento progressivo e linear na mortalidade cardiovascular. A disfunção do sistema nervoso autônomo (SNA) com hiperatividade simpática tem sido bem documentada em pacientes portadores de DRC, especialmente na população com DRCT. A isquemia renal provoca tanto a ativação exagerada do sistema renina-angiotensina-aldosterona (SRAA), através do aumento da liberação de renina, quanto do SNA simpático, por meio dos nervos simpáticos aferentes. O SRAA e o SNA simpático, ambos hiperativados, se retroalimentam mutuamente, o que contribui para a doença cardiovascular (DCV) na DRC. Apesar da clara participação destes sistemas na gênese da DCV da DRC, ensaios clínicos randomizados utilizando drogas que bloqueiam o SRAA ou SNA simpático não têm mostrado efeitos significativos na redução de eventos nessa população. Uma possível explicação para esses resultados negativos seria a heterogeneidade genética, como por exemplo, o polimorfismo no gene da enzima conversora da angiotensina (ECA). Objetivos: Investigar a associação entre polimorfismos no gene da ECA e a variabilidade da frequência cardíaca (VFC), uma ferramenta não invasiva utilizada na avaliação da atividade do SNA, em pacientes portadores de DRC em tratamento por hemodiálise. Metodologia: Estudo quasi experimental do tipo antes e depois. A amostra foi composta por 114 pacientes adultos portadores de DRC em tratamento por hemodiálise (HD) há mais de 90 dias. Foram obtidos dados sociodemográficos, história clínica pregressa e medicamentos em uso. A análise da VFC foi realizada através de aparelho de eletrocardiograma Micromed® com registro do desvio padrão de todos os intervalos normais (SDNN), raiz quadrada da média dos quadrados das diferenças entre intervalos consecutivos (RMSSD), banda de baixa frequência (LF) e de alta frequência (HF), em momento de normovolemia, após uma sessão de HD do meio da semana. A análise do estado de hidratação foi realizada através de aparelho de bioimpedância multi-frequencial. O polimorfismo do gene da ECA foi avaliado por método de reação em cadeia da polimerase em amostra de DNA de sangue periférico. A análise dos dados foi realizada por ANOVA, com comparação da razão LF/HF, componente da análise da VFC, entre os polimorfismos II, ID e DD do gene da ECA. Análise multivariada com regressão linear foi aplicada utilizando como variáveis dependentes os parâmetros da VFC e como variáveis independentes foram incluídos os polimorfismos do gene da ECA juntamente com variáveis que sabidamente influenciam a VFC, para efeito de ajuste. Os dados foram analisados através do pacote estatístico STATA 15.0.

Peptídeos bioativos são de grande interesse tanto para a indústria farmacêutica como para a de alimentos e são obtidos a partir da hidrólise enzimática de várias fontes protéicas, como as do leite, glúten de milho, soja, e músculos de suínos, aves e peixes. Estes peptídeos podem desempenhar atividades benéficas para a saúde, entre elas, a regulação ou inibição de enzimas, com destaque à inibição da enzima conversora da angiotensina I (ECA). Esta enzima é responsável pela clivagem de dois importantes substratos envolvidos na regulação da pressão arterial, a angiotensina I e a bradicinina. Neste trabalho foi estudada a atividade inibitória da ECA em hidrolisados dos peixes tilápia tailandesa (Oreochromis niloticus - linhagem tailandesa) e corvina (Micropogonias furnien) . Os \"minced\" destes peixes foram hidrolisados com pepsina e proteases de Streptomyces griséus por 5 horas em condições ideais de pH e temperatura. A atividade inibitória foi avaliada pela medida da atividade residual da enzima sobre substrato sintético fluorescente. Os hidrolisados com um grau de hidrólise de cerca de 34% apresentaram atividade inibitória semelhante, com valor de IC50 = 0,040 e 0,036 mg proteína/mL, respectivamente para a tilápia e a corvina. Observou-se aumento da atividade inibitória com o progresso da hidrólise, sendo que este aumento também está relacionado com a especificidade das enzimas proteolíticas. Em outro experimento observou-se que os peptídeos presentes no hidrolisado de tilápia inibiram indistintamente os domínios C e N terminais da ECA, não demonstrando especificidade. A atividade inibitória dos hidrolisados foi mantida após submetê-los à ação de enzimas proteolíticas gastrointestinais, indicando a sua provável estabilidade \"in vivo\". Da mesma forma, o minced de tilápia quando incubado sucessivamente com pepsina/tripsina/quimotripsina produziu peptídeos ativos (IC50 = O,025mg proteína/mL). Apesar do reduzido grau de hidrólise obtido neste ensaio (17%), os peptídeos liberados apresentaram atividade inibitória elevada, confirmando que a atividade não está relacionada apenas com o grau de hidrólise, mas também com a sequência de aminoácidos, liberados em função da especificidade das enzimas. Concluiu-se que o consumo destes peixes, ou seus respectivos hidrolisados poderá eventualmente, auxiliar na prevenção e no tratamento não medicamentoso da hipertensão, embora estudos \"in vivo\" sejam necessários para comprovar as suas funções biológicas. / Bioactive peptides have been highly valued by pharmaceutical industries and by food industries as well. These peptides can be released by the enzymatic hydrolysis of various protein sources such as milk, com gluten, soybean, muscles of pigs, chicken and fish and have been recognized to exhibit important health benefits. Among these, the regulation or inhibition of enzymes, like the inhibition of the angiotensin I-converting enzyme (ACE) have been focused. This enzyme is responsible for the cleavage of two important substrates, angiotensin I and bradykinin, both of them involved with the regulation of blood pressure. In this study the inhibitory activity of hydrolysates from tilapia tailandesa (Oreochromis niloficus - linhagem tailandesa) and corvina (Micropogonias furnien) was evaluated. The hydrolysates of the minced fishes were produced in a 5-hour lasting controlled process under optimal conditions of pH and temperature by the sequential action of pepsin and enzymes from Streptomyces griseus. Inhibitory activity was evaluated by measuring the residual activity of the enzyme on a synthetic fluorescent peptidic substrate. The extent of hydrolysis was about 34% and the hydrolysates of tilapia and corvine showed similar inhibitory activity (IC50 of 0.040 and 0.036 mg protein/mL), respectively. An íncrease of activity proportional to the degree of hydrolysis was observed, as well as a relationship with the specificity of the enzymes used. In another experiment it was observed that the bioactive peptides present in the hydrolysate of tilapia did not show specificity for the C- and N-terminal catalytic domains of the angiotensin I-converting enzyme. The inhibitory activity of the hydrolysates was still active afier a further hydrolysis by gastrointestinal enzymes, which seems to indicate an eventual activity in vivo. In a similar way, when the minced tilapia was consecutively incubated with pepsine, trypsin, chymotrypsin active peptides were produced with an activity of IC50 = 0.025 mg protein/mL. Despite the low extend of hydrolysis of about 17%, a high inhibitory activity was observed confirming that activity is not only related to the degree of hydrolysis but also to the sequence of aminoacids and therefore, to the specificity of the enzymes as well. It was concluded that the intake of fish or fish hydrolysates has the potential to help control or to prevent hypertension by a non-drug food-based treatment.

En este trabajo se evaluó la utilidad de una serie de marcadores: triglicéridos, ácido úrico, forma soluble de la tirosín kinasa 1 (sFlt-1), factor de crecimiento placentario (PlGF), múltiplo de la mediana de la proteína plasmática A asociada al embarazo (MoM PAPP-A), antígeno sérico CA125 (CA125), enzima convertidora de angiotensina (ECA) y el estudio ecográfico Doppler, como posibles predictores de preeclampsia en el primer y segundo trimestre de gestación. Se incluyeron un total de 68 gestantes con “riesgo a priori” de desarrollar preeclampsia (diabetes mellitus tipo I, enfermedad renal preexistente, hipertensión crónica sin proteinuria, etc) y un grupo control. De esas 68 gestantes, 8 desarrollaron preeclampsia. La combinación de marcadores más eficiente fue utilizando el PlGF del primer trimestre con un punto de corte ≤37,6 pg/ml, un índice de resistencia ≥0,7 y la ECA≥ 40,4 U/L del segundo trimestre, pudiendo predecir el 87,5% de las gestantes que desarrollarían preeclampsia. / This study evaluated the usefulness of several biomarkers: triglycerides, uric acid, soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), multiple of the median of pregnancy associated plasma protein-A (PAPP-A MoM), serum antigen CA125 (CA125), angiotensin converting enzyme (ACE) and the Doppler ultrasound as predictors of preeclampsia in the first and second trimester of pregnancy. We included a total of 68 pregnant women with "a priori risk" of develop preeclampsia (type I diabetes mellitus, preexisting renal disease, chronic hypertension without proteinuria, etc) and a control group. Of those 68 pregnant women, 8 developed preeclampsia. The most efficient combination of the studied biomarkers was the use of PlGF in the first-trimester with a cut-off ≤37.6 pg/ml, a resistance index ≥0.7 and ACE ≥40,4 U/L in the second trimester which can predict 87,5% of pregnant women who develop preeclampsia.

Shiga Toxin (Stx)-konvertierende Bakteriophagen besitzen eine konservierte lambdo-ide Genomstruktur, weisen aber ein hohes Maß an genetischer Diversität auf. Aus diesem Grund wurden die bereits vorhandenen Sequenzdaten der Stx1-Phagen CP-1639 des Escherichia coli O111:H- Stammes 1639/77 und BP-4795 des E. coli O84:H4 Stammes 4795/97 vervollständigt und analysiert. Im Gegensatz zu dem induzierbaren Bakteriophagen BP-4795 fehlen CP-1639 einige Gene, die für den lytischen Lebenszyklus eines Bakteriophagen notwendig sind. Daher handelt es sich bei CP-1639 um einen kryptischen Prophagen, der nicht mehr in der Lage ist das Wirtschromosom zu verlassen und intakte Phagenpartikel zu bilden. Die Integra-tionsstellen wurden innerhalb des Gens yehV für BP-4795 und ssrA für CP-1639 bestimmt. In unmittelbarer Umgebung des Integrationsortes von CP-1639 befindet sich ein eigenständiges integratives Element. Dieses besteht aus drei offenen Lese-rahmen unbekannter Herkunft, sowie einem Integrasegen und kann auch ohne Assoziation mit Phagen-DNA auftreten. Phagen können zusätzlich zu ihrem Genom Gene bakteriellen Ursprungs tragen. Diese können unter anderem infolge von Transpositionen oder einer unkorrekten Exzision der Phagen-DNA aus dem Wirtschromosom während des lytischen Lebenszyklus in das betreffende Phagengenom eingebaut werden. Eines solches Gen des Bakteriophagen BP-4795 kodiert das Typ III Effektorprotein NleA4795, dessen Funktionalität nach dem C-terminalen Einbau von neun Codons des Hämagglutinin (HA)-Epitopes des humanen Influenzavirus in die Sequenz des Gens nleA4795 überprüft wurde. Dies erfolgte unter Verwendung der Western Blot Analyse durch die Expression dieses Fusionsproteins in dem Wildtyp-Stamm 4795/97 und in der Deletionsmutante 4795escN des Stammes 4795/97. Die Typ III Sekretionssys-tem (T3SS)-inaktive Mutante 4795escN wurde im Verlauf dieser Arbeit hergestellt. Diese Untersuchungen zeigten ebenfalls, dass zur Sekretion von NleA4795 ein in-taktes T3SS notwendig ist. Weiterhin zeigte die Infektion einer HeLa-Zelllinie mit NleA4795-HA exprimierenden Bakterienstämmen und die anschließende Analyse mit Hilfe der Immunfluoreszenz, die Translokation des Proteins in eukaryotische Wirts-zellen. Darüber hinaus deuten die Ergebnisse dieser Untersuchung auf eine Lokali-sation von NleA4795-HA innerhalb des Trans-Golgi Netzwerkes hin. Die Verbreitung des Gens nleA wurde in insgesamt 170 Shiga Toxin-produzierenden E. coli und enteropathogenen E. coli Stämmen untersucht. Dies führte zur Identifika-tion von 14 verschiedenen Varianten des Gens nleA in 149 der überprüften Stämme, wobei mit Ausnahme von zwei Isolaten ebenfalls ein Markergen des T3SS nachge-wiesen werden konnte. Neben drei bereits bekannten Varianten konnten elf neue Varianten identifiziert werden, deren abgeleitete Aminosäuresequenzen sich zu 71% bis 96% glichen. Sequenzunterschiede traten insbesondere aufgrund der Deletion oder Insertion von vier bis 51 Aminosäuren im Mittelteil der potentiellen Proteine auf. Weiterhin deuten die Ergebnisse dieser Untersuchungen eine Assoziation bestimm-ter Varianten des Gens nleA mit spezifischen E. coli Serogruppen an. Southern-Blot Hybridisierungen zeigten, dass etwa ein Viertel der nleA-positiven Stämme zwei Ko-pien dieses Gens in ihrem Genom tragen. Hierbei handelt es sich meist um zwei verschiedene Varianten. In fast allen Fällen kodiert eine dieser Varianten, aufgrund einer Punktmutation oder Insertion eines IS-Elementes, ein verkürztes und vermut-lich nicht funktionelles Protein. Mit Hilfe von Transduktionsexperimenten konnten verschiedene Varianten des Gens nleA im Genom induzierbarer Phagen nachge-wiesen werden, was auf eine Verbreitung des Gens nleA durch den horizontalen Gentransfer hinweist.

Το μετατρεπτικό ένζυμο της αγγειοτενσίνης (ACE) είναι μία διπεπτιδυλκαρβοξυπεπτιδάση ψευδαργύρου που ανήκει στην οικογένεια των gluzincin πεπτιδασών της οποίας η θερμολυσίνη θεωρείται ως πρωτότυπο μέλος. Το ένζυμο πήρε το όνομά του από τη δυνατότητά του να μετατρέπει το βιολο- γικώς ανενεργό δεκαπεπτίδιο αγγειοτενσίνη-Ι στο οκταπεπτίδιο αγγειοτεν- σίνη-ΙΙ, το οποίο εμφανίζει ισχυρή αγγειοσυσπαστική δράση. Μία άλλη βασική δυνατότητα του ACE είναι η αδρανοποιήση του εννεαπεπτιδίου βραδυκινίνη που έχει αγγειοδιασταλτική δράση. Αυτές οι δύο σημαντικές ιδιότητες του ACE το καθιστούν ένα από τα σημαντικότερα συστατικά του συστήματος ρενίνης-αγγειοτενσίνης-αλδοστερόνης. Υπάρχουν δύο ισομορφές του ACE που μεταγράφονται από το ίδιο γονίδιο κατά τρόπο ιστοειδικό. Η σωματική ισομορφή του ACE, η οποία εμφανίζεται στην επιφάνεια των ενδοθηλιακών κυττάρων, είναι μία γλυκοπρωτεΐνη η οποία αποτελείται από μία ενιαία, πολυπεπτιδική αλυσίδα 1306 αμινοξέων. Η σπερματική ισομορφή που εμφανίζεται στους όρχεις και στα κύτταρα σπέρματος είναι μία χαμηλότερης-μοριακής μάζας γλυκοπρωτεΐνη 732 αμινοξέων. Η σωματική ισομορφή αποτελείται από δύο ομόλογες περιοχές (περιοχή Ν και C). Κάθε περιοχή περιέχει ένα ενεργό κέντρο με ένα συντηρημένο δεσμευτικό μοτίβο ψευδαργύρου HEXXH, όπου οι δύο ιστιδίνες είναι οι δύο πρώτοι υποκαταστάτες του ιόντος ψευδαργύρου. Μετά από 24 αμινοξέα στην αλληλουχία του μορίου, βρίσκεται ένα γλουταμινικό οξύ που είναι ο τρίτος υποκαταστάτης του ιόντος ψευδαργύρου. Η ύπαρξη αυτών των Ν- και C- περιοχών είναι πιθανότατα το αποτέλεσμα ενός αρχέγονου γεγονότος διπλασιασμού γονιδίων το οποίο έλαβε χώρα κατά την διάρκεια της εξέλιξης των σπονδυλωτών. Οι δύο περιοχές εμφανίζουν εκλεκτικότητα έναντι διαφόρων υποστρωμάτων, αναστολέων και διαφορές στην απαιτούμενη συγκέντρωση ιόντων χλωρίου προκειμένου να έχουν καταλυτική δραστικότητα. Υπάρχουν δύο υποστρώματα τα οποία εμφανίζουν εκλεκτικότητα έναντι του Ν-ενεργού κέντρου: το Ν-ακετυλ-σερυλασπαραγυλο-λυσυλ-προλυλ πεπτίδιο, το οποίο ρυθμίζει τη διαφοροποίηση και τον πολλαπλασιασμό των πολυδύναμων αιμοποιητικών κυττάρων και το πεπτίδιο αγγειοτενσίνη-(1-7) που είναι το αποτέλεσμα της δράσης της βραδυκινίνης. Αφ' ετέρου, τα ενεργά κέντρα και των δύο περιοχών καταλύουν την υδρόλυση της αγγειοτενσίνης-Ι και τη βραδυκινίνης με παρόμοια αποτελασματικότητα. Εντούτοις, η αναστολή του Ν-ενεργού κέντρου με το φωσφινικό πεπτίδιο RXP407 δεν έχει καμία επίδραση στην ρύθμιση της αρτηριακής πίεσης. Διαγονιδιακά ποντίκια τα οποία εκφράζουν μόνο το Ν-ενεργό κέντρο εμφανίζουν φαινότυπο παρόμοιο με αυτόν που εμφανίζεται σε ποντίκια στα οποία το γονιδίο του ACE έχει απαλειφθεί πλήρως. Κατά συνέπεια, το C-ενεργό κέντρο φαίνεται να είναι απαραίτητο και σημαντικό για τον έλεγχο της αρτηριακής πίεσης και της καρδιαγγειακής λειτουργίας. Η σπερματική ισομορφή του ACE είναι πανομοιότυπη με την C-περιοχή της σωματικής εκτός από μία μοναδική ακολουθία 36 αμινοξέων που βρίσκεται στο Ν-τελικό άκρο του. Επίσης έχει αποδειχθεί ότι η σπερματική ισομορφή του ACE διαδραματίζει σημαντικό ρόλο στην ωρίμανση του σπέρματος και στη δέσμευση αυτού στο επιθήλιο του ωαγωγού των ωοθηκών. Ο στόχος αυτής της διατριβής ήταν α) η υπερέκφραση, σε βακτηριακά κύτταρα, ο καθαρισμός και η λήψη σε διαλυτή μορφή δύο πεπτιδίων του ACE μεγέθους 108 αμινοξέων(Ala361-Gly468 (ACE_N), Ala959-Ser1066 (ACE_C)). Αυτή η πειραματική προσέγγιση επελέγη λόγω της ευκολίας χειρισμού και καλλιέργειας που εμφανίζουν τα βακτηριακά κύτταρα και λόγω της δυνατότητας της χρήση επισημασμένων με 15Ν ή/και 13C θρεπτικών μέσων. β) Η κατοχή ενός τόσο μεγάλου πεπτιδίου σε διάλυμα, επισημασμένο ή μη, δίνει τη δυνατότητα μελέτης του ως προς τα δομικά χαρακτηριστικά του χρησιμοποιώντας τη φασματοσκοπία κυκλικού διχροϊσμού ή/και πυρηνικού μαγνητικού συντονισμου (NMR). Τα προαναφερθέντα πρωτεϊνικά τμήματα υπερεκφράστηκαν σε βακτηριακά κύτταρα και ελήφθησαν σε καθαρή μορφή. Η καθαρότητά τους ήταν μεγαλύτερη από 99%. Η απόδοση για το πρωτεϊνικό τμήμα ACE_N ήταν 9mg και για το πρωτεϊνικό τμήμα ACE_C ήταν 6mg από 1L καλλιέργειας βακτηριακών κυττάρων. Τα τμήματα αυτά μελετήθηκαν ως προς την δευτεροταγή τους διαμόρφωση με φασματοσκοπία κυκλικού διχρωϊσμού. Τα αποτελέσματα της μελέτης αυτής έδειξαν ότι παρουσία 1,1,1-τριφθοροαιθανόλης, σε συγκέντρωση μεγαλύτερη από 60% και τα δύο τμήματα λαμβάνουν διαμόρφωση η οποία βρίσκεται σε συμφωνία με τη θεωρητικώς υπολογιζόμενη και με αυτή που έχει βρεθεί από κρυσταλλογραφικές μελέτες του ενζύμου. Το αποτέλεσμα αυτό μερικώς επιβεβαιώθηκε για το πρωτεϊνικό τμήμα ACE_N με τη μελέτη αυτού με φασματοσκοπία Πυρηνικού Μαγνητικού Συντονισμόυ. Η πλήρης επιβεβαίωση δεν κατέστει δυνατή λόγω της αδυναμίας λήψης καλής ποιότητας φάσματος 2D-NOESY. Συμπερασματικά, η περιγραφόμενη σε αυτή τη Διατριβή μεθοδολογία εμφανίζει πλεονεκτήματα όσον αφορά την ταχύτητα παραγωγής, τη δυνατότητα καθαρισμού των παραγομένων πεπτιδίων, καθώς και καλή επαναληψιμότητα. Οι in vitro επαναδιατεταγμένες ανασυνδυασμένες πρωτεΐνες εμφάνισαν χαρακτηριστικά δευτεροταγούς δομής, όμοια σχεδόν με αυτά που έχουν αποκαλυφθεί από την κρυσταλλογραφική μελέτη του ACE, έχοντας υψηλό ποσοστό σε α-έλικα. Κατά συνέπεια, αυτή η μελέτη περιγράφει ένα αποτελεσματικό σύστημα για την παραγωγή μεγάλων ποσοτήτων καθαρών πεπτιδίων του ACE που μπορούν να χρησιμοποιηθούν για διάφορες μελέτες. / Angiotensin converting enzyme (ACE) is a gluzincin zinc dipeptidyl carboxypeptidase I, of which thermolysin is considered the prototypical member. This enzyme took its name from its ability to convert the decapeptide Angiotensin-I to octapeptide Angiotensin-II, which is a highly potent vasoconstrictor. Another basic ability is to inactivate bradykinin, a vasodilatory peptide. These two major activities render ACE through the renin– angiotensin–aldosterone system. There are two isoforms of ACE that are transcribed from the same gene in a tissue-specific manner. Somatic ACE, which is present in brush-border epithelial cells and endothelial cells, exists as a glycoprotein composed of a single, large polypeptide chain of 1,306 amino acids, whereas in sperm cells it is a lower-molecular-mass glycoform of 732 amino acids. The somatic form consists of two homologous domains (N and C domain). Each domain contains an active site with a conserved HEXXH zinc binding motif, where the two histidines are zinc ligands, with a glutamate 24 residues downstream forming the third ligand. These N- and C-domains most likely are the result of an ancient gene duplication event that occurred during vertebrate evolution. The two domains differ in their substrate specificities, inhibitor, chloride activation profiles, and physiological functions. There are two N-domainspecific substrates: the peptide N-acetyl-serylaspartyl-lysyl-proline, which regulates haematopoietic stem cell differentiation and proliferation; and the bradykinin-potentiating peptide angiotensin-(1-7). On the other hand, the active sites of both domains catalyse the hydrolysis of angiotensin I and the vasodilator bradykinin with similar efficiency. However, inhibition of the N domain with a phosphinic peptide RXP407 has no effect on blood pressure regulation and expression in transgenic mice of the N domain alone produces a phenotype similar to that seen in complete ACE knockout mice. Thus, the C domain seems to be necessary and sufficient for controlling blood pressure and cardiovascular function, suggesting that the C domain is the dominant angiotensin-converting site. Testis ACE is identical to the Cterminal half of somatic ACE, except for a unique 36-residue sequence constituting its amino terminus. It has also been shown that testis ACE is thought to play a role in sperm maturation and the binding of sperm to the oviduct epithelium. Objective of this thesis was the overexpression, in bacterial cells, purification and solubilazation of two ACE peptides of 108 aa (Ala361-Gly468 (ACE_N), Ala959-Ser1066 (ACE_C)). This experimental approach was chosen because of the ease of culturing bacterial cells and the advantage of using label mediums with 15N and/or 13C. Such large peptides labelled or nonlabelled, can be studied for their structural features using circular dichroism and/or NMR spectroscopy. The above mentioned protein fragments overexpressed in bacteria and purified. Their purity was greater than 99%. The yield was 9mg for ACE_N and 6mg for ACE_C protein fragment from 1L bacterial culture. Their secondary structure was studied using circular dichroism spectroscopy. Deconvolution of ACE_N and ACE_C CD spectra had shown that the presence of trifluoroethanol, at concentrations of 60% or higher, is necessary for the correct folding of the protein. This result was partially confirmed for ACE_N protein fragment by Nuclear Magnetic Resonance spectroscopy. Complete conformation was not succeded due to the inability of recording the 2DNOESY spectrum of ACE_N. Conclusively, the described procedure in this research proved to be advantageous in speed and facility of purification. It demonstrated good reproductively for ACE peptides during purification. The in vitro refolded recombinant proteins had almost identical secondary features compared with these found using crystallographic data, with a high content in a-helix secondary structure motif. Thus, this study offers an effective system for producing large amounts of pure ACE peptides which can be used for several studies.

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