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Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-12-12T13:56:18Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_Jessica Catarine Frutuoso do Nascimento.pdf: 3226012 bytes, checksum: 795583806a66d8ac66b9fbdb738f7d93 (MD5) / Made available in DSpace on 2016-12-12T13:56:18Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_Jessica Catarine Frutuoso do Nascimento.pdf: 3226012 bytes, checksum: 795583806a66d8ac66b9fbdb738f7d93 (MD5) Previous issue date: 2015-02-24 / CAPES / FACEPE / CNPQ / O carcinoma ductal invasivo (CDI) é o tumor maligno de mama mais comum e uma das principais causas de morte relacionada ao câncer em mulheres no mundo. A alteração no padrão de glicosilação é uma característica marcante do fenótipo tumoral. Dentre as reações glicosídicas alteradas no câncer está a fucosilação. Os tetrassacarídeos fucosilados sialil Lewis X (sLex) e sialil Lewis A (sLea) são ligantes reconhecidos pelas glicoproteínas transmembrânicas selectinas envolvidos nas interações célula-célula necessárias nos processos inflamatórios, hemostase/trombose, cicatrização de feridas e metástase tumoral. A etapa final na síntese do sLex e sLea é realizada pela ação da α1,3/4-fucosiltransferase (FUT3), enzima codificada pelo gene FUT3. A expressão do sLea em carcinoma mamário está relacionada ao estágio tumoral e maiores níveis desse antígeno foram encontrados em tumores metastáticos. Níveis elevados da enzima FUT3 está relacionada ao maior poder metastático em linhagens celulares de câncer de próstata e pâncreas e sua ação é fundamental para o mecanismo de transição epitelial-mesenquimal induzido por TGF-β no câncer colorretal. Apesar da ação pró-tumoral exercida pela enzima FUT3 e seus produtos, estudos vem demonstrando sua importância para a citotoxicidade mediada pelas células NK sobre células tumorais, tanto devido ao reconhecimento do antígeno sLex pelos receptores lectina do tipo C quanto devido a fucosilação dos receptores DR4 e DR5 por essa enzima que é fundamental para o desencadeamento da via de apoptose extrínseca estimulada pelo Apo2L-TRAIL. Visando o maior conhecimento do papel dessa enzima no câncer de mama, o presente trabalho objetivou avaliar os níveis teciduais da FUT3 em tumores mamários malignos (carcinoma ductal invasivo - CDI) de pacientes do Hospital das Clínicas da UFPE (HCUFPE) e do Instituto de Medicina Integral Professor Fernando Figueira (IMIP), investigando se há correlação entre a expressão enzimática com a malignidade tumoral e o risco de metástase. A genotipagem da região promotora do gene FUT3 também foi realizada a fim de identificar possíveis SNPs relacionados à expressão dessa enzima. Para tal biópsias em parafina de carcinoma ductal invasivo (CDI) foram selecionadas no arquivo do Setor de Anatomia Patológica do HC-UFPE e do IMIP. Os níveis teciduais da FUT3 foram avaliados por imuno-histoquímica. O DNA foi extraído por metodologia adaptada de Ramalho et al. (2014), a região promotora amplificada por PCR e posteriormente sequenciada pelo método de Sanger modificado. As sequências obtidas em duplicata foram analisadas através do software CLC Main Workbench. A análise estatística foi realizada através do teste exato de Fisher para os dados de expressão e pelo teste de Qui quadrado para a análise genômica, ambas as análises utilizando o software GraphPad Prism v.5. Nossos resultados demonstraram que a ausência tecidual da enzima FUT3 está relacionada ao CDI em pacientes brasileiros, sendo mais freqüente em tumores maiores e negativos para o receptor do fator de crescimento epidérmico humano 2 (HER2). Análise genômica mostrou que duas variações localizadas na região promotora do gene FUT3 estão associadas ao CDI, embora o efeito direto desses polimorfismos na expressão da FUT3 não pode ser avaliada. O alelo T do SNP rs73920070 (-6933 C> T) está associado a ausência da neoplasia enquanto que o alelo T do SNP rs2306969 (-6951 C> T) está associado a presença do carcinoma ductal invasivo na população brasileira. / Invasive ductal carcinoma (IDC) is the most common breast malignant tumor and the mainly cause of death related to cancer among women in the world. The alteration of glycosylation pattern is a well established feature of tumor phenotype. Fucosylation is one of main glycosidic changes in cancer. The fucosylated tetrasacarides sialil Lewis X (sLex) and sialil Lewis A (sLea) are ligands recognized by the transmembrane glycoproteins selectins involved in cell-cell interactions during the inflammatory process, hemostasis/thrombosis, wound healing and tumor metastasis. The final step in sLex and sLea synthesis is done by the action of α1,3/4-fucosyltransferase (FUT3), enzyme encoded by FUT3 gene. The expression of sLea in mammary carcinoma is related to tumor stage and higher levels of this antigen were found in metastatic tumors. Higher protein expression of FUT3 were related to a bigger metastatic power in prostate and pancreas cancer cell lines and its action is primordial to epithelial-mesenchymal transition induced by TGF-β in colorectal cancer. Despite the protumoral action of FUT3 enzyme and its products, studies have shown their importance to NK cell-mediated citotoxicity against tumor cells, due to the sLex antigen recognition by type C lectin receptors and due to the fucosylation of DR4 and DR5 receptors, fundamental step to the extrinsic pathway of apoptosis stimulated by Apo2L-TRAIL. Aiming to better understand the role of this enzyme in breast cancer, the purpose of this study was evaluate the tissue protein expression of FUT3 in breast malignancies (invasive ductal carcinoma – IDC) in patients from Hospital das Clínicas da UFPE (HC-UFPE) and Instituto de Medicina Integral Professor Fernando Figueira (IMIP). We investigated whether there is correlation between the FUT3 enzymatic expression with malignancy and metastasis risk in this cancer type. The genotyping of the FUT3 promoter region was also realized in order to identify SNPs with potential to interfere on the enzyme expression. IDC formalin-fixed and paraffin-embedded biopsies were selected from pathological anatomy service from HC-UFPE and IMIP. FUT3 tissue levels were evaluated by immunohistochemistry. DNA was extracted using the adapted methodology from Ramalho et al. (2014), the promoter region was amplified by PCR and next sequenced by Sanger modified method. The sequences obtained in duplicate were analyzed using the CLC Main Workbench software. Statistical analyzes were realized using Fisher’s exact test for expression data and Qui square test for genomic data. Both analyzes were conducted using GraphPad Prism software v. 5. Our results demonstrate that the lack of FUT3 expression in breast tissues is related to the presence of IDC in Brazilian patients. No expression of FUT3 was more frequent in patients with large neoplastic lesions and tumors that do not express the human epidermal growth factor receptor 2 (HER2). Genomic analyzes showed that two variations localized in FUT3 promoter region are statistically associated to IDC, however the direct effect of these polymorphisms in FUT3 enzyme expression is still to be evaluated. The T allele of rs73920070 (-6933 C> T) SNP is associated to the neoplasia absence while the T allele of rs2306969 (-6951 C> T) SNP is associated to IDC presence in Brazilian northeastern population.

Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [to full text]

Herpes simplex virus (HSV) causes recurrent orofacial and genital infections and establishes latent infection in sensory neurons. During latency all virus genes are supressed except the latency associated transcripts which are transcribed from latency associated gene (LAT). It is established that HSV LAT promoter mutants have lower levels of spontaneous reactivation rates in small animal models compared to wild virus. However, the variation in the LAT promoter has not been studied in viruses from clinical samples in humans. The aim of the sudy was to evaluate the sequence variation in herpes simplex virus latency associated gene promoter from clinical samples by developing and applying molecular methods and correlate with herpes infection clinical features. In this study a new PCR method specific for HSV LAT promoter was developed and HSV LAT promoter DNA sequences from Lithuanian and Swedish mucocutaneous and cerebrospinal fluid clinical samples were analyzed. HSV type 2 was found to be the main cause of genital herpes in the population of the Lithuanian patients. All cases of orofacial herpes simplex infection were caused by HSV type 1. The structure of the LAT promoter region was studied in 145 HSV clinical samples. HSV LAT promoter was found to be G+C rich and contained variable homopolimer tracts. An inter- and intrastrain variability of homopolimer tracts in the promoter region was detected, potentially giving rise to a large variation at the protein level, leading to... [to full text] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą]

Bilirubin is a major product of the heme catabolism in the vascular bed with substantial antioxidant properties. These importantly contribute to pathogenesis of diseases associated with increased oxidative stress, including cardiovascular or cancer diseases. In the first part of this PhD project serum bilirubin concentrations were examined in the 1 % representative sample of the general Czech population, together with determination of the prevalence of Gilbert's syndrome. Bilirubin concentrations were determined also within individual polymorphisms of the UGT1A1 gene (OMIM*191740) responsible for bilirubin biotransformation in the liver, including their association with the basic risk factors for atherosclerosis. We also assessed the activity of the standard liver enzymes (representing another significant risk factor for the development of cardiovascular diseases) with surprisingly high proportion of subjects with elevated values. Simultaneously, we determined the concentrations of serum bilirubin in a group of patients with an acute coronary syndrome, who manifested with significantly lower concentrations as compared to general population. In the second part of this research project, the relationship between plasma concentrations of bilirubin and individual variants of UGT1A1 gene polymorphisms...

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

Niemann-Pick disease type C (NPC) is a rare, severe disease with autosomal recessive inheritance. Disease is caused by pathogenic mutations located in genes NPC1/NPC2. These genes encode lysosomal non enzymatic NPC1/NPC2 proteins that are part of lipid transport. As a result of malfunction of these proteins intracellular accumulation of lipids occurs, in particular free cholesterol and glycolipids. Causal therapy is currently still unsatisfactory therefore new therapies are evolved. However these therapies depend on whether the patient cells contain at least residual amount of transcript NPC1 gene. In a group of patiens, for which a fibroblast culture was available, I analyzed the effect of pathogenic mutations on the expression level of the transcript. Results showed that for all pathogenic mutations transcript level is low, but detectable. Moreover, I characterized the structure of the NPC1 gene promoter. By sequence analysis I found polymorphisms rs8099071, rs28403610, rs2981422, rs1652354, rs1788774, rs1788772 in promoter. On the basis of the composition of polymorphisms in individual patiens, I estimate six different haplotypes. I performed mutation analysis in DNA of recently diagnosed patient. I found only one pathogenic mutation p.I1061T (c.3182T> C) in the NPC1 gene. Therefore I tested...