Regensburg, Univ., Diss., 2009.

Aims Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). Methods and resultsCFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 25 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 824 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β1 integrin receptor blockade by Tac β1 expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. Conclusion These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line. Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally. Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line.Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally.Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

Angiotensinogen is a blood protein that plays a critical role in the regulation of blood pressure in the body. This protein exists in two forms, oxidised and reduced, determined by the presence or absence of a disulfide bridge between Cys 18 and Cys 138. The ratio of oxidised to reduced angiotensinogen is 60:40 in the blood of healthy individuals - an equilibrium that is disrupted in women who develop pre-eclampsia, leading to a higher proportion of oxidised angiotensinogen in the blood. Pre-eclampsia, one of the leading causes of premature births, is a severe and potentially fatal pregnancy condition characterised by the sudden onset of symptoms such as high blood pressure and proteinuria typically during the third trimester. This condition is responsible for an estimated 550,000 deaths globally each year, and with no available treatment or cure other than early delivery of the child, there is a desperate need for a reliable and predictive diagnostic test for this condition. Can we use angiotensinogen as a biomarker for the early diagnosis of pre-eclampsia? Being able to distinguish between reduced and oxidised angiotensinogen and determine the relative amounts of each in blood samples would be of a huge diagnostic value for this condition. This thesis outlines the expression and purification of recombinant human angiotensinogen in Escherichia coli, and the development of an antibody-based SPR assay for angiotensinogen that was subsequently used to probe whether reduced and oxidised angiotensinogen can be distinguished experimentally. The assay developed was sensitive and reproducible, and demonstrated that the reduced and oxidised forms can be distinguished experimentally. The antibody bound the two forms with differential affinity, due to differences in both the association and dissociation rates of the two forms with the monoclonal antibody. Finally, in an attempt to further elucidate the differences between the two redox states of angiotensinogen, molecular dynamic simulations were carried out on angiotensinogen in the presence or absence of the disulfide bond between Cys 18 and Cys 138. These simulations revealed some quite striking differences in the dynamics between the two forms. Reduced angiotensinogen was found to be more dynamic in regions critical for binding to renin, providing a possible explanation for the reported differential affinity that renin displays for the two forms.1 Thus, reduced and oxidised angiotensinogen show some quite distinct differences and can be distinguished in an SPR-based assay, highlighting their potential for use as a biomarker in a diagnostic bioassay.

Women seem to be largely protected against certain ‘welfare disorders’ such as cardiovacular disease and osteoporosis, during their fertile years.The metabolic changes observed during women’s non-menstrual states, i.e. during pregnancy, after the menopause and during use of oral contraceptives, indicate the importance of sex steroids and an undisturbed biological rhythm. Treatment with monophasic, combined oral contraceptives constitutes a model for the non-cyclic state.Growth hormone (GH) is a pituitary hormone that has major metabolic effects. The pattern of GH exposure to the target organ is of vital importance for the effects and changes in rhythm could possibly induce metabolic changes.Growth hormome, cholecystokinin (CCK), osteocalcin and angiotensinogen were used as markers for metabolic effects and the concentrations in serum were recorded in women during non-menstrual states. The clinical material comprised a total of 60 women: 18 healthy non-pregnant, 25 pregnant, one lactating woman and 16 postmenopausal women. Using a portable pump and a non-thrombogenic venous catheter, blood samples could be collected at 30-min intervals during 24-h periods. Furthermore, the effects of estrogen and GH in the regulation of angiotensinogen were investigated in an experimental model in the rat.Oral contraceptives were found to alter the secretion of GH towards a pattern of lower and more frequent peaks, though the total amount secreted during 24 h was unchanged. Oral contraceptives seem to induce a suppression of the 24-h concentrations of CCK, which may be important with respect to weight gain in some women. Osteocalcin in serum display a significant circadian variation. This emphasizes the need for careful timing of single point measurements and the value of continuous blood sampling. Oral contraceptives may reduce osteocalcin serum concentrations. The long-term effects on bone are unknown. During late pregnancy osteocalcin levels are extremely low, which could indicate osteoblast inhibition and reduced bone turnover. The mode of GH administration is important for the plasma concentration of angiotensinogen in the non-pregnant rat. Estrogen effects on this protein may be mediated via a modification of GH secretion. Oral contraceptives not only increase angiotensinogen concentrations in serum but also markedly enhance their variability. Further studies are needed to elucidate the relation between the individual pattern of angiotensinogen and hypertension. / <p>S. 1-42: sammanfattning, s. 43-88: 6 uppsatser</p> / digitalisering@umu

[Truncated abstract] Our understanding of the actions of oestrogen on the vasculature has recently been questioned following the results of large clinical trials revealing a negative effect of hormone replacement therapy (HRT) on cardiovascular disease (CVD) risk amongst postmenopausal women. It is important to determine how a hormone with numerous positive effects on intermediate pathways of atherosclerosis fails to offer cardioprotection. Further investigation into the actions of oestrogen in the vasculature may add to our current understanding of the pathogenesis of atherosclerosis and oestrogen biology. The primary aim of this thesis was to investigate involvement of the oestrogen receptors (ERs) in atherosclerotic CVD and to provide further insight into the actions of oestrogen on the vasculature by studying the actions of oestrogen on the regulation of an oestrogen-responsive gene within human vascular cells. Following confirmation of ERa and ERß expression at the RNA and protein level in human aorta sections, correlations of receptor expression with age and atherosclerosis were examined. Significantly strong negative relationships of ERa, androgen receptor (AR), and progesterone receptor (PR) with age in both males and females were detected. No trend was detected between ERß expression and age. These findings suggest that the receptor-mediated actions of hormones in the vasculature may change with age. Further, this thesis compared for the first time sex hormone receptor expression in normal and adjacent atherosclerotic aortic tissue providing a critical assessment of receptor differences due to atherosclerosis. Results revealed reductions of all hormone receptors in early atherosclerotic versus normal aorta tissue. ... These results suggest that the 3'-UTR SNPS may have more of an influence on carotid thickening when oestrogen levels are lower, suggesting the importance of both genetic variation of the ERß gene and oestrogen status on carotid thickening. Finally, this was the first study to investigate oestrogen-induced regulation of angiotensinogen (AGT), a candidate gene for CVD, in human vascular cells. Oestrogen influenced AGT transcription in a cell specific manner. The overall influence of oestrogen on AGT transcription in the vasculature is unknown. This thesis adds to the knowledge of oestrogen and atherosclerosis by suggesting the involvement of the sex hormone receptors (ERa, ERß, PR and AR) in atherosclerosis, presenting ERß as a potentially important candidate gene for atherosclerosis, revealing interactions between estrogen status and associations of ERß SNPs with carotid thickening, and demonstrating vascular cell-specific actions of oestrogen on the regulation of a candidate gene for CVD. These factors may have contributed to the lack of cardio-protection following HRT, as revealed by large clinical trials.

Orientador: Marcio Ajudarte Lopes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:31:33Z (GMT). No. of bitstreams: 1 Flores_IsadoraLuana_D.pdf: 2280685 bytes, checksum: 4c70e1fe2979897700a7a4161710b445 (MD5) Previous issue date: 2014 / Resumo: OBJETIVO: A leucoplasia verrucosa proliferativa (LVP) é uma variante rara e ainda pouco compreendida de leucoplasia oral com um comportamento de progressão persistente para malignidade apresentando uma taxa de malignização entre 40-100%. Além disso, a detecção precoce da LVP às vezes é um desafio para os clínicos, porém desempenha um papel crucial para estabelecer um contínuo e rigoroso acompanhamento. Aspectos moleculares subjacentes são relevantes e nenhum estudo anterior investigou a saliva de pacientes com LVP. O aumento do interesse no estudo do proteoma salivar ocorre porque as proteínas são consideradas as moléculas mais importantes do fluido salivar com potencial para atuar como biomarcador para o diagnóstico de várias doenças sistêmicas e locais. Com base nestes aspectos, o presente estudo teve como objetivo traçar o perfil do proteoma salivar de pacientes com LVP, a fim de identificar potenciais biomarcadores para a melhor compreensão desta entidade visando o possível uso clínico. MATERIAIS E MÉTODOS: A saliva total não estimulada foi coletada de 30 voluntários (15 pacientes com LVP e 15 controles). Uma abordagem proteômica baseada na associação de cromatografia líquida acoplada à espectrometria de massa foi realizada para análise de 20 µg de proteínas das amostras. Os testes de qui-quadrado, análise de variância e regressão logística foram utilizados na análise estatística. RESULTADOS: Um total de duzentas e oitenta e três proteínas foram identificadas. Entre estas, 31 proteínas apresentaram diferença estatisticamente significativa em relação à abundância, sendo 25 proteínas com maior abundância no grupo controle e 6 proteínas com maior abundância no grupo LVP. A combinação das proteínas angiotensinogênio e dipeptidil peptidase 1 criaram um modelo de diferenciação de grupo com um índice de concordância de 94,2% revelando ambas as proteínas como potenciais biomarcadores para o diagnóstico de LPV. CONCLUSÕES: Apesar deste estudo ser o primeiro a avaliar o proteoma salivar em pacientes com LVP, os resultados mostraram que a triagem da saliva pode ser um teste útil no diagnóstico de indivíduos com risco para o desenvolvimento de LPV / Abstract: OBJECTIVE: Proliferative verrucous leukoplakia (PVL) is a rare variant and still poorly understood of oral leukoplakia with a behavior of persistent progression to malignancy showing a rate of malignancy between 40-100%. Moreover, the early detection of PVL is sometimes challenging for clinicians, but plays a crucial role to establish a continuous and rigorous follow-up. Underlying molecular aspects are relevants and no previous study investigated the saliva of PVL patients. The increased interest in the salivary proteome study is because proteins are considered the most important molecules in the salivary fluid with potential to act as biomarker for diagnosis of various systemic and local diseases. Based on these aspects, the present study aimed to draw the salivary proteome profile of patients with PVL in order to identify potential biomarkers to better understanding of this entity targeting the possible clinical use. MATERIALS AND METHODS: Unstimulated whole-mouth saliva was collected of 30 voluntaries (15 PVL patients and 15 controls). Proteomic approach based to liquid chromatography coupled to tandem mass spectrometry was performed to 20 µg of proteins of the samples. Chi-Square, analysis of variance and logistic regression test were used in the statistical analysis. RESULTS: A total of two hundred eighty-three proteins were identified. Among of them, 31 proteins showed statistical significance difference in relation to abundance, being 25 proteins with higher abundance in control group and 6 proteins with higher abundance in PVL group. The combination of angiotensinogen and dipeptidyl peptidase 1 created a model for group differentiation with a concordance index of 94.2% revealing both proteins as potential biomarkers for diagnosis of PVL. CONCLUSIONS: Although this study is the first to evaluate the salivary proteome in PVL patients, the results showed that saliva screening may be helpful test to diagnosis of individuals with risk to PVL development / Doutorado / Patologia / Doutora em Estomatopatologia

No description available.

No description available.