Orientador: Anibal Eugenio Vercesi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T13:52:03Z (GMT). No. of bitstreams: 1 Paim_BrunoAlves_D.pdf: 3170914 bytes, checksum: d4f5cbbb9bfc262aef0879eaf9d377d6 (MD5) Previous issue date: 2008 / Resumo: Neste trabalho, o estresse oxidativo em mitocôndrias isoladas de fígados de camundongos knockout para o receptor de LDL (LDLr-/-) foi caracterizado por apresentar um menor conteúdo de isocitrato mitocondrial, uma reduçãoda razão GSH/GSSG e um maior conteúdo de proteínas oxidadas quando comparados a camundongos controles. A maior produção de EROs pelas mitocôndrias de camundongos LDLr-/- foi prevenida quando as mesmas foram incubadas na presença de isocitrato exógeno, o qual é capaz de manter NADPH completamente reduzido. Resultados semelhantes foram obtidos quando os animais foram tratados com solução de citrato solução de citrato de sódio 55 mM/ ácido cítrico 67 mM durante duas semanas, onde houve uma redução na produção de EROs pelas mitocôndrias isoladas de fígado assim como uma recuperação parcial no consumo de oxigênio mantido por substratos endógenos bem como no perfil de oxidação dos nucleotídeos de piridina. Os linfócitos de camundongos LDLr-/- também apresentaram elevação na produção de EROs, celular e mitocondrial bem como uma elevação nos níveis de Ca2+, citosólico e mitocondrial, quando comparados a camundongos controles. Uma vez que [Ca2+] intracelular tem demonstrado participar na modulação da resposta Th1 e Th2 e no processo de ativação linfocitária, decidimos estudar algumas citocinas em camundongos LDLr-/-. As citocinas Th1 (IL-2 e TNF- a) e Th2 (IL-4 e IL-10) se mostraram elevadas quando comparadas a camundongos controles. A elevação das citocinas Th1 e Th2, a geração de EROs e as elevações das concentrações de Ca2+ foram parcialmente inibidas após tratamento dos camundongos com verapamil, um antagonista de canal de cálcio tipo L. Os resultados nos levam a concluir que o estresse oxidativo observado em mitocôndrias isoladas de fígados de camundongos LDLr-/- ocorre em resposta a uma redução no conteúdo de substratos endógenos redutores de NADPH e que pode, ao menos em parte, ser corrigido após suplementação oral com solução de citrato. Além do mais, foi demonstrado que o Ca2+, em linfócitos isolados de baços de camundongos LDLr-/-, possui papel fundamental modulando a geração de EROs e a produção de citocinas. Estas alterações são provavelmente relacionadas com o processo de ativação linfocitária, em razão das lesões ateroscleróticas observadas no arco aórtico dos camundongos LDLr-/-, formadas espontaneamente / Abstract: In this work, oxidative stress in liver mitochondria isolated from LDLr-/- mice was further characterized by showing a lower mitochondrial isocitrate content, lower matochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to control mice. The higher rate of ROS production in LDLr -/- mitochondria was also prevented by the presence of exogenous isocitrate which maintains NADP+ fully reduced. Similar results were obtained when the annimals were treated with drinking water containing 55 mM sodium citrate/67 mM citric acid during two weeks, when was observed lower levels of ROS production, an improve in the rate of oxygen consumption supported by NADP-linked substrates as well as a higher capacity to sustain reduced NAD(P)H in liver mitochondria. The spleen lymphocytes isolated from k/o mice also showed higher ROS generation by lymphocyte mitochondria and whole cell, and higher levels of cytosolic and mitochondrial Ca2+ compared to control mice. As intracellular Ca2+ has been shown to affect Th1 and Th2 priming and lymphocyte activation we evaluated some cytokines in LDL receptor k/o mice compared to controls. Th1 cytokines (interleukin-2 and TNF-a), as well as Th2 cytokines (interleukin-10 and interleukin-4) were higher in k/o mice than in control. The increase of Th1 and Th2 cytokines, ROS generation, and Ca2+ concentration were partially inhibited by treatment of the k/o mice with verapamil, an L-type Ca2+ channel antagonist. In conclusion, the data demonstrate that the liver mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate. Also, we demonstrate that Ca2+ presents a central role in spleen mononuclear cell from hypercholesterolemic LDL receptor knockout mice modulating ROS generation and cytokines production. These alterations are probably related to lymphocyte activation due the expontaneous atherosclerotic lesion observed in aortic arch of k/o mice / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

The expression of two phenotypically-contrasting LDL receptor mutations was characterized in cultured fibroblasts from the genetically-homozygous Afrikaner subjects, FH1a and lb, and FH3a and 3b, respectively. Surface receptor expression and functional activity were studied by ligand (¹²⁵I-LDL) and monoclonal antibody (¹²⁵I-IgG-C7) binding, and c35s]-methionine pulse-chase experiments were used to analyze biosynthesis, processing and degradation of IgG-C7- immunoprecipitable mutant receptors. Cells from the "receptor-negative" subjects, FH3a and 3b exhibited reduced, but significant (40-60% of normal) LDL receptor synthesis rates. Newly-synthesized precursors were processed slowly (t½ 1.5 hours versus normal t½ of approximately 15 minutes) to mature receptors which reached the cell-surface, but were rapidly degraded thereafter with a half-life of approximately 1.7 hours (normal value 12.6 hours) thus representing a new type of LDL receptor defect. Lysosomotropic weak bases such as ammonium chloride partially inhibited rapid degradation of the mutant receptors, suggesting the involvement of proteolysis in acidic compartments such as lysosomes or endosomes. Fibroblasts from FH1a and lb exhibited normal synthesis rates of LDL receptor precursors that were processed at a severely reduced rate (t½ approximately 5 hours) to functionally heterogeneous mature surface receptors. Onethird of the receptors (20% of normal levels) bound ¹²⁵I-LDL with normal affinity at 4°C and 37°C, whereas the majority were able to recognize only ¹²⁵I-IgG-C7, and apparently showed defective internalisation and subsequent degradation of the bound IgG-C7 at 37°C. The existence of the two receptor populations was further supported by selective intracellular trapping and degradation of only the active, LDL-binding population, in the presence of ammonium chloride and LOL. The abnormal form predominated even in newly-synthesized receptors and reached a maximum of 50-70% of normal levels after 48 hours of upregulation. Upregulation kinetics and degradation rates (t½ = 10-11 hours) of both functionally-active and abnormal receptor populations were similar to normal. A progressive increase in apparent molecular weight of the slowly-processed precursor receptors suggested a possible role for abnormal glycosylation in the formation of both "normal" and abnormal conformations of the same receptor molecule.

An animal model to study complications resulting from childhood obesity is lacking. Our objective was to develop a porcine model for studying mechanisms underlying diet-induced childhood obesity. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing tallow and refined sugars, or a control corn-based diet (n = 11) for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but, by wk 5, consumed more (P < 0.001) energy per kg body weight. At wk 15 and 22, pigs were subjected to an oral glucose tolerance test (OGTT); blood glucose increased (P < 0.05) in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01), even 3 h post-challenge. During OGTT, glucose area under the curve was higher and insulin area under the curve was lower in HED pigs compared to controls (P = 0.001). Pigs given 6 wk of dietary intervention, consuming a control diet, marginally improved glucose area under the curve and LDL-cholesterol although insulin area under the curve was unaffected. Chronic HED intake increased (P < 0.05) subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia; however, a 6 wk dietary intervention partially recovered a normal physiology. These data suggest pre-pubertal pigs fed HED are a viable animal model for studying childhood obesity. / Master of Science

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Hypercholesterolemia is a significant risk factor in the development of cardiovascular disease and is associated with chronic leukocyte adhesion in the microvasculature. While the underlying mechanisms behind this have yet to be determined, it may be possible that hypercholesterolemia impairs the fluid shear stress (FSS) inactivation of neutrophils through the rigidifying effect of cholesterol on membrane fluidity. FSS restricts surface expression of CD18 integrins through cathepsin B (ctsB) proteolysis, which minimizes neutrophil adhesivity. If hypercholesterolemia blocks FSS mechanotransduction, then the inhibition of CD18 cleavage may link pathologic blood cholesterol elevations with dysregulated neutrophil adhesion. We hypothesized that elevated cholesterol contributes to dysregulated neutrophil adhesion by impairing ctsB FSS-induced CD18 cleavage through membrane fluidity changes. In the first part of this study, we demonstrated that FSS-induced CD18 cleavage is a robust response of neutrophils and involves selective cleavage of macrophage 1-antigen (Mac1) through ctsB proteolysis. The second part of this study confirmed that ctsB regulates neutrophil adhesion through its proteolytic actions on Mac1, an important integrin involved in adhesion and chemotaxis. Specifically, ctsB accelerated neutrophil motility through an effect on Mac1 integrins during pseudopod retraction. Furthermore, by using a flow-based assay to quantify the mechanoregulation of neutrophil adhesivity, we demonstrated that FSS-induced ctsB release promoted neutrophil detachment from platelet-coated substrates and unstimulated endothelium. For the third part of this study, we linked cholesterol-related membrane fluidity changes with the ability of FSS to restrict neutrophil adhesion through Mac1. We also determined that pathologic cholesterol elevations associated with hypercholesterolemia could block FSS-induced Mac1 cleavage and were linked to disrupted tissue blood flow. This was accomplished using low-density lipoprotein receptor deficient (LDLR-/-) mice fed a high-fat diet. Ultimately, the results provided in the present study confirmed that cholesterol-related changes in membrane fluidity blocked the ability of ctsB to regulate neutrophil adhesion through FSS-induced Mac1 cleavage. This implicates an impaired neutrophil FSS mechanotransduction response in the dysregulation of neutrophil adhesion associated with hypercholesterolemia. Since dysregulated adhesion may be one of the earliest upstream features of cardiovascular disease associated with hypercholesterolemia, the present study provides a foundation for identifying a new mechanobiological factor in the pathobiology of microcirculatory dysfunction.

Introdução: Doenças cardiovasculares constituem importante causa de morte em todo mundo e a hipercolesterolemia está diretamente relacionada como fator agravante desta morbidade. A dieta desempenha papel importante neste processo e alguns alimentos como o amaranto, especialmente sua proteína, tem mostrado capacidade de redução do colesterol plasmático. Estudos sugerem que este efeito está relacionado a peptídeos formados durante a digestão da sua proteína, os quais desempenham um papel importante na regulação e modulação do metabolismo lipídico. Os efeitos hipocolesterolêmicos, já observados, indicam o uso da proteína do amaranto como um composto bioativo direcionado para a promoção da saúde. Considerando que os efeitos hipocolesterolêmicos destes peptídeos são complexos e há diversas hipóteses formuladas, torna-se importante a realização de estudos visando avaliar a interação dos peptídeos na absorção intestinal do colesterol e da sua modulação genética. Objetivos: Verificar os efeitos do hidrolisado da farinha do grão de amaranto na absorção de colesterol e modulação de genes ABCA1, ABCG1, NPC1L1, AMPK, HMGR e SREBP-2em células Caco-2, e modulação dos genes ABCG8, HMGR, SREBP-2 e AMPKem enterócitos de hamsters. Metodologia: O amaranto foi triturado, sua farinha desengordurada e sua proteína isolada, com posterior digestão in vitro e filtração dos peptídeos. Três experimentos in vitro foram conduzidos com as células: permeação de hidrolisado, permeação de colesterol e de efeito sob a expressão gênica. No primeiro, o hidrolisado proteico de amaranto foi permeado em culturas celulares de Caco-2 no tempo de 2 horas. O permeato foi coletado e analisado por LC/MS/MS. No segundo, o hidrolisado de amaranto foi incorporado a micelas de colesterol e incubados em culturas celulares, nas concentrações de 1,0 mg/ml, e 3,0 mg/ml em tempos de 2h. Também em concentrações de 3,0 mg/ml foi adicionado albumina e caseína para efeito comparativo. O conteúdo de colesterol na porção apical e basolateral foi analisado em HPLC. O terceiro experimento foi avaliaçãoda exposição do hidrolisado, em concentrações de 0,5 mg/ml, 1,0 mg/ml e 3,0 mg/ml, em tempos de 2h e 12h. Após este período, foi realizada a extração de RNA total, avaliação de rendimento e integridade do material; medida quantitativa de expressão de RNAm por RT-PCR e quantificação relativa da expressão por ?CT dos genes ABCA1, ABCG1, ABCG8, NPC1L1, AMP1, HMGR e SREBP-2das células Caco-2 e tecido intestinal de hamsters, coletados em ensaios anteriores. Resultados: Na permeação de colesterol não houve diferença entre as concentrações dos hidrolisados proteicos e controle, porém o hidrolisado de amaranto em 1,0 mg/ml demonstrou uma tendência em diminuir a absorção de colesterol (p = 0,05). Na exposição das células Caco-2 aos peptídeos por 2h, houve uma diminuição nas concentrações de RNAm dos genes ABCA1, NPC1L1, AMPK, HMGR e SREBP-2 nas concentrações de 3,0 mg/ml. O tempo de exposição de 12h apresentou resultados semelhantes ao tempo de 2h. Somente a expressão gênica de ABCG8foi influenciada pelo isolado proteico de amaranto no experimento in vivo. Conclusão: A partir do exposto, podemos concluir que os peptídeos do grão de amaranto influenciam o metabolismo de colesterol por mecanismos genéticos. Portanto, torna-se uma alternativa a ser introduzida na dieta de indivíduos saudáveis e em pacientes com hipercolesterolemia, visando a prevenção de agravos e como estratégia de terapia adicional no controle dos níveis de LDL-c plasmático. Contudo, mais experimentos in vivo e em humanos são necessários para estabelecer a dose efetiva para consumo. / Introduction: Cardiovascular diseases are an important cause of death worldwide and hypercholesterolemia is directly related as an aggravating factor of this morbidity. Diet plays an important role in this process and some foods such as amaranth, especially its protein, have shown ability to lower plasma cholesterol. Studies suggest that this effect is related to peptides formed during the digestion of their protein, which play an important role in the regulation and modulation of lipid metabolism. The hypocholesterolemic effects, already observed, indicate the use of amaranth protein as a bioactive compound aimed to promoting health. Considering that the hypocholesterolemic effects of these peptides are complex and there are several hypotheses formulated, it is important to carry out studies to evaluate the interaction of peptides in the intestinal absorption of cholesterol and its genetic modulation. Objectives: To verify the effects of amaranth grain flour hydrolyzate on cholesterol uptake and ABCA1, ABCG1, NPC1L1, AMPK, HMGR and SREBP-2 genes modulation in Caco-2 intestinal cells, and modulation of ABCG8, HMGR, SREBP-2 genes and AMPK in hamster intestinal cells. Methodology: Amaranth was crushed, the created flour was defatted and its protein isolated, with subsequent in vitro digestion and filtration of the peptides. Three in vitro experiments were conducted with the cells: hydrolyzate permeation, cholesterol permeation and genetic expression. In the first, the amaranth protein hydrolyzate was permeated in Caco-2 cell cultures in the time of 2 hours. The permeate was collected and analyzed by LC/MS/MS. In the second, the amaranth hydrolyzate was incorporated into cholesterol micelles and incubated in cell cultures at concentrations of 1.0 mg/ml and 3.0 mg/ml in times of 2 h. Also, at concentrations of 3.0 mg/ml albumin and casein were added for comparison. Cholesterol content in the apical and basolateral portion was analyzed by HPLC. The third experiment was to evaluate the exposure of the hydrolyzate at concentrations of 0.5 mg/ml, 1.0 mg/ml and 3.0 mg/ml, in times of 2 h and 12 h. After this period, the extraction of total RNA, evaluation of yield and integrity of the material was performed; quantitative measurement of mRNA expression by RT-PCR and relative quantification of ?CT expression of the ABCA1, ABCG1, ABCG8, NPC111, AMPK, HMGR and SREBP-2 genes from Caco-2 cells and hamster intestinal tissue, collected in previous assays, were finalized. Results: In cholesterol permeation there was no difference between the concentrations of the protein hydrolysates and control, but the amaranth hydrolyzate at 1.0 mg/ml showed a tendency to decrease the cholesterol absorption (p = 0.05). Exposure of Caco-2 cells to peptides for 2 h resulted in a decrease in ABCA1, NPC111, AMPK, HMGR and SREBP-2 mRNA levels at concentrations of 3.0 mg/ml. The exposure time of 12h presented results similar to the time of 2h. Only the gene expression of ABCG8 was influenced by the amaranth protein isolate in the in vivo experiment. Conclusion: From the above, we can conclude that amaranth peptides influence the metabolism of cholesterol by genetic mechanisms. Therefore, it becomes an alternative to be introduced in the diet of healthy individuals and in patients with hypercholesterolemia, aiming at the prevention of aggravations and as a strategy of additional therapy in the control of plasma LDL-c levels. However, more studies should bedone with animals and humans to define the dose-efficiency for diet.

Familial hypercholesterolemias (FH) are inherited mutations that cause elevated total cholesterol and low-density lipoprotein cholesterol levels (LDL-C) which lead to premature coronary heart diseases. Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect the patient’s response to the drug. Single Nucleotide Morphism (SNP) mutations in the LDLR, apoB, LDRAP1, and PCSK9 genes are linked to familial hypercholesterolemia. The mutations in the LDLR gene are the most common while mutations in the apoB and PCSK9 genes are the least common in hypercholesterolemia patients. My research will study how pharmacogenetics can be used to diagnose and prescribe patients with FH who have apoB and PCSK9 double gene mutations. I will genotype and sequence the PCR amplified gene segments of the patients with FH to identify any of the 6 apoB SNPs and any of the 3 PCSK9 SNPs that are known. Then, I will provide 4 different treatments: placebo, antisense therapy (mipomersen), PCSK9 inhibitor (alirocumab), and a combination of mipomersen + alirocumab, and I will measure the LDL-C levels before and after a 12-week trial. I hypothesize that individuals with both apoB and PCSK9 gene mutations with the known SNPs that cause loss of function will be more responsive when given both treatments by observing a significant decrease in LDL-C levels.

Background: Recently across the United States, chronic diseases have been becoming more prevalent and compliance rates to recommendations have been declining. Non-adherence to health professionals recommendations creates a greater risk of complications for the patient. Objective: The objective of this study is to discover which populations have the highest prevalence of chronic disease and compare adherence to physicians’ recommendations throughout those populations. If a physician tells a patient to complete a certain behavioral change to improve health, a physician would assume that the adherence level should be nearly 100%, but it is clear adherence levels do not reach those levels. Different demographic factors play a role in adherence: gender, age, race, socioeconomic status, education status, marital status, medical insurance coverage, and comorbidity of chronic diseases. Methods: The 2007-2008 National Health and Nutrition Examination Survey (NHANES), a secondary data source, was used for data collection. The total number of people who participated in the 2007-2008 NHANES study was 5,687. Data analysis was performed with the statistical software program SPSS 19.0. A number of descriptive analyses, cross tabulations, correlations, and binary logistic regression were used to conduct a univariate and multivariate analysis of the subjects. The chronic diseases chosen to assess were hypercholesterolemia, hypertension, and diabetes. The different recommendations were made to all the patients included: eating less fat, control weight, increase exercise, and take a prescription medication for each specific condition Results: The 5,687 participants were included in the study. The prevalence of hypertension was 21.2%, hypercholesterolemia was 19.3%, and diabetes was 8.7% among those who were surveyed. Among those who were told to eat less fat, control weight, increase exercise and take prescription to control their chronic disease condition, adherence levels ranged greatly. The significance of a physician recommending a behavioral change had the biggest impact on whether a patient would adhere. No significance was seen between any of the demographic variables except for marital status for those who were told to take a prescription to control hypercholesterolemia. Discussion: The study has proven physician recommendations to control chronic disease are usually to take a medication. An individual’s demographics have a small impact on whether he or she will adhere to the advice of the physician. Additional research needs to be completed to understand the patient to physician relationship, which seems to have the biggest impact on behavioral change. Furthermore, new interventions are needed to increase adherence to 100%. Increasing chronic disease adherence across the United States will result in decrease spending in health care costs in the United States.

Cardiovascular disease (CVD) is ranked as one of top leading causes of death in most industrialized countries. Recent research suggests that fruit vinegar beverages (FVB) possess beneficial effects such as antihypertensive properties, reduction of serum cholesterol and triacylglycerols (TAG). FVB made using apple, blueberry, cranberry and tomato were evaluated for their sensory, antioxidant, antihypertensive and lipid lowering properties. All four treatments demonstrated very high in vitro antioxidant and antihypertensive properties. These FVB were further evaluated for their hypolipidemic and antihypertensive properties using a spontaneously hypertensive rats (SHR) model with diet-induced hyperlipidemia. All four FVB significantly reduced serum TAG, elevated the high density lipoprotein (HDL)-cholesterol compared to the control. Further, all four FVB demonstrated a reduction in the diastolic blood pressure after four weeks of supplementation. Overall, the FVB exhibited lipid lowering effects and antihypertensive properties in vivo. Confirmation of the beneficial effects of FVB using a clinical trial is needed.

Thèse (Med.Exp.) -- Université du Québec à Chicoutimi, programme en extension de l'Université Laval, 2006. / La p. de t. porte en outre: Mémoire présenté à la faculté des études supérieures de l'Université Laval comme exigence partielle du programme de maîtrise en médecine expérimentale - génétique des populations humaines offert à l'Université du Québec à Chicoutimi en vertu d'un protocole d'entente avec l'Université Laval pour l'obtention du grade de maître ès sciences (M.Sc.). CaQCU Bibliogr.: f. 63-101. Document électronique également accessible en format PDF. CaQCU