Abstract not available

Skin is the largest organ in the body that protects the organism from environmental, chemical and physical traumas of each passing day. The protective skin epidermal permeability barrier (EPB) is formed within the exterior layers of the epidermis, which are regularly sloughed off and repopulated by movement of inner cells. The epidermal permeability barrier is established during in utero development and maintained through lifetime. Impaired epidermal barrier formation is one of the major features of several dermatoses such as psoriasis and atopic dermatitis. Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (Ctip2), also known as Bcl11b, is a C���H��� zinc finger protein expressed in many organs and tissues. It has been shown to regulate the development of thymocyte, tooth and corticospinal motor neurons. Ctip2 is highly expressed in mouse epidermis during skin organogenesis and in adulthood. It is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Germline (Ctip2- null mice) and selective ablation of Ctip2 in mouse epidermis (Ctip2[superscript ep-/-] mice) leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation and terminal differentiation as well as altered lipid distribution during embryogenesis. Sphingolipids account for ~50% of total skin lipids by weight and are crucial components of epidermal barrier. We have recently identified Ctip2 as a key regulator of skin lipid metabolism. Germline deletion of Ctip2 in mouse embryos leads to altered lipid composition in the developing mouse epidermis by modulating the expression levels of key enzymes involved in lipid metabolism (bio-synthesis and catabolism). We also demonstrated that Ctip2 is recruited to the promoter regions of several genes involved in the ceramide and sphingomyelin biosynthesis pathways and could directly regulate their expression. Thus, we have identified Ctip2 as a key regulator of several lipid metabolizing genes and hence epidermal sphingolipid biosynthesis during skin development. To study the role of Ctip2 in adult skin homeostasis, we have utilized Ctip2[superscript ep-/-] mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. We showed that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as high infiltration of T lymphocytes and immune cells. We have also observed increased expression of Th2-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression is significantly upregulated in the mutant epidermis as early as postnatal day 1 and Ctip2 was recruited to the promoter region of the TSLP gene in mouse epidermal keratinocytes. The results suggest that upregulation of TSLP expression in the Ctip2[superscript ep-/-] epidermis could be due to a derepression of gene transcription in absence of Ctip2. Thus, our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult skin, as well as a non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines in adult mouse skin. Present results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism mediated by Ctip2 in mouse epidermal keratinocytes. Altogether, our study indicates that Ctip2 could be involved in a diverse range of biological events in skin including barrier formation, maintenance and epidermal homeostasis. Ctip2 appears to be a master regulator in skin barrier functions by directly regulating the transcription of a subset of genes involved in lipid metabolism and inflammatory responses. / Graduation date: 2013

This study was conducted to determine the influence of factors on the efficiency of protein utilization and the rate of lipid deposition and withdrawal in growing pigs. Two experiments were conducted in total. The first experiment involved fifty-two crossbred entire Large White x Landrace male pigs, individually penned, which were used to test the proposition that the efficiency of protein utilisation is influenced by the body composition of the pig at the start of the trial. The experiment was divided into two phases: in the first period, starting at 20kg liveweight, when 3 pigs were slaughtered to determine the initial body composition of the pigs on the trial, the remaining 48 pigs were divided into three groups, two of which were fed ad libitum, with 11 pigs being offered a feed high in crude protein (HP, 1979 CP/kg) and 19 pigs being offered a low CP (LP, 166g/kg) feed. The remaining 19 pigs were fed HP on a restricted basis; the daily allowance being 0.7 of the mean intake of those pigs fed HP ad libitum. The objective of this initial period was to create three groups of pigs differing in body lipid content. As each pig achieved a protein weight of approximately 5.9kg, predicted to occur when the pigs on the three treatments reached live weights of 35, 39 and 34kg respectively, the pig entered phase 2 of the trial. At this stage three pigs from each treatment (a total of 9 pigs) were slaughtered for carcass analysis, the protein contents being approximately 5.9kg, and lipid contents being 85,98 and 87g/kg for the 3 treatments respectively. During phase 2, the 8 pigs fed HP in phase 1 continued to be fed HP in phase 2; 8 pigs were chosen at random from those fed LP in phase 1 and were allocated the high CP basal feed, while the remaining 8 were given LP; and 8 of the pigs feed-restricted in phase 1 were randomly chosen and fed HP, while the remaining 8 were given LP. All pigs were fed ad libitum during phase 2. Four pigs from each treatment in phase 2 were slaughtered after 1 week and the remaining 4 a week later for analysis of body composition In the first week of the second phase of the trial protein gain was highest (264g/d) on the pigs previously restricted and then fed HP, followed by those previously fed LP and then HP (242g/d), with pigs previously restricted and then fed LP depositing the least amount of protein (192g/d). Pigs fed LP or HP throughout, had protein gains of 217 and 210g/d, respectively. Efficiencies of utilization of dietary protein did not differ significantly between treatments, however, the highest being measured in pigs fed LP throughout (461g/kg), followed in order by those fed LP and then HP (457g/kg), those fed HP throughout (404g/kg), those previously restricted and then fed LP (394g/kg), with those previously restricted and then fed HP being the least efficient (372g/kg). The second experiment involved twenty-six male and twenty-six female crossbred Large White x Landrace pigs, individually penned, which were used to determine the maximum rate at which growing pigs can gain lipid. The experiment was divided into three phases: In the first, starting at 20kg live weight (56 days old), when two males and 2 females were slaughtered to determine the initial body composition of the pigs on the trial, the remaining 24 males and 24 females were randomly allocated to their various treatments. The treatments consisted of a feed high in crude protein (H, 197g/kg), a feed low in CP (L, 166g/kg) and three blends, namely 5OH/5OL (180g/kg) (male diet), 30H/70L (167g/kg) (both male and female diets) and 20H/80L (162g/kg) (female diet). Six pigs from each sex were allocated to each treatment. The EFG Pig Growth Model was used to determine the fat contents (lipid index) on the two feeds available and the three blends, to estimate the best times to sample pigs. It was estimated that phase 1 would terminate at 63 d, phase 2 at 70 d and phase 3 at 77 d of age. At the end of each phase two pigs from each sex and treatment were slaughtered. The lipid contents differed significantly between treatments at the end of phase 2 for the male pigs, with the highest being measured in pigs fed L (108g/kg), followed in order by those fed 70L/30H (86g/kg), those fed 5OL/5OH (74g/kg), and those fed H (68g/kg) with the least lipid content. The lipid contents of the female pigs were highly significantly different at the end of phase 3, with the highest being measured in pigs fed L (147g/kg), followed in order by those fed 80L/20H (124g/kg), those fed 70L/3OH (116g/kg) and the least lipid content from those fed H (115g/kg). As estimated by the EFG Pig Growth Model, the male and female pigs fed L treatment had the highest lipid content and those fed H treatment, achieving their target rate of lipid deposition, with the lowest lipid content. This study indicates that the response in protein gain and in efficiency of utilization of protein of pigs to a given feed is dependent on the amount and quality of the feed given to the animals previously. Also, the maximum rate of lipid deposition can be achieved by monitoring the changes in lipid deposition over a period of time, which enables an enhanced understanding of the theory of food intake regulation in a growing pig. As a result, accurate changes can be made when designing a phase-feeding program for growing pigs. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Orientador: Mara Patricia Traina Chacon Mikahil / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Educação Fisica / Made available in DSpace on 2018-08-14T05:19:49Z (GMT). No. of bitstreams: 1 Salgado_JoseVitorVieira_M.pdf: 867713 bytes, checksum: 6908f05e1d80d8d4a637179e624366e2 (MD5) Previous issue date: 2009 / Resumo: O avançar da idade aliado à inatividade física contribuem para o surgimento de uma série de doenças metabólicas dentre as quais se destacam a hipertensão arterial, obesidade, síndrome da resistência à insulina e dislipidemia, levando a um quadro de síndrome metabólica (SM) aumentando o risco de desenvolvimento de diabetes mellitus e doenças cardiovasculares. O presente estudo objetivou investigar se períodos curtos de intervenção com treinamento aeróbio de baixa a moderada intensidades podem promover alterações nos marcadores de risco de doenças cardiovasculares em homens de meia-idade. Além disso, procurou comparar essas possíveis alterações com indivíduos de mesma faixa etária praticantes de treinamento aeróbio regular há alguns anos (corredores de longa distância). Compuseram a amostra dois grupos de indivíduos do sexo masculino de meia idade, sendo que um dos grupos (n=15, 46,45±4,50 anos, 173,59±7.52 cm) submetido a um treinamento aeróbio com intensidades entre 60% e 80% do VO2pico durante 16 semanas com 3 sessões semanais e avaliado nos dois momentos distintos: chamados de pré treinamento aeróbio (PRÉ-TA) e pós treinamento aeróbio (PÓS-TA): massa corporal total 80,77±8,67kg e PÓS-TA: massa corporal total 78,83±9,01kg. Um segundo grupo estudado foi o de corredores de longa distância (C) (n=21, 47,43± 5,77 anos, massa corporal total 66,97±10,56 kg, 171,05±7,47 cm), com um tempo médio de treinamento 15,38 ± 8,94 anos percorrendo semanalmente 62,95 ± 32,04 km e com tempo médio de performance para 10km de 45'36" ± 6'32". Foi avaliado diretamente o consumo máximo de oxigênio (VO2pico), composição corporal, perfil lipídio, PCR, hemoglobina glicada, glicose de jejum, insulina basal; a resistência à insulina foi estimada por meio do método Homeostasis Model Assessment (HOMA-IR). A análise estatística foi realizada intergrupos e intragrupos adotando um valor de p<0,05. Foi observada diferença significante entre o grupo PRÉ-TA e PÓS-TA, com redução dos valores para as variáveis: massa corporal total (p=0,011), IMC (p=0,011), % de gordura (p=0,023), massa gorda (p=0,012), circunferência de cintura (p=0,001), colesterol total (p=0,009), LDL (p=0,038), insulina basal (p=0,036) e hemoglobina glicada (p=0,031) e aumento no consumo máximo de oxigênio (p=0,000). Contudo, para a massa magra, HDL, triglicérides, glicemia, HOMA e PCR não foram observadas diferenças estatísticas significantes. Quando comparados PÓS-TA e corredores somente não foram observadas diferenças estatísticas para a MM, colesterol total, LDL, triglicérides, glicemia e PCR, com menores valores para o grupo de corredores com exceção do maior VO2pico e HDL. Indivíduos de meiaidade apresentaram alterações positivas nos marcadores de risco de doenças cardiovasculares após a realização de apenas 16 semanas de treinamento aeróbio. No entanto, indivíduos que realizam TA durante anos, em intensidades superiores de treino demonstraram ter benefícios ainda maiores decorrentes do exercício físico, evidenciando o importante papel do exercício físico, em menor ou maior quantidade e intensidade, como sinalizador do controle de fatores de risco para o desenvolvimento de doenças associadas à síndrome metabólica / Abstract: The advancement of age combined with physical inactivity contribute to the emergence of a number of metabolic diseases among which are included; arterial hypertension, obesity, syndrome of insulin resistance and dyslipidemia. These could lead to a metabolic syndrome (MS) and increase the risks of developing diabetes and cardiovascular diseases. Thus, this study aims at investigating whether short periods of intervention with aerobic training of low to moderate intensities can promote changes in markers of risks of cardiovascular diseases in middle-aged men. Furthermore, we try to compare these changes with individuals of the same age who have been practitioners of regular aerobic training for several years (long-distance runners). The sample was composed of two groups of males of middle age, with one group (n = 15) 46,45 ± 4,50 years, 173,59 ± 7,52 cm subjected to an aerobic training with intensities between 60% and 80% of VO2peak for 16 weeks with 3 sessions per week and assessed in two distinct moments: pre-aerobic training (PRE-TA) and post-aerobic training (POST-TA). The total body mass for the PRE-TA sample populations is 80,77 ± 8,67 kg and that for POST-TA is 78,83 ± 9,01 kg. A second group studied was the long-distance runners (C) (n = 21), 47,43 ± 5,77 years, total body mass 66,97 ± 10,56 kg, 171,05 ± 7,47 cm, with an average of 15,38 ± 8,94 years of weekly training across an estimated distance of 62,95 ± 32,04 kilometers with an average time of performance for 10km of 45'36"± 6'32". The study directly assessed the maximum oxygen uptake (VO2peak.), body composition, lipid profile, CRP, glycated hemoglobin, fasting glucose, basal insulin and insulin resistance which was estimated using the Homeostasis Model Assessment method (HOMA-IR). Statistical analysis was performed between groups and within groups, and adopted a value of 0,05. A significant difference was observed between the group PRE and POST-TA-TA, with reduction of the values for the variables: total body mass (p=0,011), BMI (p=0,011),% fat (p=0,023), fat mass (p=0,012), waist circumference (p=0,001), total cholesterol (p=0,009), LDL (p=0,038), basal insulin (p=0,036) and glycated hemoglobin (p=0,031) and increase in the maximum oxygen (p=0,000). However, for lean mass, HDL, triglycerides, blood glucose, HOMA and CRP were not statistically significant difference. When compared POST-TA and runners not only statistical differences were observed for the MM, total cholesterol, LDL, triglycerides, glucose and CRP, with lower values for the group of runners with the exception of higher VO2peak and HDL. Middle-aged subjects showed positive changes in markers of risk of cardiovascular disease after the completion of only 16 weeks of aerobic training. However, individuals who perform TA for years in higher training intensities have demonstrated benefits of exercise even greater. These data indicate the important role of physical exercise, to a lesser or greater quantity and intensity, as flags of the control of risk factors for the development of diseases associated with metabolic syndrome. / Mestrado / Ciencia do Desporto / Mestre em Educação Física

Thesis (D. Tech.) -- Central University of Technology, Free State, 2009

Thesis (M. Tech.) -- Central University of Technology, Free State, 2007 / Cerebrovascular disease and coronary heart disease (CHD) are of the most important causes of morbidity and mortality amongst South Africans. The risk factor prevalence for stroke and CHD becomes altered by changes in lifestyle, including diet. In general it is suggested that lifestyle management should be the first choice when having to treat patients with increased cardiovascular risk. The prudent low-fat, high-fibre diet is regarded as an apparently healthy diet. It is suspected that this diet is effective for the control of known coronary risk factors as well as raised clotting factors. Research studies have shown the addition of dietary fibre to the diet as a promising therapeutic agent for the limited control of known coronary risk factors. The physiological effects of dietary fibre in humans are significantly influenced by the degree to which fibre is fermented in the colon. Fermentation results in the production of short-chain fatty acids (SCFAs); acetate, propionate and butyrate. The aim of this study was to examine the possible effects of different combinations of short-chain fatty acids on some metabolic risk markers. In this study a group of westernised African male volunteers was recruited and randomly assigned to three groups. Group one received a placebo. Group two received a supplement containing 50% acetate and 50% propionate. Group three received a SCFA supplement in the ratio of 70% acetate, 15% propionate and 15% butyrate. Supplementation was sustained for a period of six weeks. Blood samples were drawn during the different visits. At baseline the study group represented a group of black African men without any apparent metabolic or physical abnormalities. All measured variables fell within the normal range. In the placebo group, there was a statistically significant decrease in plasma fibrinogen levels from baseline to the end of supplementation. In the acetatepropionate supplement study group a statistically significant decrease in factor VIII (from 91.1 ± 11.2 to 90.9 ± 8.3%, respectively), and ATIII (from 114.3 ± 13.1 to 108.34 ± 9.5%), as well as a statistically significant decrease in low-density lipoprotein cholesterol (LDL-C) from 3.10 ± 0.79 to 2.64 ± 0.73 mmol/L. The significant increase in %HDL-C from 26.3 ± 6.5 to 30.2 ± 9.3% should also be noted. Both triglycerides (8%) and plasma fibrinogen (2%) showed a statistically significant increase. However, these changes are of no clinical significance. For the high-acetate supplement study group (with the addition of butyrate), a statistically significant decrease in factor VII (from 102.5 ± 13.7 to 101.1 ± 6.4%), VIII (from 92.6 ± 12.8 to 87.6 ± 6.0%), ATIII (from 109.2 ± 16.0 to 103.0 ± 9.9%) as well as fibrin monomer concentration (from 13.9 ± 2.2 to 12.1 ± 3.6 mg/L), were measured. Fibrin network compaction increased significantly from 14.2 ± 4.6 to 13.7 ± 4.0%. Other changes include a statistically significant increase in the serum-TC of 4.2%. From the results it is evident that the acetate-propionate supplement, with exclusion of butyrate, has a beneficial effect on metabolic parameters when compared to a highacetate- propionate supplement. The results do provide evidence of a possible therapeutic application for the propionate-acetate containing supplement. The specific mechanism should, however, still be investigated. It can be concluded from this study that acetate, propionate and butyrate each have different effects on human metabolism. It is evident that the use of a mixture of acetate and propionate may have a beneficial effect on patients at risk of developing CVD. Further studies that investigate the optimum ratio of these two products may lead to the development of a naturally derived therapeutic product for the prevention or treatment of CVD in black African men, as well as the population at large.

Background. The kidney is one of the major organs involved in whole-body homeostasis and it is well understood that chronic renal impairment is further complicated with deranged carbohydrate metabolism, dyslipidemia, altered abdominal fat distribution and the activation of renin-angiotensin system (RAS). Recently, RAS blockades of angiotensinconverting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB) have been noticed for their potential effects on improve glucose and lipid metabolisms and lowering the risk of new-onset diabetes. However, underlying cellular and molecular mechanisms are not fully established. / Conclusions. (1) UNX induces progressive renal impairment and dysregulation of pancreatic and renal RAS in rats. (2) Pancreatic RAS activation leads to intra-islet fibrosis, insulin-secreting beta-cell deficit and insulin secretory deficiency. (3) Renal cortex RAS dysregulation induces ectopic adipocyte differentiation and lipid infiltration, in combination with lipodystrophy and lipid peroxidation, results to insulin resistance. (4) Pancreatic insulin-secretion deficit and insulin resistance contribute to the development of glucose intolerance and hyperglycemia. (5) Kidney impacting on glucose and lipid metabolism by affecting pancreatic islet and adipocyte, suggesting an essential role of the kidney in maintaining the whole-body homeostasis. (6) RAS blockade with ACEI or ARB may prevent the development of chronic renal impairment and glucose and lipid dysmetabolisms in UNX rats. (7) Common pathways modulating blood pressure, glucose and lipid metabolism warrant future studies for the better management of the global epidemic of metabolic syndrome. / Materials and methods. Chronic renal impairment and RAS disturbance were induced by unilateral nephrectomy (UNX) in adult Sprague-Dawley rats undergoing as long as 10 months of observation. Three-month old male rats were randomized into 4 groups: (1) sham operated control rats (n=10), (2) untreated UNX model rats (n=10), (3) ACEI---lisinopril treated UNX rats (n=10), and (4) ARB-olmesartan treated UNX rats (n=10). Blood glucose levels during fasting and oral glucose tolerance test (OGTT) conditions, lipids, insulin and renal function were measured at 3, 6, 8 and 10 months after operation. Histological changes of kidney, pancreas, liver, and adipose tissue were examined at 10 months post-operation. / Objectives. (1) To set up a rat model with persistent chronic renal impairment and RAS activation. (2) To examine changes of fasting blood glucose, glucose tolerance, blood lipids and insulin sensitivity. (3) To examine changes of pancreatic islets and the factors contributing to pancreatic islet damage such as RAS, transforming growth factor (TGF)-beta and alpha-smooth muscle actin (SMA). (4) To examine changes of systemic and renal adipose tissue and the factors contributing to adipopathy such as RAS, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and hydroxy-3-methylglutary coenzyme A reductase (HMGCR). (5) To investigate preventive effect of RAS blockades by the ACEI-lisinopril (4 mg/kg body weight) and ARB-olmesartan (4 mg/kg body weight) on the rat model of progressive renal deficiency. / Results. (1) UNX rats developed time-dependent progressive renal functional impairment and marked glomerulosclerosis and tubulointerstitial lesions. (2) UNX rats showed fasting hyperglycemia, progressive glucose intolerance, hyperlipidemia and insulin resistance. (3) UNX rats demonstrated insulin secretory deficiency in parallel to pancreatic islet fibrosis, beta-cell deficit, and overexpression of RAS components, TGF-beta, and alpha-SMA. (4) UNX rats displayed adipopathy evidenced by shifts the subcutaneous and visceral fats to the ectopic fat with lipid accumulation, lipofuscin pigmentation and adipocytes transformation. The adipopathy associated with down-regulation of AT1R and over-expression of angiotensin, AT2R, PPAR-gamma and HMGCR in the remnant kidney. (5) Treatment with lisinopril and olmesartan significantly attenuated the development of chronic renal impairment, RAS dysregulation and aberrant proteins expression, islet damage, adipose redistribution, and glucose and lipid dysmetabolism. / Sui, Yi. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3422. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 195-220). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Indiana University-Purdue University Indianapolis (IUPUI) / An investigation of the lipid metabolism pathway in the yeast Yarrowia lipolytica was conducted. Yarrowia is an oleaginous ascomycete that is capable of growing on many different substrates, which derives its name from its high efficiency of growth on lipids. Once the exogenous lipids are converted into free fatty acids and internalized by the yeast, the primary mode of degradation is through β-oxidation mediated by the peroxisomal oxidases, or POX genes. These enzymes catalyze the formation of a trans double bond, producing the trans-2-enoyl product. Our study looked at the comparison of the Y. lipolytica prototrophic strain against a knockout of the Pox2 gene on the uptake, incorporation, and degradation of relevant fatty acids. To construct this gene knockout, a novel gene deletion method using a combination of Cre recombinase and the AHAS* gene was synthesized, developed, and tested successfully. This knockout system allows for serial deletion of genes with the use of only one resistance marker, with excision of the marker after selection.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.

Lípides nitrados (NO2-FA) são apontados como uma nova classe de mediadores lipídicos, podendo atuar como reservatórios endógenos de óxido nítrico (&#8226NO) bem como moduladores pluripotentes de sinalização celular. Recentemente, tem sido sugerido que os doadores de &#8226NO estariam envolvidos na regulação da angiogênese. Evidências contundentes indicam ainda que o processo de neovascularização poderia contribuir para a patogênese de uma serie de condições clínicas, entre elas a aterosclerose. Contudo, apesar de diversos estudos terem explorado os efeitos biológicos dos NO2-FA, os efeitos destes compostos sobre o processo de angiogênese não haviam sido descritos. Dessa maneira, o presente trabalho investigou os efeitos dos NO2-FA (derivados da nitração do ácido linoléico e oléico) noprocesso de angiogênese. Demonstrou-se que os NO2-FA podem atuar como mediadores pró-angiogênicos. Este efeito foi caracterizado em células endoteliais humanas, assim como, em modelos ex vivo e in vivo. Nas células endoteliais, observou-se que os No2-FA não influenciaram a proliferação ou a viabilidade celular, ao passo que estimularam a migração. Demonstrou-se também que os NO2-FA podem modular o brotamento ex vivo de novos vasos, em cultura de anéis de aorta de rato, bem como o processo angiogênico in vivo observado na membrana corioalantóica de embrião de galinha. Adicionalmente, os NO2-FA induziram a expressão do fator de crescimento endotelial vascular (VEGF), que é o principal mediador do processo de angiogênese. Em relação ao mecanismo de ação, os achados sugerem que os efeitos demonstrados seriam via mecanismos dependentes de &#8226NO, uma vez que foram abolidos na presença de um seqüestrador de &#8226NO, enquanto concentrações equivalentes dos lípides precursores não demonstraram qualquer influência nas condições experimentais utilizadas neste estudo. Por fim, os efeitos pró-angiogênicos dos NO2-FA foram mediados pela estabilização da proteína do fator induzível por hipóxia -1&#945; (HIF-1&#945;), uma vez que estes compostos promoveram acúmulo desta proteína e falharam em demonstrar efeitos indutores em células knockdown para o gene HIF-1&#945;. Em conjunto, estes resultados indicam que os NO2-FA podem modular a migração de células endoteliais e estimular o processo de angiogênese resultante da ativação de HIF-1a via mecanismo dependente de &#8226NO. / Nitrated lipids (NO2-FA) are described as a new class of Iipid mediators that are able to act as endogenously nitric oxide (&#8226NO) reservoirs as well as pluripotent cell signaling modulators. Furthermore, recent findings suggest that &#8226NO donors could be involved in the regulation of angiogenesis. Compelling evidence also indicate that the neovascularization process might contribute to the pathogenesis of many clinical conditions, such as atherosclerosis. However, although several studies have explored the NO2-FA biological properties, the effects of these compounds on the angiogenic process remain unknown. Hence, the present study investigated the effects of the NO2-FA (derivates from the nitration of Iinoleic and oleic acids at physiological concentrations) on angiogenesis processo It is demonstrated that the No2-FA could act as pro-angiogenic mediators. This effect was observed not only in human endothelial cells but also in ex vivo and in vivo models. Using endothelial cells, it is showed that NO2-FA failed to affect cell proliferation ar influence cellular viability, but significantly stimulated cell migration. It was also found that the NO2-FA might modulate the ex vivo sprouting of new vessels as well as the in vivo angiogenic process, while inducing the expression of the vascular endothelial growth factor, the main mediator of angiogenesis. The data are consistent with the hypothesis that the observed effects mediated by NO-dependent mechanisms, since the presence of a &#8226NO scavenger abrogated the induced effects, whereas equimolar concentrations of its precursors, showed no effect on angiogenesis under our experimental conditions. Finally, the pro-angiogenic effects of NOrFA were mediated by the stabilization of the hypoxia inducible factor-1&#945; (HIF-1&#945;) protein, because these compounds increased the protein amount and failed to show inductive effects in HIF-1&#945; knockdown cells. Taken together, these findings indicated that NO2-FA might modulate the endothelial cell migration and stimulate the process of angiogenesis by the HIF-1&#945; induction through a &#8226NO-dependent mechanism.