Procurando compreender o envolvimento da aldosterona (Aldo) na injúria renal, o objetivo deste projeto foi avaliar o efeito do tratamento crônico com Aldo sobre a função renal e a histologia das arteríolas renais, procurando correlacionar os achados com a expressão de genes reguladores do processo de fibrose e apoptose. A Aldo não alterou os parâmetros fisiológicos, PA, ritmo de filtração glomerular (estimado pela depuração plasmática de creatinina), proteinúria e a morfologia das arteríolas corticais. No entanto, aumentou a expressão do RNAm para TGF-β1, PAI-1 e BAX no tecido renal, além da contagem de células TUNEL positivas nos glomérulos. O antagonismo ao receptor MR (pelo uso da espironolactona) aboliu o efeito hormonal somente sobre a expressão do RNAm para BAX e a marcação do DNA degradado (TUNEL), enquanto que o antagonismo ao GR (pelo uso do RU 486) reduziu ou aboliu todos os efeitos da Aldo. Os resultados indicam que a Aldo pode induzir respostas precoces sobre o remodelamento do tecido renal, sem ainda comprometer a função renal ou alterar a PA. Essas respostas foram independentes da sobrecarga de sal e ocorreram por um mecanismo que envolveu os receptores MR e GR. / In order to understand the aldosterone (Aldo) involvement in renal injury, the objective of this project was to evaluate the effect of Aldo chronic treatment on renal function and renal arterioles histology, trying to correlate the findings with the regulatory genes of fibrosis and apoptosis. Aldo did not change the physiological parameters, BP, glomerular filtration rate (estimated by creatinina clearance), proteinuria and cortical arterioles morphology. However, Aldo increased mRNA expression for TGF-β1, PAI-1 and BAX in renal tissue, as well as TUNEL-positive cell count in glomeruli. MR receptor antagonism (by spironolactone) abolished only the hormonal effect on the mRNA expression for BAX and degraded DNA labeling (TUNEL); whereas GR antagonism (by RU 486) reduced or abolished all Aldo effects. The results indicated that Aldo can induce early responses on renal tissue remodeling, without altering renal function or BP. These responses were salt independent and involved MR and GR receptors.

Procurando compreender o envolvimento da aldosterona (Aldo) na injúria renal, o objetivo deste projeto foi avaliar o efeito do tratamento crônico com Aldo sobre a função renal e a histologia das arteríolas renais, procurando correlacionar os achados com a expressão de genes reguladores do processo de fibrose e apoptose. A Aldo não alterou os parâmetros fisiológicos, PA, ritmo de filtração glomerular (estimado pela depuração plasmática de creatinina), proteinúria e a morfologia das arteríolas corticais. No entanto, aumentou a expressão do RNAm para TGF-β1, PAI-1 e BAX no tecido renal, além da contagem de células TUNEL positivas nos glomérulos. O antagonismo ao receptor MR (pelo uso da espironolactona) aboliu o efeito hormonal somente sobre a expressão do RNAm para BAX e a marcação do DNA degradado (TUNEL), enquanto que o antagonismo ao GR (pelo uso do RU 486) reduziu ou aboliu todos os efeitos da Aldo. Os resultados indicam que a Aldo pode induzir respostas precoces sobre o remodelamento do tecido renal, sem ainda comprometer a função renal ou alterar a PA. Essas respostas foram independentes da sobrecarga de sal e ocorreram por um mecanismo que envolveu os receptores MR e GR. / In order to understand the aldosterone (Aldo) involvement in renal injury, the objective of this project was to evaluate the effect of Aldo chronic treatment on renal function and renal arterioles histology, trying to correlate the findings with the regulatory genes of fibrosis and apoptosis. Aldo did not change the physiological parameters, BP, glomerular filtration rate (estimated by creatinina clearance), proteinuria and cortical arterioles morphology. However, Aldo increased mRNA expression for TGF-β1, PAI-1 and BAX in renal tissue, as well as TUNEL-positive cell count in glomeruli. MR receptor antagonism (by spironolactone) abolished only the hormonal effect on the mRNA expression for BAX and degraded DNA labeling (TUNEL); whereas GR antagonism (by RU 486) reduced or abolished all Aldo effects. The results indicated that Aldo can induce early responses on renal tissue remodeling, without altering renal function or BP. These responses were salt independent and involved MR and GR receptors.

L’identification de marqueurs pronostiques et prédictifs du cancer du sein est un facteur important pour une meilleure compréhension du processus évolutif et le développement de thérapies ciblées. Les récepteurs des oestrogènes (RE) représentent ainsi à la fois un marqueur pronostique mais aussi prédictif du traitement par le tamoxifène ou les anti-aromatases. Cependant, un certain nombre de patientes vont évoluer en dépit de traitements anti-hormonaux adaptés. L’objectif de notre travail, a été d’évaluer la méthode de mesure des RE, l’apport des protéases dans la distinction de profils tumoraux pronostiques et prédictifs. Nous avons démontré l’influence du mode de mesure des RE et en particulier de l’expression quantitative sur l’interprétation pronostique et sur une meilleure détermination du bénéfice du traitement en fonction du niveau d’expression des RE. Nous avons montré l’intérêt de l’évaluation des protéases tissulaires uPA, PAI-I et cathépsine-D, pour caractériser l’hétérogénéité des tumeurs en complément des RE. Particulièrement, chez les patientes RE+, des taux élevés de cathépsine-D et de PAI-1 étaient un indicateur de mauvais pronostic. Nous avons développé un nomogramme combinant RE et le statut ganglionnaire à 3 types de protéases : PAI-1, cathépsine-D et la thymidine kinase, pour déterminer la probabilité de survie à 2 et 5 ans. De plus, ces protéases évaluées dans les tumeurs infectées par l’Epstein-Barr virus (EBV), témoignaient de tumeurs biologiquement agressives avec des taux plus élevés de thymidine kinase. Notre travail a contribué à améliorer l’identification de profils des tumeurs en fonction des RE et des protéases et de caractériser les tumeurs viro-induites. / The identification of prognostic and predictive markers is important for a better understanding of the evolutionary process and the development of targeted therapies. Thus estrogen receptors (ER) represent both an important prognostic marker but also predictive of therapies using tamoxifen or aromatase inhibitors. However, a number of patients will evolve despite hormonotherapy. The objective of our work was to evaluate the method for measuring ER, the contribution of proteases in the distinction of prognostic and predictive tumor profiles. In our work, we demonstrated the influence of the mode of measure of ER and in particular its quantitative expression on the prognostic interpretation and a better determination of benefit of treatment depending on the level of expression of ER. We show the interest of the evaluation of tissue proteases uPA, PAI-I and cathepsin-D, to characterize the heterogeneity of tumors in addition to ER. Specifically, in ER + patients, high levels of cathepsin-D and PAI-1 are an indicator of poor prognosis. We developed a nomogram combining ER and nodal status, to 3 types of proteases: PAI-1, cathepsin-D and thymidine kinase, to determine the probability of survival at 2 and 5 years. In addition, these proteases are evaluated in tumors infected with the Epstein-Barr virus (EBV) and shows high rates of thymidine kinase in EBV + BC, reflecting biologically aggressive tumors. Our work has helped to improve the identification of profiles of tumors according to ER and proteases and characterize virus-associated tumors.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-12-15T14:53:15Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) VersaoAtualizada2016 - Tese Georgge Gomes Oliveira - varsão para biblioteca UFPE.pdf: 3290385 bytes, checksum: 2ddc23c4486bcc121a9ea222566d1f09 (MD5) / Made available in DSpace on 2016-12-15T14:53:15Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) VersaoAtualizada2016 - Tese Georgge Gomes Oliveira - varsão para biblioteca UFPE.pdf: 3290385 bytes, checksum: 2ddc23c4486bcc121a9ea222566d1f09 (MD5) Previous issue date: 2015-04-27 / Ministério da Saúde do Brasil / Estudos recentes mostram que a síndrome metabólica (SM) é freqüente nas pessoas vivendo com HIV/AIDS (PLWHA). A importância na identificação da SM baseia-se no aumento do risco em cinco vezes de desenvolver diabetes mellitus tipo 2 (DM2) e em duas vezes de apresentar doença cardiovascular (DCV) trombóticas, embora os fatores de hipercoagulabilidade não estejam incluídos nos critérios de definição da síndrome. A SM é caracterizada pela presença concomitante de fatores reconhecidamente aterogênicos em um mesmo indivíduo. A freqüência de DCV em PLWHA vem aumentando ao longo dos anos. O PAI-1 é uma proteína importante na cascata de fibrinólise e seu aumento está associado ao estado de hipercoagulabilidade. Sua regulação depende de fatores genéticos, dentre eles, destaca-se o polimorfismo 4G5G do gene SERPINE1. A participação de substâncias protrombóticas na doença cardiovascular é conhecida em pessoas sem HIV, porém menos elucidada em PLWHA. Diante disto o objetivo deste trabalho foi determinar a freqüência do polimorfismo 4G5G em pessoas que vivem com HIV e verificar se o polimorfismo tem associação com a expressão do PAI-1 plasmático, com SM e com risco cardiovascular (RCV) estimado pelo escore de Framingham. Também objetivamos verificar associação dos níveis de PAI-1 com RDC e com SM. Para tanto foi desenvolvido estudo transversal para determinação da freqüência do polimorfismo 4G5G do PAI-1 e estudo tipo caso-controle para verificar associações entre polimorfismos com níveis plasmáticos de PAI-1 com SM e depois com RCV. Também foram testadas associações com fatores de risco tradicionais. Para primeiro estudo a amostra foi 185 pessoas sorteadas de um grupo de 2074 participantes da Cohort AIDS-PE Study Group. A prevalência de heterozigose foi de 86,8% e homozigose para 4G4G de 4,4%. A média de idade foi de 40,5 (DP ± 9,9 anos). A mediana de PAI-1 ativado foi de 13,6 ng/mL (IQ: 10,8-17,5). A freqüência de SM foi de 37,9% e de dislipidemia de 82,4%. Não encontramos associação do polimorfismo com os níveis plasmáticos de PAI-1, nem com SM. Para o segundo estudo houve perda de 23, restando 162 pessoas das quais 72,8% era do sexo feminino e a média de idade foi de 40 anos. A freqüência de RDCV estimado > 10% foi de 10,5%. O alelo 4G esteve presente em 91,0% das pessoas (genótipos 4G4G e 4G5G). Não houve associação entre polimorfismo e RDCV estimado > 10% (OR=0,6; IC95% 0,1 – 3,7), nem diferença dos níveis de PAI-1 em relação ao RDCV estimado (RCV>10% 14,6 ng/ml x RDCV < 10% 14,1 ng/ml; ρ=0,9). Hipercolesterolemia foi associada com genótipo 5G5G do polimorfismo (OR: 3,3; IC95%: 1,25 – 10) e com níveis plasmáticos mais elevados do PAI-1 (colesterol não HDL (CNHDL) > 130 mg/dl = 15,6 ng/ml versus CNHDL < 130 ng/ml = 13,8 ng/ml; ρ=0,04). Nesse estudo, encontramos alta prevalência do heterozigose para o polimorfismo 4G5G em pessoas vivendo com HIV/AIDS, no nordeste do Brasil. Entretanto, não encontramos associação entre o polimorfismo estudado com níveis plasmáticos de PAI-1 nem com SM. Também não verificamos associação do polimorfismo 4G5G do PAI-1 nem dos níveis plasmáticos de PAI-1 com RCV>10% pelo escore de Framingham, mas houve com hipercolesterolemia. / Recent studies show that the metabolic syndrome (MS) is common in people living with HIV / AIDS (PLWHA). The importance of identifying MS is based on an increased risk of developing fivefold type 2 diabetes mellitus (T2DM) and twice presenting cardiovascular disease (CVD) thrombotic, although hypercoagulability factors are not included in the definition of criteria syndrome. MS is characterized by the concomitant presence of known atherogenic factors in the same individual. The frequency of CVD in PLWHA has increased over the years. The PAI-1 is an important protein in the fibrinolytic cascade and its increase is associated with the hypercoagulable state. Its regulation depends on genetic factors, among them stands out the 4G5G polymorphism SERPINE1 gene. The participation of prothrombotic substances in cardiovascular disease is known in people without HIV, but less elucidated in PLWHA. In view of this the objective of this study was to determine the frequency of 4G5G polymorphism in people living with HIV and verify that polymorphism is associated with the expression of PAI-1 plasma with MS and cardiovascular risk (RCVD) estimated by the Framingham score . We aim to also assess the association of PAI- 1 levels with CVD and with MS. For this cross-sectional study was developed to determine the frequency of 4G5G polymorphism of PAI- 1 and case-control study to examine associations between polymorphisms and plasma levels of PAI- 1 with SM and then RCVD. Associations were also tested with traditional risk factors. For the first study sample was randomly selected 185 people of a group of participants 2074 of AIDS-PE Cohort Study Group. The prevalence of heterozygosity was 86.8% and homozygous for 4G4G 4.4%. The average age was 40.5 (SD ± 9.9 years). The PAI-1 activated median was 13.6 ng/mL (CI: 10.8 to 17.5). The frequency of MS was 37.9% and 82.4% dyslipidemia. We did not find polymorphism association with plasma levels of PAI-1 or with SM. For the second study, there was loss of 23, leaving 162 people of which 72.8% were female and the average age was 40 years. The frequency of RDCV estimated> 10% was 10.5%. The 4G allele was present in 91.0% of people (4G4G and 4G5G genotypes). There was no association between polymorphism and RDCV estimated> 10% (OR = 0.6; 95% CI 0.1 to 3.7), or difference of PAI-1 levels relative to estimated RDCV (RCV> 10% 14.6 ng / ml x RDCV <10% 14.1 ng/ml; ρ = 0.9). Hypercholesterolemia was associated with 5G5G genotype polymorphism (OR: 3.3; 95% CI: 1.25 to 10) and higher plasma levels of PAI-1 (non-HDL cholesterol (CNHDL)> 130 mg / dl = 15.6 ng/ml CNHDL versus <130 ng/ml = 13.8 ng/ ml; ρ = 0.04). In this study, we found a high prevalence of heterozygous for the polymorphism 4G5G in people living with HIV/AIDS, in northeastern Brazil. However, we found no association between the polymorphism studied with plasma levels of PAI-1 or with SM. Nor do we find polymorphism association 4G5G of PAI-1 or plasma levels of PAI-1 with RCV> 10% by Framingham score, but happened to hypercholesterolemia.

Le facteur 4 plaquettaire (PF4/CXCL4) est un tétramère constitué de quatre sous-unités identiques de 7,8 kDa qui est libéré en grande quantité par les plaquettes lors de l’hémostase primaire (ensemble des phénomènes permettant un colmatage initial d’une lésion vasculaire). L’étude de la formation d’un caillot de fibrine en présence de PF4 montre une augmentation de la turbidité finale du caillot : le PF4 modifie le réseau formé. Etant donné que la plupart des acteurs de la fibrinolyse se lie au caillot de fibrine et que le PF4 modifie sa structure, nous avons pensé qu’il serait intéressant de rechercher si le PF4 influençait aussi la fibrinolyse. La lyse d'un caillot est effectuée par la plasmine issue de l'activation du plasminogène par son activateur d’origine tissulaire (t-PA) en présence d’un cofacteur qui n'est autre que la fibrine. Nous avons étudié la lyse de caillots de plasma, obtenus par activation de la cascade de la coagulation, en condition statique et à l'aide d'un modèle de thrombose artérielle (système Chandler loop). Dans les deux cas, une diminution du temps de demi-lyse a été observée en présence de PF4. Cependant, la lyse de caillots préparés par simple ajout de thrombine sur du fibrinogène ne permet pas de retrouver cet effet du PF4. Ceci suggère que l’influence du PF4 sur la structure du caillot n’est pas à l’origine de l’effet sur sa lyse et que le PF4 n’influence pas (ou très peu) l'activation du plasminogène, ainsi que l'activité de la plasmine résultante. Cette hypothèse a été confirmée par l’étude de l’activité amydolytique du t-PA et de la plasmine (quelle soit ajoutée ou générée). En système purifié, les inhibiteurs plasmatiques de la fibrinolyse sont absents. Les deux principaux sont l'inhibiteur de l'activateur du plasminogène de type 1 (PAI-1) et l’α2-antiplasmine (α2-AP). La lyse de caillots préparés à partir de plasma déficient en α2-AP montre une diminution du temps de demi-lyse en présence de PF4 (comme pour le plasma normal), alors qu’avec le plasma dépourvu de PAI-1 le temps de demi-lyse n'est plus influencé. De plus, l’ajout de PAI-1 dans le système purifié entraine une diminution du temps de demi-lyse en présence de PF4. Ceci suggère que le PF4 prévient directement ou indirectement l'inhibition du t-PA par PAI-1. L’étude de la cinétique d'inhibition de l’activité amidolytique du t-PA par le PAI-1, la détermination de la stœchiométrie de cette inhibition, et l’analyse de ces cinétiques par immuno-empreinte montrent que le PF4 est un modulateur de la fibrinolyse qui agit en retardant la formation d'un complexe initial entre le t-PA et le PAI-1. Cette nouvelle fonction du PF4 est cohérente, et vient en complément de celle décrite récemment d’inhibiteur de l'activation du TAFI. / Platelet factor 4 (PF4/CXCL4) is a tetramer constituted of four identical 7,8 kDa subunits released in large quantities by platelets during primary heamostasis (allowing initial clogging of a vascular injury). Study of fibrin clot formation in the presence of PF4 shows an increase of the final clot turbidity: PF4 modifies the formed network. Given that most fibrinolysis actors are bound to the fibrin clot and that PF4 modifies its structure we thought it would be interesting to investigate if PF4 also influences fibrinolysis. Clot lysis is performed by plasmin originating from activation of its precursor by tissue plasminogen activator (t-PA) with fibrin itself as cofactor of the reaction. We have studied lysis of plasma clots formed by activation of the coagulation cascade in static condition and in a Chandler loop model mimicking arterial thrombosis. Half-times of lysis decreased in the presence of PF4 in both systems. However, PF4 had no longer detectable influence on the half-time of lysis with clots formed by direct addition of thrombin on purified fibrinogen. Observation suggested that the observed decrease of the half-time of lysis induced by PF4 did not originate from its influence on fibrin clot formation and that PF4 had little effect if any on plasminogen activation or plasmin activity. We confirmed this hypothesis by comparing amydolytic activities of t-PA and plasmin (added or generated through plasminogen activation). In purified system, fibrinolysis inhibitors are absent. The two main inhibitors are plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin (α2-AP). Lysis of clots obtained from α2-AP deficient plasma showed a decrease of the half-time of lysis in the presence of PF4 (as in normal plasma), whereas in PAI-1 deficient plasma half-time of lysis was unchanged. Moreover if PAI-1 was added to the purified system, half-time of lysis decreased in the presence of PF4. Observations therefore suggested that PF4 prevented directly or indirectly t-PA inhibition by PAI-1. Kinetics of the amidolytic activity of t-PA inhibition by PAI-1 in the presence or not of PF4, determination of its stoichiometry and Western blot analysis of these inhibition kinetics revealed that PF4 is a fibrinolysis modulator which delays formation of the initial (Michaelis) complex between t-PA and PAI-1. This new feature of PF4 is consistent and complementary with its recently described role as a modulator of TAFI activation.

Background: Cardiovascular disease (CVD) is the leading global cause of death. CVD risk factors are considered intermediaries for the association between dietary fatty acids and CVD. Raised plasma total cholesterol, low density lipoprotein (LDL) cholesterol, raised triglycerides and decreased levels of high density lipoprotein (HDL) cholesterol, as well as reduced fibrinolytic potential (measured as increased clot lysis time) are known risk factors for CVD. Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of the fibrinolytic process and an elevated PAI-1 level is therefore considered to be a potential risk factor for CVD. The growing number of controversies around the role that fat intake (more specifically the type of dietary fat) plays in CVD risk, is making it increasingly difficult for consumers and practitioners alike to form conclusions, and make recommendations and decisions regarding fat intake. Knowledge of the intake of individual fatty acids, fatty acid status (as opposed to subgroups of fat such as polyunsaturated fatty acids) and their associations with blood lipids, PAI-1act and fibrinolytic potential is lacking in black South Africans and other populations. Therefore we aimed to investigate dietary fatty acid intake, as well as plasma phospholipid fatty acid status and their associations with blood lipids, PAI-1act and clot lysis time, as a marker for fibrinolytic potential. Methods: Cross-sectional data analysis within the Prospective Rural Urban Epidemiology (PURE) baseline study of apparently healthy black South African men and women (n=1950, 35– 70 years) from rural and urban areas in the North West Province, from whom dietary data were collected. Blood lipid analyses, as well as laboratory analyses of fibrinolysis markers such as PAI-1act and clot lysis time were also performed. Plasma phospholipid fatty acid extraction and isolation were performed on a random subsample (n = 716). Results: The intake of individual fatty acids was significantly higher in urban than rural dwellers. However, the intake of omega-3 polyunsaturated fatty acids was below recommendations in all groups (rural and urban males, and rural and urban females). Total cholesterol and LDL cholesterol were higher in females than in males, with no rural‒urban differences. Intake of alpha-linolenic acid was positively associated with total cholesterol (β=0.143) and triglycerides (β=0.256) in males. The risk of having elevated LDL cholesterol also increased with increased intake of alpha-linolenic acid (OR 1.49, 95% CI 1.04, 2.14). In females, dietary arachidonic acid and eicosapentaenoic acid (EPA) were positively associated with total cholesterol and LDL cholesterol, whereas docosahexaenoic acid (DHA) was negatively associated with total cholesterol and LDL cholesterol. Dietary alpha-linolenic acid was positively correlated with plasma EPA (males r = 0.19, p = 0.002, females r = 0.25, p < 0.001) and DHA (males r = 0.33, p < 0.001, females r = 0.30, p < 0.001). Plasma DHA was positively associated with triglycerides in males (β = 0.410, p< 0.001) and in females (β = 0.379, p< 0.001). PAI-1act was positively associated with clot lysis time, and plasma myristic acid and DHA were positively associated with PAI-1act in females. However, these fatty acids were not associated with clot lysis time. Different types of plasma fatty acids were associated with PAI-1act than with clot lysis time. Plasma alpha-linolenic acid (β = 0.123, P = 0.037), mead acid (β = 0.176, P = 0.019), arachidonic acid (β = 0.253, 0.025) and omega-3 docosapentaenoic acid (omega-3 DPA) (β = 0.224, P = 0.002) were positively associated with clot lysis time, while both myristic acid (β = - 0.130, P = 0.016) and EPA (β = -0.131, P = 0.021) were negatively associated with clot lysis time in male subjects. Plasma oleic acid (C18:1n9) (β = -0.411, P = 0.001) and omega-6 DPA (C22:5n6) (β = -0.285, P = 0.001) were negatively associated with clot lysis time, while dihomogamma- liolenic acid (DGLA) (C20:3n6) were positively associated (β = 0.178, P = 0.001) with clot lysis time in females. Conclusions: These results suggest that specific individual dietary fatty acids might be associated with blood lipids in males differently than in females, irrespective of rural or urban dwelling. It is not known however, if associations would still be present under conditions of greater intake of alpha-linolenic acid. Our results further suggest that a higher percentage of alpha-linolenic acid might be converted to DHA in this population with low intake of essential and long-chain polyunsaturated fatty acids compared to populations with a high intake of these fatty acids. These results suggest that plasma phospholipid fatty acids should not be used in isolation as biomarkers for intake of fat, without taking dietary intake data into consideration also. Associations between fatty acids and clot lysis time might be independent from PAI-1act. The association between mead acid and clot lysis time indicates that clot lysis time might increase with an essential fatty acid deficiency. This may be of particular concern in this population with a documented lower fat intake. Because the study design of this study is crosssectional, it is not able to determine cause-and-effect, and results should therefore be verified with a randomised controlled trial. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

Background: Cardiovascular disease (CVD) is the leading global cause of death. CVD risk factors are considered intermediaries for the association between dietary fatty acids and CVD. Raised plasma total cholesterol, low density lipoprotein (LDL) cholesterol, raised triglycerides and decreased levels of high density lipoprotein (HDL) cholesterol, as well as reduced fibrinolytic potential (measured as increased clot lysis time) are known risk factors for CVD. Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of the fibrinolytic process and an elevated PAI-1 level is therefore considered to be a potential risk factor for CVD. The growing number of controversies around the role that fat intake (more specifically the type of dietary fat) plays in CVD risk, is making it increasingly difficult for consumers and practitioners alike to form conclusions, and make recommendations and decisions regarding fat intake. Knowledge of the intake of individual fatty acids, fatty acid status (as opposed to subgroups of fat such as polyunsaturated fatty acids) and their associations with blood lipids, PAI-1act and fibrinolytic potential is lacking in black South Africans and other populations. Therefore we aimed to investigate dietary fatty acid intake, as well as plasma phospholipid fatty acid status and their associations with blood lipids, PAI-1act and clot lysis time, as a marker for fibrinolytic potential. Methods: Cross-sectional data analysis within the Prospective Rural Urban Epidemiology (PURE) baseline study of apparently healthy black South African men and women (n=1950, 35– 70 years) from rural and urban areas in the North West Province, from whom dietary data were collected. Blood lipid analyses, as well as laboratory analyses of fibrinolysis markers such as PAI-1act and clot lysis time were also performed. Plasma phospholipid fatty acid extraction and isolation were performed on a random subsample (n = 716). Results: The intake of individual fatty acids was significantly higher in urban than rural dwellers. However, the intake of omega-3 polyunsaturated fatty acids was below recommendations in all groups (rural and urban males, and rural and urban females). Total cholesterol and LDL cholesterol were higher in females than in males, with no rural‒urban differences. Intake of alpha-linolenic acid was positively associated with total cholesterol (β=0.143) and triglycerides (β=0.256) in males. The risk of having elevated LDL cholesterol also increased with increased intake of alpha-linolenic acid (OR 1.49, 95% CI 1.04, 2.14). In females, dietary arachidonic acid and eicosapentaenoic acid (EPA) were positively associated with total cholesterol and LDL cholesterol, whereas docosahexaenoic acid (DHA) was negatively associated with total cholesterol and LDL cholesterol. Dietary alpha-linolenic acid was positively correlated with plasma EPA (males r = 0.19, p = 0.002, females r = 0.25, p < 0.001) and DHA (males r = 0.33, p < 0.001, females r = 0.30, p < 0.001). Plasma DHA was positively associated with triglycerides in males (β = 0.410, p< 0.001) and in females (β = 0.379, p< 0.001). PAI-1act was positively associated with clot lysis time, and plasma myristic acid and DHA were positively associated with PAI-1act in females. However, these fatty acids were not associated with clot lysis time. Different types of plasma fatty acids were associated with PAI-1act than with clot lysis time. Plasma alpha-linolenic acid (β = 0.123, P = 0.037), mead acid (β = 0.176, P = 0.019), arachidonic acid (β = 0.253, 0.025) and omega-3 docosapentaenoic acid (omega-3 DPA) (β = 0.224, P = 0.002) were positively associated with clot lysis time, while both myristic acid (β = - 0.130, P = 0.016) and EPA (β = -0.131, P = 0.021) were negatively associated with clot lysis time in male subjects. Plasma oleic acid (C18:1n9) (β = -0.411, P = 0.001) and omega-6 DPA (C22:5n6) (β = -0.285, P = 0.001) were negatively associated with clot lysis time, while dihomogamma- liolenic acid (DGLA) (C20:3n6) were positively associated (β = 0.178, P = 0.001) with clot lysis time in females. Conclusions: These results suggest that specific individual dietary fatty acids might be associated with blood lipids in males differently than in females, irrespective of rural or urban dwelling. It is not known however, if associations would still be present under conditions of greater intake of alpha-linolenic acid. Our results further suggest that a higher percentage of alpha-linolenic acid might be converted to DHA in this population with low intake of essential and long-chain polyunsaturated fatty acids compared to populations with a high intake of these fatty acids. These results suggest that plasma phospholipid fatty acids should not be used in isolation as biomarkers for intake of fat, without taking dietary intake data into consideration also. Associations between fatty acids and clot lysis time might be independent from PAI-1act. The association between mead acid and clot lysis time indicates that clot lysis time might increase with an essential fatty acid deficiency. This may be of particular concern in this population with a documented lower fat intake. Because the study design of this study is crosssectional, it is not able to determine cause-and-effect, and results should therefore be verified with a randomised controlled trial. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

<p>Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A. </p><p>There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form.</p><p>The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography.</p><p>The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one. </p><p>To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form. </p>

<p>In this thesis, different aspects of the insulin resistance syndrome in relation to plasminogen activator inhibitor-1 (PAI-1) activity are investigated in a longitudinal population-based study. Participants were men investigated at ages 50 and 70 with follow-up data on mortality.</p><p>High PAI-1 activity was associated with low insulin sensitivity, high concentrations of serum triglycerides, high body mass index and high waist/hip ratio, independently of each other and of potential confounders. Low birth weight predicted high blood pressure, insulin resistance, truncal obesity and high PAI-1 activity but not the abdominal obesity or dyslipidaemia present in the insulin resistance syndrome. Increased physical activity level between 50 and 70 years of age, in the absence of active intervention, was associated with improved glucose, insulin, proinsulin and lipoprotein metabolism. Insulin and proinsulin seemed to be important factors that mediate much of the association between a sedentary lifestyle and increased risk of cardiovascular disease. The reported dietary intake of both mono- and polyunsaturated fatty acids was positively associated with PAI-1 activity, whereas saturated fatty acid intake displayed no association. The associations present between PAI-1 activity and the fatty acid proportions in serum cholesterol esters were partly influenced by factors related with the insulin resistance syndrome.</p><p>This thesis provides further knowledge to the epidemiological view of the interrelations of the insulin resistance syndrome, PAI-1, birth weight, and lifestyle factors as physical activity and dietary habits. PAI-1 is a part of the insulin resistance syndrome and is associated both with modifiable and non-modifiable factors related with this syndrome.</p>

Des données antérieures obtenues dans notre laboratoire ont montré le rôle essentiel de PAI-1 dans la croissance de cellules cancéreuses dorigine murine et linduction dune angiogenèse concomitante (Bajou et al, 1998;Bajou et al, 2001). Bien que ces résultats aient été confirmés par dautres groupes dans différents modèles expérimentaux (Gutierrez et al, 2000;McMahon et al, 2001), très peu dinformations concernant limplication de PAI-1 dans la progression des carcinomes humains étaient disponibles dans la littérature. Cest pourquoi, dans le premier chapitre de ce travail, nous nous sommes intéressés au comportement de différentes lignées de carcinomes humains lorsquelles sont placées dans un environnement dépourvu en PAI-1. Lensemble des résultats obtenus est décrit dans la première publication intitulée : « Host plasminogen activator inhibitor-1 promotes human skin carcinoma progression in a stage-dependent manner » (Maillard et al, 2005, Neoplasia, 7: 57-66) La synthèse des différentes études utilisant différents modèles expérimentaux in vivo (voir introduction, Tableau 6) a montré quelques divergences quant à linfluence de PAI-1 dans la progression tumorale et la formation de métastases. Ces résultats contradictoires pourraient sexpliquer en partie par la grande diversité des modèles expérimentaux utilisés. Les lignées de cellules tumorales utilisées, le nombre de cellules implantées, ainsi que le site dimplantation chez les animaux sont quelques uns des facteurs pouvant influencer les résultats. Dans ce contexte, lutilisation danimaux transgéniques développant spontanément des lésions tumorales est une alternative attrayante pour étudier limpact dun facteur sur lévolution de ces lésions. A lheure actuelle, un seul groupe sest intéressé à limpact dun déficit en PAI-1 dans un modèle de souris développant de manière spontanée des adénocarcinomes mammaires qui métastasent dans les poumons, et na pas observé de différences entre les animaux déficients en PAI-1 et leurs contrôles de type sauvage (Almholt et al, 2003). Ce deuxième chapitre est consacré à létude de limplication de PAI-1 dans un autre modèle murin de cancérogenèse spontanée, à savoir, les souris TRP-1/SV40 Tag caractérisées par un développement de tumeurs oculaires qui forment des métastases dans le cerveau. Ce travail a été réalisé en collaboration avec le Laboratoire de Vectorologie et de Transfert de Gènes (Docteur M. Perricaudet, UMR8121, Villejuif, France), au cours dun séjour de plus de 6 mois. Lensemble des résultats obtenus est décrit dans la seconde publication intitulée : « Reduction of brain metastases in transgenic ocular tumor in plasminogen activator inhibitor-1 deficient mice » (Maillard et al, soumis pour publication) Les résultats présentés dans le troisième chapitre mettent en avant lintérêt des chambres de transplantation in vivo de kératinocytes tumoraux comme modèle détude des interactions hôte-tumeur. Dans un premier temps, ce modèle nous a permis de déterminer limportance de lorigine cellulaire de PAI-1 (cellules tumorales ou cellules hôtes) sur son effet pro-angiogène. Dautre part, des effets opposés de PAI-1 en fonction de sa concentration ont été mis en évidence et apportent des éléments de réponse quant aux effets pro- ou anti-angiogènes de cet inhibiteur des activateurs du plasminogène. Les résultats sont repris dans la troisième publication intitulée : « Host-derived plasminogen activator inhibitor-1 (PAI-1) concentration is critical for in vivo tumoral angiogenesis and growth » (Bajou et al, 2004, Oncogene, 23:6986-6990) Dans un second temps, en collaboration avec léquipe du Professeur J. Boniver (Laboratoire dAnatomie et de Cytologie Pathologiques, ULg, Liège), les chambres de transplantation ont été utilisées pour évaluer in vivo lefficacité thérapeutique du GM-CSF comme cytokine favorisant la migration intratumorale de cellules dendritiques matures. Ces travaux sont présentés dans la quatrième publication intitulée : « Delivery of granulocyte-macrophage colony-stimulating factor in bioadhesive hydrogel stimulates migration of dendritic cells in models of human papillomavirus-associated (pre)neoplastic epithelial lesions » (Hubert et al, 2004, Antimicrobial agents and chemotherapy, 48: 4342-4348)