Os membros da família paraoxonase (PON1, PON2 e PON3) tem sido objeto de grande interesse por prevenir o estresse oxidativo e o processo inflamatório, condições que estão em evidência em pacientes que possuem anemia falciforme. A anemia falciforme é causada por uma mutação pontual no gene ? globina que resulta em uma alteração na estrutura da molécula, gerando a HbS. Sua fisiopatologia envolve múltiplas alterações nos eritrócitos falcêmicos, hemólise, ativação de mediadores inflamatórios, disfunção das células endoteliais, episódios vaso-oclusivos e estresse oxidativo. Deste modo, o objetivo do presente estudo foi avaliar a atividade da enzima PON1, relacionando-as com os polimorfismos PON1 192 e 55, PON2 311 e 148 e PON3 10340, 2115, 45486 e 55146, bem como avaliar marcadores de inflamação e o perfil lipídico em pacientes portadores de anemia falciforme. A casuística foi composta por 43 indivíduos com anemia falciforme e 43 indivíduos saudáveis. O sangue foi coletado, para as determinações bioquímicas e determinação das atividades arilesterase e paraoxonase da PON1. O DNA foi extraído de leucócitos do sangue periférico pelo método de extração salina. A análise dos polimorfismos foi realizada por PCR/RFLP e por PCR em tempo real. A pesquisa de autoanticorpos anti-LDL oxidada foi feira por ELISA. Em relação ao polimorfismo do gene da PON1 192QR foi encontrada uma diferença significativa entre o genótipo 192RR, que apresentou maior frequencia no grupo caso (32,6%) do que no grupo controle (13,9%) (p=0,0064). A atividade arilesterase apresentou valores significativamente menores nos pacientes com anemia falciforme (p<0,001). Houve correlação positiva entre atividade arilesterase e as variáveis apolipoproteína A1(Apo A1) (p=0,0042), colesterol total (CT) (p=0,0066) e lipoproteína de alta densidade (HDL) (p=0,0283) e correlação negativa com leucócitos (p=0,04). Em relação à atividade paraoxonase foi encontrada uma correlação positiva e significativa entre a atividade da enzima e a transferrina (p=0,0094). Os títulos de anticorpos anti-oxLDL diferiram significativamente entre os grupos, grupo caso apresentando valores maiores quando comparados ao grupo controle (p<0,001). O mesmo aconteceu para a dosagem dos níveis séricos de Proteína C Reativa (p<0,001). Concentrações significantemente diminuídas de CT, lipoproteína de baixa densidade (LDL), HDL e Apo A1 e B foram observadas nos pacientes com anemia falciforme em relação ao grupo controle (p< 0,01). Conclusões: Os pacientes falciformes estão apresentaram mais estresse oxidativo que os indivíduos saudáveis, o que poderia influenciar na gravidade da doença / The members of paraoxonase family (PON1, PON2 and PON3) have been the subject of great interest by preventing oxidative stress and inflammation, conditions that are evident in patients who have sickle cell anemia. Sickle cell anemia is caused by a point mutation in the ? globin gene that results in a change in structure of the molecule, generating the HbS. Its pathophysiology involves multiple changes in sickle cell erythrocytes, hemolysis, activation of inflammatory mediators, endothelial cell dysfunction, vaso-occlusive episodes and oxidative stress. Thus, the objective of this study was to evaluate the activity of PON1 enzyme, associating them to the PON1 192 e 55, PON2 311 e 148 e PON3 10340, 2115, 45486 e 55146 polymorphisms, as well as evaluating inflammation markers and lipid profile in patients with sickle cell anemia. The casuistry has consisted of 43 individuals with SCD and 43 healthy. Blood was collected for biochemical studies and determination of arylesterase and paraoxonase activities of PON1. DNA was extracted from peripheral blood leukocytes by extraction saline. The analysis of the polymorphisms was performed by PCR / RFLP and real time PCR. The determination of autoantibodies anti-oxidized LDL was performed by ELISA. Regarding the PON1 192QR gene polymorphism was found a significant difference between genotype 192RR, with the highest frequency in the case group (32.6%) than in the control group (13.9%) (p = 0.0064). The arylesterase activity values were significantly lower in patients with sickle cell anemia (p <0.001). A positive correlation between arylesterase activity and variables apolipoprotein A1 (Apo A1) (p = 0.0042), total cholesterol (TC) (p = 0.0066) and high density lipoprotein (HDL) (p = 0.0283) and negative correlation with leukocytes (p = 0.04). Regarding paraoxonase activity was found a positive and significant correlation between the enzyme activity and transferrin (p = 0.0094). The titers of anti-oxLDL differed significantly between groups, with higher values in the case group compared to the control group (p <0.001). The same happened to the determination of serum C-reactive protein (p <0.001). Significantly decreased concentrations of TC, low density lipoprotein (LDL), HDL and Apo A1 and B were observed in patients with sickle cell disease in the control group (p <0.01). Conclusions: Patients with sickle cell disease presented more oxidative stress than healthy ones, which could influence in the severity of the disease

INTRODUÇÃO. Os genes da família paraoxonase (PON1, PON2 e PON3) apresentam grande homologia estrutural. PON1 está associada à molécula de HDL e possui funções fisiológicas, sendo a principal a de lactonase. PON1 também pode proteger as moléculas de LDL de modificações oxidativas. Embora o papel biológico mais conhecido das paraoxonases seja a prevenção da aterosclerose, elas também atuam sobre o estresse oxidativo envolvido na patogênese de outras condições como doenças inflamatórias, infecções e neoplasias. Toda a família PON parece estar implicada no desenvolvimento de linfomas. O polimorfismo L55M de PON1 foi relacionado a um maior risco para linfomas em indivíduos da população geral, enquanto PON3 e PON2 foram relacionadas à sobrevida de células tumorais. A Imunodeficiencia comum variável (ICV) é uma doença heterogênea caracterizada pela redução dos niveis de IgG, IgA e/ou IgM e da função de anticorpo. As manifestações clínicas incluem a presença de infecções recorrentes ou crônicas, doenças inflamatórias/autoimunes e incidência aumentada de malignidades como linfomas não-Hodgkin (LNH) e câncer gástrico. OBJETIVO: estudar os polimorfismos de PON1 e PON2 bem como a atividade arilesterase de PON1 e sua relação com o perfil lipídico, morbidade, mortalidade e presença de fatores de risco para linfoma LNH em pacientes com ICV. MÉTODOS/RESULTADOS: Foram avaliadas as frequências alélicas dos polimorfismos de PON1 e PON2, o perfil lipídico e a atividade arilesterase da PON1 em 63 pacientes com ICV e 130 controles saudáveis. No grupo de pacientes foi analisada a presença de fatores de risco para LNH e parâmetros de morbidade e gravidade da doença. O polimorfismo Q192R da PON1 e os polimorfismos de PON2 (S311C e A148G) não diferiram entre os grupos e não apresentaram relação com os parâmetros analisados. O genótipo 55MM e o alelo 55M foram mais frequentes no grupo ICV em relação ao grupo controle. A atividade arilesterase foi similar em pacientes e controles apresentando correlação positiva com os níveis de HDL. Pacientes com o genótipo 55MM apresentaram menor atividade de PON1 associada a maior morbidade da doença representada pela maior frequência de infecções de vias aéreas e maior taxa de internações. O genótipo 55MM também apresentou relação com a presença de fatores de risco para LNH como hiperplasia nodular linfoide (HNL) e linfonodomegalias. Por outro lado, a análise dos alelos demonstrou que a menor morbidade da doença foi associada à presença do alelo 55L, que apresentou relação com menor frequência de HNL e linfonodomegalia e menor ocorrência de óbitos. O alelo 55M apresentou relação com história familiar de imunodeficiências e neoplasias hematológicas. CONCLUSÃO: Este constitui o primeiro relato demonstrando maior frequência do genótipo 55MM e do alelo 55M em pacientes com ICV. Nossos resultados são sugestivos de que a presença do alelo 55L possa estar associado a um melhor prognóstico da doença. Inversamente, sugerem que pacientes com o genótipo 55MM apresentem maior morbidade e, possivelmente, maior risco para LNH / INTRO: The paraoxonase gene family (PON1, PON2 and PON3) has great structural homology. PON1 is associated with the HDL molecule and possess many physiological roles, the major one being of a lactonase. PON1 also protects LDL molecules against oxidative modifications. Although the best known biological role of PONs is the prevention of atherosclerosis, they also act on the oxidative stress involved in the pathogenesis of different conditions such as inflammatory diseases, infections and malignancies. The whole PON family appears to be implicated in the development of lymphomas. The L55M polymorphism of PON1 was related with a higher risk for lymphoma in the general population while PON3 and PON2 were related to survival of tumor cells. The Common Variable Immunodeficiency (ICV) is a heterogeneous disease characterized by reduced levels of IgG, IgA and/or IgM and antibody function. Clinical manifestations include the presence of chronic or recurrent infections, inflammatory/autoimmune diseases and increased incidence of malignancies such as non-Hodgkin lymphoma (NHL) and gastric cancer. OBJECTIVE: to study the PON1 and PON2 polymorphisms and the arylesterase activity of PON1 and its correlation with the lipid profile, morbidity, mortality and the presence of risk factors for NHL in CVID patients. METHODS/RESULTS: We evaluated the allele frequencies of polymorphisms of PON1 and PON2, lipid profile and arylesterase activity of PON1 in 63 patients with CVID and 130 healthy controls. In the group of patients we analyzed the presence of risk factors for NHL and parameters of morbidity and disease severity. The Q192R polymorphism of the PON1 and PON2 polymorphisms (A148G and S311C) did not differ between groups and did not correlate with the parameters analyzed. The 55MM genotype and the 55M allele were more frequent in the CVID group than in control group. The arylesterase activity was similar in patients and controls showing a positive correlation with HDL levels. Patients with genotype 55MM had lower PON1 activity, associated with increased morbidity of the disease represented by the higher frequency of respiratory infections and a higher rate of hospitalization. The 55MM genotype also was correlated with the presence of risk factors for NHL, such as lymphoid nodular hyperplasia (HNL) and lymphadenopathy. Moreover, analysis of the alleles showed that less morbidity of the disease was associated with the presence of the allele 55L, which was correlated with a lower frequency of HNL and lymphadenopathies and fewer deaths. The 55M allele was correlated with a family history of immunodeficiency and hematological malignancies. CONCLUSION: This is the first report showing a greater frequency of 55MM genotype and 55M allele in patients with CVID. Our results suggest that the presence of 55L allele may be associated with a better prognosis. Conversely, these results suggest that patients with the 55MM genotype show higher morbidity and, possibly, higher risk for NHL

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2018-06-04T16:46:17Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_carlos_ferreira.pdf: 752331 bytes, checksum: 1791efb2b3372ab1fb6e88d0ec2a0e2f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-05T11:53:24Z (GMT) No. of bitstreams: 2 tese_carlos_ferreira.pdf: 752331 bytes, checksum: 1791efb2b3372ab1fb6e88d0ec2a0e2f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-05T11:53:24Z (GMT). No. of bitstreams: 2 tese_carlos_ferreira.pdf: 752331 bytes, checksum: 1791efb2b3372ab1fb6e88d0ec2a0e2f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-22 / A fertilidade dos reprodutores é de suma importância para a maximização das taxas de prenhez e melhoramento genético. O exame andrológico é uma importante ferramenta para a identificação de reprodutores sub férteis e para o monitoramento da qualidade seminal. Porém, poucas características do exame estão correlacionadas com a fertilidade in vivo. Assim, é fundamental a identificação de métodos que possam auxiliar na seleção mais precisa dos futuros reprodutores. Os objetivos do primeiro trabalho foram avaliar as acurácias e o viés das predições genômicas, utilizando diferentes métodos, para a característica perímetro escrotal ajustado à idade e ajustado à idade e peso em touros das raças Hereford e Braford. Os valores estimados para a herdabilidade apresentaram magnitude moderada a alta (0,39 a 0,48), demonstrando que é possível a obtenção de ganhos genéticos a partir da seleção. A predição de valores genéticos utilizando informações genômicas pelos métodos índice de seleção e single-step possibilitou o aumento de acurácia (em torno de 30%) para as características estudadas. Os ganhos de acurácia obtidos com os métodos, combinando as informações tradicionais com a genômica em relação ao método BLUP tradicional indicam que as predições genômicas podem ser usadas como ferramenta para melhorar os ganhos genéticos e reduzir o intervalo de gerações. Já o segundo artigo teve como objetivos: determinar a expressão de RNAm das paraoxonases (PON) 1, 2 e 3 no parênquima testicular, vesículas seminais e epidídimo de touros, avaliar a atividade de PON1 na circulação sanguínea e no plasma seminal; e correlacionar esta atividade com características avaliadas durante o exame andrológico. A caracterização da expressão dos genes foi realizada por qRT-PCR e a determinação da atividade de PON1 foi realizada em amostras de soro e plasma seminal provenientes de 110 touros. As PON 1, 2 e 3 foram expressas no parênquima testicular dos animais analisados. Observou-se uma correlação positiva entre a atividade sérica e seminal de PON1 com diversos indicadores de fertilidade. / Male fertility is important to optimize pregnancy rates and genetic improvement. Breeding soundness evaluations are key tools to identify sub fertile males and to survey characteristics of sperm quality. However, few sperm quality traits are correlated with fertility in vivo. Thus, it is essential to identify methods that can assist in more precise selection of future bulls. The goals of the first study were to evaluate the accuracy and the bias of the genomic predictions, using different methods, for the characteristic scrotal perimeter adjusted to age and adjusted to age and weight in Hereford and Braford bulls. The estimates for the heritability showed moderate to high magnitude (0.39 to 0.48), demonstrating that it is possible to obtain genetic gains from selection. The prediction of genetic values using genomic information by methods of selection index and single-step made possible the increase of accuracy (around 30%) for the characteristics studied. The gains in accuracy obtained with the methods, combining the traditional with the genomic information compared to traditional BLUP method indicate that the genomic predictions can be used as a tool to improve the genetic gains and reduce the range of generations. The second article had as its objectives: to determine the mRNA expression of paraoxonases (PON) 1, 2 and 3 in the testicular parenchyma, seminal vesicles, and epididymis of bulls, to evaluate the activity of PON1 in the bloodstream and in seminal plasma; and to correlate that activity with characteristics of breeding soundness. Characterization of gene expression by qRT-PCR and the determination of PON1 activity were performed in serum and seminal plasma from bulls 110. The PON 1, 2 and 3 were expressed in testicular parenchyma of the animals examined. There was a positive correlation between activity and serum PON1 seminal with different breeding soundness estimators.

Stroke is an important public health problem and the third leading cause of death after coronary heart diesase and all cancers in all over the world. Free radicals and oxidative stress play important role in the pathogenesis of several diseases including atherosclerosis, stroke, cancer, neurodegenerative diseases such as Alzheimer&#039 / s dementia. The activity of paraoxonase (PON1) aganist phenylacetate is known as arylesterase (ARE). Paraoxonase is an esterase associated with high-density lipoprotein (HDL) and contributes to the protective role of this lipoprotein on low-density lipoprotein (LDL) oxidation. Oxidized LDL is known to play a central role in early events in the progression of atherosclerosis which is a risk factor for stroke. Glutathione S-transferases (GSTs) catalyze the conjugation of nonpolar compounds to reduced glutathione (GSH) and detoxify toxic metabolites produced within the cell by oxidative stress to protect cells from oxidant injury. v The maximum ARE enzyme activity was detected at 10 mM Tris-HCl buffer, pH 8.0 and at 45 &deg / C. ARE enzyme was saturated with its substrate phenylacetate around 20 mM concentration. The apparent Km and Vmax values of human blood serum ARE for phenylacetate were found as 1.66 mM and 3300 nmol/min/mg, respectively. The maximum GST enzyme activity was detected at 2 mM potassium phosphate buffer, pH 5.5 and at 65 &deg / C. GST enzyme was saturated with its substrate, CDNB around 4.5 mM concentration and with its cofactor, GSH around 8 mM concentration. The apparent Km and Vmax values of human blood serum GST for CDNB substrate were found as 2.8 mM and 0.43 nmol/min/mg and for GSH were found as 4.11 mM and 0.23 nmol/min/mg, respectively. In addition, effects of three different heavy metal ions, Cd+2, Hg+2 and Ni+2, on human blood serum ARE and GST activity were studied and half maximal inhibitory concentrations (IC50) were determined. The main objective of this study was to investigate the human blood serum ARE and GST activities in patient and control groups using the optimized conditions. For this purpose, blood samples were collected from 172 ischemic stroke patients and 105 controls. Then serum obtained from blood samples were used to determine ARE and GST activities. The mean of ARE activity in patient group (n=172, 109.9 &plusmn / 32.5 U/mL ) was insignificantly lower than the mean of ARE activity in control group (n=105, 113.5 &plusmn / 33.1 U/mL, P=0.284). GST activity of the patients (10.8 &plusmn / 4.4 U/L) was insignificantly higher than that of controls (10.5 &plusmn / 4.2 U/L, P=0.483 ). In addition, statistical analysis showed hypertension, diabetes and HDL as significant risk factors of stroke.

Thesis (M.S.)--Mississippi State University. in Veterinary Medical Science in the Department of Basic Sciences. / Title from title screen. Includes bibliographical references.

Lycopene is a potent carotenoid antioxidant shown to decrease the risk of chronic diseases associated with oxidative stress and has recently begun to be studied in relation to osteoporosis. However, studies specifically associating intervention with lycopene and a decreased risk for osteoporosis have not yet been conducted, and the mechanisms by which lycopene affects bone have yet to be elucidated. The purpose of this thesis was to explore the hypotheses that supplementation with lycopene would increase antioxidant capacity while decreasing oxidative stress parameters; subsequently decreasing bone turnover markers, and thus the risk of osteoporosis in postmenopausal women. Specifically, experiments were designed to determine whether lycopene acts in its antioxidant capacity to improve bone health, and to delineate the mechanisms of these effects. These hypotheses were investigated through a cross-sectional study, a randomized controlled clinical study, and in vitro studies on human osteoblast cells. The results presented in this thesis demonstrate that intervention with the potent antioxidant lycopene significantly increased concentrations of the 5-cis isomer and resulted in significantly decreased oxidative stress parameters in postmenopausal women. This decrease in oxidative stress parameters resulted in significantly decreased concentrations of the bone resorption marker crosslinked N-telopeptides of type I collagen (NTx). The typical diet of participants included a relatively low intake of lycopene, and the corresponding serum lycopene concentrations were not as effective in decreasing biomarkers of oxidative stress and bone resorption as those obtained from supplementation with lycopene to increase 5-cis serum lycopene. Studies on the paraoxonase enzyme suggest that lycopene is most effective in quenching oxidative stress to decrease bone turnover markers when the internal antioxidant defenses are insufficient or decremented. Mechanisms demonstrated by the in vitro findings suggest that cis lycopene is capable of both preventing and repairing the damaging effects of oxidative stress in osteoblasts. Overall, this thesis provides evidence that lycopene acts through its antioxidant capacity to decrease oxidative stress parameters and bone turnover markers, and may, therefore, reduce the risk for osteoporosis. Based on these findings, the consumption of lycopene by women to improve overall bone health should be considered.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Paraoxonase (PON) 1 is a high density lipoprotein (HDL) - bound antioxidant enzyme that was originally discovered and better known for its role in protecting against organophosphate (OP) - induced neurotoxicity. In the past two decades, the enzyme has gained prominence as a protective agent against atherosclerosis on account of increasing evidence that it accounts for many of the anti-atherogenic roles attributed to HDL. PON1 is a polymorphic enzyme displaying a high variability in human populations which is associated with a considerable degree of inter-individual differences in enzyme phenotype that translates to differential risk for OP toxicity and cardiovascular disease (CVD). In a series of studies and analyses, this thesis describes investigations regarding the possible involvement of PON 1 in the risk for CVD in a mixed ancestry population from Bellville, Western Cape, South Africa. This was done by evaluating the distribution of PON1 coding region polymorphisms (Q192R and L55M) and their influence on PON1 phenotype as well as the latter‟s relation to CVD risk factors (oxidative stress, inflammation and atherogenic dyslipidemia) and possible involvement in early CVD assessed by measuring intima media thickness of the carotid artery (CIMT). Since PON1 is increasingly measured in samples that have been stored for varied periods of time, the main study was preceded by a pilot study evaluating the influence of baseline conditions on the stability of PON 1 activity and antioxidant status in human sera stored for up to 12 months. It was shown that baseline glycemic status enhances the degradation of antioxidants in stored samples with indications of also accelerating the decline of PON1 levels and activity. Thus baseline glycemic status should be a factor to be considered in analyses involving stored samples. The Q192R polymorphism was found to be the functional variant influencing both concentration and activity of plasma PON1. Contrary to expectation, the L55M was nonfunctional, possibly due to its unusual distribution in this population where the 55M (83%) allele overwhelmingly predominated over the L55 allele. The R allele was the more frequent (60.4%) of the 192 polymorphism. The R allele has previously been associated with less efficient breakdown of lipid peroxides and a subsequent higher risk for atherosclerotic heart disease while the 55M is recognized as a “low concentration/activity” variant. Thus the predominant PON1 genotype distribution in this population constitutes a risk profile that may relate to increased risk for CVD. The risk for CVD was confirmed to be very high in this population indicated by high prevalence of the metabolic syndrome (48%) and its key components (and CVD risk factors) diabetes (28%), obesity (53%) and high blood pressure (57%). Paraoxonase activity associated inversely with indices of inflammation (high sensitive C- reactive protein [hs-CRP] and leptin) and oxidative stress (oxidized low density lipoprotein [LDL]) and directly with adiponectin and markers of systemic antioxidant status. These findings suggest that low paraoxonase-I activity contributes to increased cardiovascular risk possibly via involvement in early atherogenesis. However, only a modest inverse relation was observed between PON1 phenotype and CIMT thus suggesting that PON1 may not play a major role in early atherosclerosis. Taken together, the findings presented in this thesis demonstrate the presence of a risk PON1 genotypic profile and indication that the enzyme may play a role in the enhanced CVD risk in this population possibly via interactions with inflammation and oxidative stress. However, conclusive evidence for the involvement of PON1 in early CVD was not demonstrated indicating a need to explore the participation of PON1 in later stages of CVD. / AFRIKAANSE OPSOMMING: Paraoksonase (PON) 1 is 'n antioksidant ensiem wat aan HDL gebind is. Oorspronklik is dit ontdek en het bekend geword as 'n beskermer teen organofosfaat (OF)-gedrewe neurotoksisiteit. In die afgelope twee dekades het die ensiem belangrik geraak as 'n beskermer teen arterosklerose as gevolg van toenemende bewyse dat dit 'n belangrike rol speel in die beskermende effekte van HDL teen arterosklerose. PON1 is 'n polimorfiese ensiem wat groot variasie toon in verskillende populasies. Daar is ook inter-individuele verskille in ensiem fenotipe wat uitloop op 'n differensiele risiko vir OF toksisiteit en kardiovaskulêre hartsiekte (KVH). Hierdie tesis beskryf 'n reeks analises en ondersoeke betreffende die moontlike betrokkenheid van PON1 in die risiko vir KVH in 'n gemengdeafkoms populasie van Bellville, Wes-Kaap, Suid Afrika. Dit was gedoen deur die evaluering van die verspreiding van die PON-1 koderende omgewing polimorfismes (Q192R en L55M), hulle invloed op PON1 fenotipe en laasgenoemde se verhouding tot KVH risikofaktore (oksidatiewe stress, inflammasie en arterogeniese dislipedimie) en moontlike voorkoms in vroeë kardiovaskulêre siekte bepaal deur die meting van die intima media dikte van die karotied slagaar. Aangesien PON1 al hoe meer gemeet word in monsters wat vir verskeie tydperke gestoor word, was die hoofstudie voorafgegaan deur 'n loodsstudie wat die invloed van basislyn kondisies op die stabiliteit van PON1 aktiwiteit en antioksidant status in menslike sera wat vir tot 12 maande gestoor was, bepaal het. Dis is duidelik aangetoon dat basislyn glisemiese status die afbraak van antioksidante in gestoorde monsters verhoog het, asook aanduidings van die afname van PON1 vlakke en aktiwitetit. Basislyn glisemiese status behoort dus ook as 'n faktor ingereken te word in analises van gestoorde monsters. Die Q192R polimorfisme is aangetoon om 'n funksionele variant te wees wat beide die konsentrasie asook die aktiwiteit van PON1 beïnvloed het. Anders as wat verwag is, was die L55M polimorfisme nie-funksioneel, moontlik as gevolg van sy ongewone distribusie in hiedie populasie waar die voorkoms van die 55M (83%) alleel die L55 alleel oorheers het. Die R alleel was die mees algemene (60.4%) van die 192 polimorfisme. Die R alleel is voorheen reeds geassosieer met minder effektiewe afbraak van lipied peroksides en gevolglike hoër voorkoms van arteriosklerotiese hartsiekte, terwyl die 55M erken word as 'n “lae konsentrasie/aktiwiteit” variant. Die oorheersende PON1 genotipe distribusie in hierdie populasie behels dus 'n risikoprofiel wat betrekkking mag hê op verhoogde KVH. Die risiko vir KVH was bevestig om baie hoog te wees in hierdie populasie, soos aangedui deur 'n hoë voorkoms van die metaboliese sindroom (48%) en die sleutelkomponente daarvan (insluitend KVH risikofaktore), diabetes (28%), obesiteit (53%) en hipertensie (57%). Paraoksinase aktiwiteit was omgekeerd geassosieer met indekse van inflammasie (hoë C-reaktiewe proteïen [hs-CRP] en leptien) en oksidatiewe stres (geoksideerde lae digtheid lipoproteïen [LDL], en direk geassosieer met adiponektien en merkers van sistemiese antioksidantstatus. Hierdie bevindings mag aandui dat lae paraoksonase-1 aktiwiteit bydra tot verhoogde kardiovaskulêre risiko, moontlik via betrokkenheid in vroeë arterogenese. Slegs 'n klein omgekeerde verhouding is egter waargeneem tussen die PON1 fenotipe en karotied intima media dikte, wat mag aandui dat PON1 nie 'n beduidende rol speel in vroeë arterosklerose nie. In geheel, die bevindinge voorgedra in hierdie tesis demonstreer die voorkoms van 'n risiko PON1 genotipiese profiel wat 'n aanduiding mag wees dat die ensiem 'n rol mag speel in die verhoogde KVH risiko in hierdie populasie, moontlik deur interaksies met inflammasie en oksidatiewe stress. Afdoende bewys van die betrokkenheid van PON1 in vroeë KVH was egter nie gedemonstreer nie, wat die nodigheid aandui om die deelname van PON1 in latere stadiums van KVH te ondersoek.

Lycopene is a potent carotenoid antioxidant shown to decrease the risk of chronic diseases associated with oxidative stress and has recently begun to be studied in relation to osteoporosis. However, studies specifically associating intervention with lycopene and a decreased risk for osteoporosis have not yet been conducted, and the mechanisms by which lycopene affects bone have yet to be elucidated. The purpose of this thesis was to explore the hypotheses that supplementation with lycopene would increase antioxidant capacity while decreasing oxidative stress parameters; subsequently decreasing bone turnover markers, and thus the risk of osteoporosis in postmenopausal women. Specifically, experiments were designed to determine whether lycopene acts in its antioxidant capacity to improve bone health, and to delineate the mechanisms of these effects. These hypotheses were investigated through a cross-sectional study, a randomized controlled clinical study, and in vitro studies on human osteoblast cells. The results presented in this thesis demonstrate that intervention with the potent antioxidant lycopene significantly increased concentrations of the 5-cis isomer and resulted in significantly decreased oxidative stress parameters in postmenopausal women. This decrease in oxidative stress parameters resulted in significantly decreased concentrations of the bone resorption marker crosslinked N-telopeptides of type I collagen (NTx). The typical diet of participants included a relatively low intake of lycopene, and the corresponding serum lycopene concentrations were not as effective in decreasing biomarkers of oxidative stress and bone resorption as those obtained from supplementation with lycopene to increase 5-cis serum lycopene. Studies on the paraoxonase enzyme suggest that lycopene is most effective in quenching oxidative stress to decrease bone turnover markers when the internal antioxidant defenses are insufficient or decremented. Mechanisms demonstrated by the in vitro findings suggest that cis lycopene is capable of both preventing and repairing the damaging effects of oxidative stress in osteoblasts. Overall, this thesis provides evidence that lycopene acts through its antioxidant capacity to decrease oxidative stress parameters and bone turnover markers, and may, therefore, reduce the risk for osteoporosis. Based on these findings, the consumption of lycopene by women to improve overall bone health should be considered.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-10-19T16:18:49Z No. of bitstreams: 1 Tese Elizabete Inês 02012016.pdf: 4733623 bytes, checksum: 8a1ffca0e2467119048af81a914bba45 (MD5) / Made available in DSpace on 2016-10-19T16:18:49Z (GMT). No. of bitstreams: 1 Tese Elizabete Inês 02012016.pdf: 4733623 bytes, checksum: 8a1ffca0e2467119048af81a914bba45 (MD5) / prevalência da infecção por Strongyloides stercoralis é elevada em pacientes alcoolistas. O consumo excessivo de álcool eleva os níveis de corticosteroides endógenos, os quais estimulam a fecundidade da fêmea e a diferenciação das larvas rabditoides em filarioides infectantes, mimetizando o efeito do hormônio parasitário ecdisona,levando a autoinfecção interna. Além disso, as alterações da barreira intestinal e da resposta imune do hospedeiro, pelo uso crônico do álcool, contribuem para o desenvolvimento da hiperinfecção e estrongiloidíase grave. A atividade da paraoxonase (PON1) tem sido implicada na patogênese de doenças inflamatórias, além de doenças causadas por bactérias, vírus eparasitos. A PON1 é predominantemente sintetizada pelos hepatócitos e liberada na circulação associada com as lipoproteínas de alta densidade (HDL), que é a principal fonte de colesterol, para a síntese de esteroides. Os pacientes infectados com parasitos intestinais têm alterações no seu perfil lipídico e na atividade da PON1. Neste estudo, o diagnóstico da estrongiloidiase atraves da detecção de IgG anti-S. stercoralis demonstrou 88,6% de sensibilidade e 98,4% de especificidade. Através da analise proteômica é possívele estabelecerproteínas com maior especificidade a serem utilizadas para em ensaios de diagnóstico e/ ou imunomodulação. Este trabalho teve como objetivo identificar a infecção por S. stercoralis e determinar os níveis de cortisol endógeno, o perfil lipídico e a atividade da paraoxonase nos pacientes alcoolistas, internados no Centro de Acolhimento e Tratamento de Alcoolistas (CATA), pertencente às Obras Sociais Irmã Dulce. Além, de realizar o estudo da proteômica de S. stercoralis e de S. venezuelensis. A frequência da infecção por S. stercoralis foi avaliada em 332 pacientes alcoolistas e 92 pacientes que não fazem uso crônico de álcool. A determinação dos níveis de cortisol foi realizada em 78 pacientes alcoolistas infectados com S. stercoralis, 80 pacientes alcoolistas não infectados e 76 pacientes não alcoolistas com resultados de três exames parasitológicos negativos para S. stercoralis. A frequência da infecção por S. stercoralis foi maior nos pacientes alcoolistas, 23,5% (78/332) do que nos pacientes que não faziam uso crônico de álcool, 5,4% (5/92) (p < 0,05). Os níveis de cortisol endógeno foram 3,7 vezes mais elevados nos pacientes alcoolistas comparado com os não alcoolistas (p < 0,05). Níveis elevados de cortisol endógeno não foi um fator que predispôs à infecção por S. stercoralis nos pacientes alcoolistas, entretanto uma vez infectado, o aumento dos níveis deste hormônio tiveram associados com a elevação da carga parasitária. A determinação da atividade da Paraoxonase (PON1) foi realizada em 202 alcoolistas e em 74 não alcoolistas infectados ou não com S. stercoralis. A atividade da PON1 nos indivíduos infectados com S.stercoralis foi mais baixa, tanto no grupo dos alcoolistas (1,4 vezes), quanto no grupo dos não alcoolistas (6,4 vezes) (p < 0,05). Uma correlação positiva foi observada entre a atividade da paraoxonase e a concentração de cortisol em indivíduos alcoolistas não infectados com S. stercoralis (R= 0,258; p < 0,05), enquanto uma correlação negativa ocorreu com indivíduos não alcoolistas infectados com S. stercoralis (R= -0,60; p < 0,05). Os níveis de triglicérides foram 1,3 vezes menores em indivíduos alcoolistas infectados, assim como o LDL-C e VLDL-C foram respectivamente 1,1 e 6,4 mais baixos nesse mesmo grupo (p < 0,05). No entanto, os níveis de HDL foram 1,3 vezes maior nos alcoolistas do que nos não alcoolistas (p < 0,05), independentemente da infecção. Indivíduos alcoolistas infectados com S. stercoralisapresentaram um perfil lipídico compatível com um padrão anti-aterogênico. A análise proteômica de antígenos brutos de larvas filarioides de S. stercoralis, S. venezuelensis e de produtos excretados / secretados de Strongyloides venezuelensis foi realizada utilizando a plataforma shotgun (LC-MS/MS) para separação e identificação dos péptideos trípticos. Em seguida os dados foram analisados por meio do COMET.Para a distribuição do gene ontology terms, foi utilizado o programa Blast2go. Um total de 272 proteínas do parasito foram identificados sendo 158, 62 e 52 encontradas em antígeno bruto de S.stercoralis (CSS), antígeno bruto de S.venezuelensis (CSV) e produtos secretados /excretados de S. venezuelensis (ESPSV), respectivamente. Dentre as 108 proteínas encontradas apenas no CSS, oito apresentaram homologia com Strongyloides sp. - proteínas foram compartilhadas entre os três antígenos. A ubiquitina foi a proteína mais representativa dos produtos secretados/excretados de S. venezuelensis, no entanto não apresentou peptidio de sinal sugerindo que múltiplas vias de secreção pode ser utilizada para proteínas homologas. A análise proteômica pode ser utilizada como uma ferramenta para identificar proteínas para testes de diagnóstico principalmente, para a estrongiloidíase que ainda carece de diagnóstico de elevada sensibilidade e especificidade.

Abstract Dietary vegetables and fruit may play a significant role in atherosclerosis. We investigated the effects of a high intake of vegetables, berries, and citrus fruit along with a diet low in total and saturated fat on plasma concentrations of lipids, lipoprotein(a), antioxidants, oxidised LDL (OxLDL), folate, homocysteine, and on serum paraoxonase-1 activity. We also determined whether gene polymorphisms affect diet response of plasma homocysteine and serum paraoxonase-1 activity. Thirty-seven healthy females consumed two diets (low and high vegetable diets) in a controlled crossover intervention. The plasma measurements were determined at the baseline and at the end of diet periods. The average plasma concentrations of total, LDL, and HDL cholesterol were 5.0 mmol/l, 2.8 mmol/l, and 1.7 mmol/l, respectively, on the low vegetable diet, and decreased by 8%, 8%,and 5%, respectively, in response to the high vegetable diet. The high vegetable diet increased the plasma concentrations of alpha-carotene, beta-carotene, lutein-zeaxanthin, beta-cryptoxanthin, and vitamin C by 133%, 134%, 107%, 65%, and 25%, respectively, compared with the low vegetable diet. There were no differences in the plasma concentrations of OxLDL between the low and high vegetable diets. The mean serum paraoxonase-1 activity was lower at the end of the high vegetable diet (226 U/l) than at the end of the low vegetable diet (240 U/l). Subjects having a genotype with high baseline paraoxonase-1 activity showed the most extensive reduction in their serum enzyme activities. The high vegetable diet enhanced the serum and erythrocyte folate concentrations by 78% and 14%, respectively, and reduced the plasma homocysteine by 13% compared with the low vegetable diet. The dietary treatment was effective even among subjects homozygous for C677T mutation in methylenetetrahydrofolate reductase gene, who are susceptible to high homocysteine levels. In conclusion, a high intake of vegetables, berries, and citrus fruit resulted in reduced plasma total and LDL cholesterol concentrations and enhanced plasma antioxidant levels. The high vegetable diet also effectively increased blood folate concentrations and reduced plasma homocysteine concentration.