Patients on anti-retroviral (ARV) drug treatment are sometimes simultaneously taking other prescribed drugs and/or over-the-counter drugs and/or herbal remedies. Pharmacokinetic drug-drug or herb-drug interactions can occur in these patients, which might be synergistic or antagonistic in nature leading to increased or decreased bioavailability of the ARV. Consequences of bioavailability changes may either be adverse effects due to increased plasma levels, or lack of pharmacological responses due to decreased plasma levels. The aim of this study is to determine if pharmacokinetic interactions exist between selected commercially available herbal products, namely Linctagon Forte®, Viral Choice® and Canova® and the ARV, indinavir, in terms of transport and metabolism in cell culture models. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in four experimental groups, namely indinavir alone (200 μM, negative control group), indinavir in combination with Linctagon Forte®, indinavir in combination with Viral Choice® and indinavir in combination with Canova® at three different concentrations. Verapamil (100 μM), a known P-gp inhibitor, was combined with indinavir in the positive control group. Samples obtained from the transport studies were analysed by means of a validated high performance liquid chromatography (HPLC) method. The apparent permeability coefficient (Papp) values were calculated from the transport results in both directions and the efflux ratio (ER) values were calculated from these Papp values. The metabolism of indinavir was determined in LS180 cells in the same groups as mentioned for the transport study but with ketoconazole (40 μM), a known CYP3A4 inhibitor, as the positive control group. Indinavir and its predominant metabolite (M6) were analysed in the metabolism samples by means of liquid chromatography linked to mass spectroscopy (LC/MS/MS) to determine the effect of the herbal products on the biotransformation of indinavir. The BL-AP transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone (control group). Canova® only slightly affected the efflux of indinavir compared to that of the control group. Noticeable increases in the efflux ratio values of indinavir were found for Linctagon Forte® and Viral Choice®, whilst the effect of Canova® on the efflux ratio value was negligible. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Patients on anti-retroviral (ARV) drug treatment are sometimes simultaneously taking other prescribed drugs and/or over-the-counter drugs and/or herbal remedies. Pharmacokinetic drug-drug or herb-drug interactions can occur in these patients, which might be synergistic or antagonistic in nature leading to increased or decreased bioavailability of the ARV. Consequences of bioavailability changes may either be adverse effects due to increased plasma levels, or lack of pharmacological responses due to decreased plasma levels. The aim of this study is to determine if pharmacokinetic interactions exist between selected commercially available herbal products, namely Linctagon Forte®, Viral Choice® and Canova® and the ARV, indinavir, in terms of transport and metabolism in cell culture models. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in four experimental groups, namely indinavir alone (200 μM, negative control group), indinavir in combination with Linctagon Forte®, indinavir in combination with Viral Choice® and indinavir in combination with Canova® at three different concentrations. Verapamil (100 μM), a known P-gp inhibitor, was combined with indinavir in the positive control group. Samples obtained from the transport studies were analysed by means of a validated high performance liquid chromatography (HPLC) method. The apparent permeability coefficient (Papp) values were calculated from the transport results in both directions and the efflux ratio (ER) values were calculated from these Papp values. The metabolism of indinavir was determined in LS180 cells in the same groups as mentioned for the transport study but with ketoconazole (40 μM), a known CYP3A4 inhibitor, as the positive control group. Indinavir and its predominant metabolite (M6) were analysed in the metabolism samples by means of liquid chromatography linked to mass spectroscopy (LC/MS/MS) to determine the effect of the herbal products on the biotransformation of indinavir. The BL-AP transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone (control group). Canova® only slightly affected the efflux of indinavir compared to that of the control group. Noticeable increases in the efflux ratio values of indinavir were found for Linctagon Forte® and Viral Choice®, whilst the effect of Canova® on the efflux ratio value was negligible. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

In this thesis the enriched stable isotope marking technique was examined to determine its validity and effectiveness in marking of Lake Sturgeon fin rays. The enriched stable isotope marking technique uses stable isotopes of elements that replace calcium in boney tissue, which are fixed in nature, however in laboratory settings can be easily manipulated. Manipulated ratios of naturally occurring isotopes can be determined from a small sample of the fin ray, thus allowing for the discrimination of fish stocks. Further, the thesis examined the underlying assumptions for strontium and barium into the Lake Sturgeon. For the marking technique to work, strontium and barium must be taken up from the environment and accumulated long-term in boney tissue. Net flux and tissue retention of strontium is reported and the research has demonstrated that enriched stable isotopes can be successfully used to mark the fin ray of Lake Sturgeon for long-term stock discrimination. / October 2015

El objetivo central del presente trabajo de Tesis Doctoral fue restablecer in vitro la susceptibilidad antimicrobiana de cepas comensales de Escherichia coli (con fenotipo multirresistente-MDR-) aisladas de explotaciones pecuarias, mediante la asociación de diferentes antimicrobianos con el inhibidor de bombas de eflujo 1-(1-naphthylmethyl)-piperazine (NMP). De esta manera, se pretende contribuir al desarrollo de planes de dosificación de máxima eficacia antimicrobiana minimizando el riesgo de emergencia y diseminación de resistencia bacteriana; permitiendo así la obtención de productos de origen animal de excelencia sanitaria, en función de la correcta interpretación de los parámetros farmacocinéticos/farmacodinámicos (modelización PK/PD) para una correcta dosificación, del mecanismo de resistencia y del eventual bloqueo de éste último. Se obtuvieron muestras de materia fecal mediante hisopado rectal de vacas en ordeñe, terneros y animales de compañía, pertenecientes a tambos de la provincia de Buenos Aires; así como también de pozos sépticos, agua de consumo y bombas estercoleras. Se determinaron los perfiles de sensibilidad, mediante el método estandarizado de Kirby-Bauer de difusión en agar, frente a ocho antimicrobianos a todas las cepas de E.coli aisladas. Se hallaron diez cepas de E. coli multirresistentes a partir de los animales muestreados en el estudio de tipo transversal. Se emplearon cepas isogénicas de E. coli con diferentes grados de expresión de bombas de eflujo como control de calidad (AG100A con deleción total de bombas de eflujo, AG100 como cepa “normal” y AG112 con sobreexpresión de bombas). Se determinó la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) de florfenicol, ciprofloxacina, tetraciclina y ampicilina por el método de microdilución seriada en caldo Luria-Bertani (LB) con y sin NMP. Para la determinación de las interacciones entre los tres antimicrobianos seleccionados (florfenicol, ciprofloxacina y tetracilina) y el inhibidor de bombas se calculó el Índice de Concentración Inhibitoria Fraccionaria (CIF) a fin de evaluar la manifestación o no de efecto sinérgico. Para evaluar la cinética de muerte bacteriana se realizaron curvas de muerte bacteriana de las cepas de referencia y las cepas de campo MDR con y sin NMP. Con los datos obtenidos se realizó el análisis estadístico correspondiente. Por último, a partir de la información farmacodinámica obtenida se realizó una modelización farmacocinética/farmacodinámica (PK/PD), para lo cual se utilizaron datos obtenidos de estudios farmacocinéticos de florfenicol, danofloxacina y oxitetraciclina realizados previamente por el grupo de investigación de la Cátedra de Farmacología. Se aislaron cepas resistentes y multirresistentes con altos niveles de resistencia frente a tetraciclina y ampicilina. Todas las combinaciones de resistencia múltiple siempre incluyeron en su perfil a la tetraciclina. El NMP asociado a ampicilina tuvo un efecto nulo tanto en las cepas isogénicas como en las de campo. Sin embargo, ciprofloxacina, florfenicol y tetraciclina, demostraron ser claros sustratos de las bombas. Se evidenció la ocurrencia de sinergismo de potenciación ya que con una concentración de antimicrobiano varias veces inferior a la de su CIM, se logró un efecto antibacteriano mejorado con la incorporación de NMP. Se comprobó que es posible disminuir la concentración de los antimicrobianos -florfenicol, ciprofloxacina y tetraciclina- con la incorporación de NMP, sin modificar de manera importante la “cinética de muerte bacteriana”, tanto en las curvas de muerte de las cepas de referencia como en las cepas problema. En cuanto a la relación PK/PD, en la cepa AG112 se mejoraron notablemente los parámetros predictores de eficacia con la adición del inhibidor, con la consecuente disminución de la concentración de los antimicrobianos. Resulta prometedor el efecto de la combinación de un fármaco inhibidor de bombas de eflujo como coadyuvante de aquellos antimicrobianos sustratos de las bombas de eflujo sobreexpresadas como mecanismo inespecífico de resistenca bacteriana.

La déficience en phosphore est l’un des facteurs limitant la production de légumineuses à graines, dont le haricot (Phaseolus vulgaris). La diversité génétique de cette espèce en Amérique latine a permis d'identifier avec le CIAT, des lignées recombinantes du croisement de DOR364 et BAT477 à fort pouvoir fixateur d'azote (FSN) et à forte efficacité d'utilisation de phosphore (EUP). Les résultats obtenus au cours de ce travail nous ont permis de répondre à notre objectif principal de thèse qui consistait à vérifier la pertinence des critères d'adaptation de la fixation symbiotique de l'azote chez le haricot à la disponibilité de P que nous avons déterminé. Nous avons démontré, d'une part, en milieu hydroaéroponique, que les racines nodulées du haricot, excrètent dans leur rhizosphère une quantité de H+ qui est corrélée à la perméabilité nodulaire. Ce qui laisse penser qu'une part d'H+ excrétée par les racines nodulées est liée à la fixation symbiotique de N2. D'autre part, nous avons démontré que sous déficience en P les lignées tolérantes acidifient plus leur rhizosphère que les sensibles. En multisites les facteurs les plus déterminants de la hiérarchisation des sites sont N-total et P-total. Deux groupes de lignées se distinguent, les tolérantes versus les sensibles à la faible fertilité des sols en P. La lignée locale se distingue des groupes précédents. Elle est capable de croître dans différents sols, notamment les contraignants avec une grande capacité à noduler, par conséquent, à compenser la déficience en N. Nous avons également pu démontrer que les biomasses nodulaire et aérienne sont corrélées au P Olsen du sol / Phosphorus deficiency is one of the limiting factor for grain legume production like bean (Phaseolus vulgaris). The results obtained during this work enabled us to confirm our main aim of this work which is checking the relevance of the criteria of adaptation of the symbiotic nitrogen fixation of bean to P availability. We showed, in controlled conditions that the nodulated roots of bean, release in their rhizosphere a quantity of H+ which is correlated with the nodul permeability. What lets think that a share of H+ released is related to the symbiotic N2 fixing. In addition, we showed that under P deficiency the tolerant lines acidify more their rhizosphere that the sensitive ones. Into multisite trial the most determining factors of the hierarchisation of the sites are total-N and total-P. Two groups of lines are distinguished: tolerant versus sensitive to P deficiency. The local farmer line is distinguished from the studied lines. It is able to grow in various soil fertility, in particular the constraining soils. ones with a great capacity with noduler, consequently, to compensate for deficiency in NR. We also could show that shoot and nodule biomass are correlated with the Olsen-P Olsen of the soil

The crucial role of antibiotics in modern medicine, in curing infections and enabling advanced medical procedures, is being threatened by the increasing frequency of resistant bacteria. Better understanding of the forces selecting resistance mutations could help develop strategies to optimize the use of antibiotics and slow the spread of resistance. Resistance to ciprofloxacin, a clinically important antibiotic, almost always involves target mutations in DNA gyrase and Topoisomerase IV. Because ciprofloxacin is a substrate of the AcrAB-TolC efflux pump, mutations causing pump up-regulation are also common. Studying the role of efflux pump-regulatory mutations in the development of ciprofloxacin resistance, we found a strong bias against gene-inactivating mutations in marR and acrR in clinical isolates. MIC and fitness measurements revealed that amino acid substitutions conferred smaller susceptibility reductions and smaller fitness costs than gene-inactivating mutations, suggesting that resistance mutations in clinical isolates are selected for high fitness rather than high resistance (Paper I and II). We asked whether the high fitness costs of marR-inactivating mutations could be ameliorated without affecting the resistance phenotype. Multiple independent lineages were experimentally evolved to select for improved growth fitness. Whole genome sequencing revealed mutations affecting marA, lon and arcA as potential compensatory pathways. For the marA and lon mutations the improved growth rate was associated with an increased susceptibility (arcA is being investigated). (Paper III). An evolution experiment selecting for ciprofloxacin resistance revealed upon whole genome sequencing the expected mutations in drug target and efflux-regulatory genes, but also in genes encoding aminoacyl-tRNA synthetases. We investigated two independently selected leuS mutations, and concluded that they contributed to ciprofloxacin resistance by activating the stringent response that in turn caused up-regulation of genes involved in efflux. However, these leuS mutations incur a high fitness cost (Paper IV). To summarize, the research findings in this thesis suggest that the potential ciprofloxacin resistome may include more genes than previously thought, but a strong selection for high fitness selectively purifies many resistance mutations from clinical isolates. In conclusion, selection for high relative fitness constrains the spectrum of mutations that survive and get fixed in clinical populations of bacteria.

Acinetobacter baumannii is an important nosocomial pathogen characterized by the ability to acquire and develop complex resistance to antimicrobial agents. This capability is caused by eflux systems removing molecules of antibiotics from bacterial intracellular space. AdeABC is an RND-type chromosomal eflux system specific for A. baumannii which has a broad substrate spectrum. In this work, we focused on functional analysis of AdeABC to define its role in the resistance development to aminoglycosides in genetically different strains. We studied a set of 15 epidemiologically and genotypically well characterized strains of A. baumannii which were fully susceptible to aminoglycosides and other antibiotics primarily effective against this species. We determined genotyp of AdeABC for these strains and performed a selection for resistant variants in the presence of netilmicin. Using real-time qRT-PCR we compared the expression of the transporter gene adeB in originally sensitive strains and selected variants. The obtained results confirmed that the increased expression of AdeABC significantly reduces susceptibility to aminoglycosides and other antibiotics. The results also suggest that the efflux system provides a significant selective advantage for nosocomial strains of A. baumannii.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos. / The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

Os óxidos de colesterol modulam o desenvolvimento da aterosclerose por mediarem a síntese, captação e exportação de colesterol, além de inflamação e citotoxicidade na parede arterial. O exercício físico regular previne e regride a lesão aterosclerótica, por melhorar o perfil lipídico, transporte reverso de colesterol e defesas antioxidantes. A proteína cinase ativada por AMP (AMPK) é um importante mediador dos efeitos metabólicos do exercício físico. Em macrófagos, sua ativação vincula-se ao aumento no efluxo de colesterol e diminuição na captação de LDL. Entretanto, não está claro se o treinamento físico modula as concentrações de óxidos de colesterol, refletindo seu benefício sobre a prevenção da aterosclerose, e se esses efeitos podem ser mediados pela AMPK. O objetivo do presente estudo foi avaliar, em camundongos dislipidêmicos, o papel de 6 semanas de treinamento físico aeróbio sobre: o infiltrado de colesterol arterial e a distribuição de óxidos de colesterol no arco aórtico e no plasma; a expressão gênica de proteínas envolvidas na metabolização de óxidos de colesterol na parede arterial; e o efeito da ativação da AMPK em macrófagos, in vitro, sobre a concentração dos óxidos de colesterol e expressão de genes envolvidos na metabolização de óxidos de colesterol. Para tanto, camundongos machos knockout para apolipoproteína E, com 16 semanas de idade, alimentados com dieta padrão, foram incluídos no estudo. O treinamento físico foi realizado em esteira, 15 m/min, por 60 min, 5 dias/semana, durante 6 semanas. Lípides plasmáticos e glicose foram determinados por ensaio enzimático e glicosímetro, respectivamente, antes e após o treinamento físico. Colesterol arterial e óxidos de colesterol foram avaliados por cromatografia gasosa acoplada à espectrometria de massa. A expressão de genes envolvidos no metabolismo de lípides foi avaliada RT-qPCR. Os resultados foram comparados por ANOVA de um fator com pós-teste de Newman-Keuls ou teste t de Student. Peso corporal, colesterol total, TG, HDL-c, glicose e óxidos de colesterol no plasma foram semelhantes entre os grupos. O treinamento físico aumentou a concentração de 7alfa-OH C (70%) e reduziu a de colesterol (32%) na aorta. Além disso, o exercício físico aumentou a expressão gênica da Cyp27a1 (54%), Cd36 (75%), Cat (70%), Prkaa1 (AMPKalfa1) (40%) e Prkaa2 (AMPKalfa2) (51%) e reduziu Abcg1 (31%), Olr1 (LOX-1) (65%), Cyp7b1 (35%) e Ch25h (48%). Nenhuma alteração foi observada na expressão de Abca1, Nr1h3 (LXRalfa) e Nr1h2 (LXRbeta). Nos macrófagos, a ativação da AMPK por AICAR, reduziu o conteúdo de 7alfa-OH C após estimulo com HDL2. O tratamento com AICAR aumentou a expressão gênica de Abca1 (52%) e Cd36 (220%) e diminuiu Prkaa1 (19%) e Cyp27a1 (47%), e não alterou Abcg1, Nr1h3 e Nr1h2. Em conclusão, em camundongos dislipidêmicos, o treinamento físico aeróbio, por 6 semanas, aumentou a concentração de 7 beta -OH C, o que se vincula à maior expressão de Cd36 no arco aórtico. A rápida difusão de óxidos de colesterol, como via complementar ao transporte reverso de colesterol, pode também ser favorecida pelo aumento e redução, respectivamente, na expressão de Cyp27a1 e Cyp7b1, favorecendo maior liberação de 27-OH C das células. Juntamente com suas ações diretas que beneficiam o transporte reverso de colesterol, previamente descritas o treinamento físico diminui a concentração de colesterol na parede arterial, prevenindo a aterosclerose. Baseado nos ensaios in vitro a ativação da AMPK não parece contribuir para o aumento das concentrações de óxidos de colesterol após treinamento físico / Oxysterols modulate the development of atherosclerosis by mediating cholesterol synthesis, uptake and exportation as well as inflammation and cytotoxicity in the arterial wall. Regular physical exercise prevents and regresses atherosclerosis by improving lipid metabolism, reverse cholesterol transport and antioxidant defenses. AMP-activated protein kinase (AMPK) plays an important role in the beneficial metabolic adaptations of physical exercise. In macrophages, its activation is related to the enhancement in cholesterol efflux and reduction in LDL uptake. However, it is not clear whether exercise training benefits in atherosclerosis is mediated by its action in oxysterols concentrations, and whether this can be modulated by AMPK. The aim of this study was to evaluate the role of a 6-week aerobic exercise training program in dyslipidemic mice in the arterial and plasma accumulation of cholesterol and oxysterols subspecies; expression of genes related to oxysterols metabolisms in the aortic arch, and the effect of AMPK activation in macrophage on the concentration of oxysterols and expression of genes linked to oxysterols metabolism. Sixteen-week-old male apoE knockout mice fed a chow diet were included in the protocol. Animals were trained in a treadmill running, 15 m/min, 60 min, 5 days/week, during 6 weeks. Plasma lipids and glucose were determined by enzymatic techniques and glycosometer, respectively. Cholesterol in aortic arch and oxysterols were measured by gas chromatography/mass spectrometer. The expression of genes involved in lipid metabolism was determined by RT-qPCR. Results (mean ± SD) were compared by one-way ANOVA with Newman-Keuls posttest or Student\'s t-test. Body weight and plasma total cholesterol, TG, HDL-c, glucose, and oxysterols were similar among groups. The exercise training enhanced 7beta-hydroxycholesterol (70%) and reduced cholesterol (32%) in the aortic arch. In addition, exercise increased Cyp27a1 (54%), Cd36 (75%), cat (70%), Prkaa1 (AMPKalpha1) (40%) and Prkaa2 (AMPKalpha2) (51%) mRNA. No changes were observed in the expression of Abca1, Nr1h3 (LXRalpha) and Nr1h2 (LXRbeta). In macrophages, the activation of AMPK by AICAR, reduced 7beta-hydroxycholesterol level after stimulation by HDL2. Treatment with AICAR increased Abca1 (52%) and Cd36 (220%), decreased Prkaa1 (19%) e Cyp27a1 (47%), and did not change Abcg1, Nr1h3 e Nr1h2. In conclusion, in dyslipidemic mice aerobic exercise training increases the nonenzymatic-driven oxysterol, 7beta-hydroxycholesterol, which is related to the enhanced expression of Cd36. The rapid diffusion of oxysterols, as a complementary pathway for the reverse cholesterol transport, may also be favored by the increase and reduction of Cyp27a1 and Cyp7b1 expressions, respectively, which in turns favors 27-OH C desorption from cells. Together with its direct role in improving reverse cholesterol transport as previously reported, aerobic exercise training diminishes cholesterol accumulation in the arterial wall preventing atherosclerosis. Based on in vitro assays, the AMPK activation does not seem to contribute to the effect of exercise in increasing oxysterols

Os óxidos de colesterol modulam o desenvolvimento da aterosclerose por mediarem a síntese, captação e exportação de colesterol, além de inflamação e citotoxicidade na parede arterial. O exercício físico regular previne e regride a lesão aterosclerótica, por melhorar o perfil lipídico, transporte reverso de colesterol e defesas antioxidantes. A proteína cinase ativada por AMP (AMPK) é um importante mediador dos efeitos metabólicos do exercício físico. Em macrófagos, sua ativação vincula-se ao aumento no efluxo de colesterol e diminuição na captação de LDL. Entretanto, não está claro se o treinamento físico modula as concentrações de óxidos de colesterol, refletindo seu benefício sobre a prevenção da aterosclerose, e se esses efeitos podem ser mediados pela AMPK. O objetivo do presente estudo foi avaliar, em camundongos dislipidêmicos, o papel de 6 semanas de treinamento físico aeróbio sobre: o infiltrado de colesterol arterial e a distribuição de óxidos de colesterol no arco aórtico e no plasma; a expressão gênica de proteínas envolvidas na metabolização de óxidos de colesterol na parede arterial; e o efeito da ativação da AMPK em macrófagos, in vitro, sobre a concentração dos óxidos de colesterol e expressão de genes envolvidos na metabolização de óxidos de colesterol. Para tanto, camundongos machos knockout para apolipoproteína E, com 16 semanas de idade, alimentados com dieta padrão, foram incluídos no estudo. O treinamento físico foi realizado em esteira, 15 m/min, por 60 min, 5 dias/semana, durante 6 semanas. Lípides plasmáticos e glicose foram determinados por ensaio enzimático e glicosímetro, respectivamente, antes e após o treinamento físico. Colesterol arterial e óxidos de colesterol foram avaliados por cromatografia gasosa acoplada à espectrometria de massa. A expressão de genes envolvidos no metabolismo de lípides foi avaliada RT-qPCR. Os resultados foram comparados por ANOVA de um fator com pós-teste de Newman-Keuls ou teste t de Student. Peso corporal, colesterol total, TG, HDL-c, glicose e óxidos de colesterol no plasma foram semelhantes entre os grupos. O treinamento físico aumentou a concentração de 7alfa-OH C (70%) e reduziu a de colesterol (32%) na aorta. Além disso, o exercício físico aumentou a expressão gênica da Cyp27a1 (54%), Cd36 (75%), Cat (70%), Prkaa1 (AMPKalfa1) (40%) e Prkaa2 (AMPKalfa2) (51%) e reduziu Abcg1 (31%), Olr1 (LOX-1) (65%), Cyp7b1 (35%) e Ch25h (48%). Nenhuma alteração foi observada na expressão de Abca1, Nr1h3 (LXRalfa) e Nr1h2 (LXRbeta). Nos macrófagos, a ativação da AMPK por AICAR, reduziu o conteúdo de 7alfa-OH C após estimulo com HDL2. O tratamento com AICAR aumentou a expressão gênica de Abca1 (52%) e Cd36 (220%) e diminuiu Prkaa1 (19%) e Cyp27a1 (47%), e não alterou Abcg1, Nr1h3 e Nr1h2. Em conclusão, em camundongos dislipidêmicos, o treinamento físico aeróbio, por 6 semanas, aumentou a concentração de 7 beta -OH C, o que se vincula à maior expressão de Cd36 no arco aórtico. A rápida difusão de óxidos de colesterol, como via complementar ao transporte reverso de colesterol, pode também ser favorecida pelo aumento e redução, respectivamente, na expressão de Cyp27a1 e Cyp7b1, favorecendo maior liberação de 27-OH C das células. Juntamente com suas ações diretas que beneficiam o transporte reverso de colesterol, previamente descritas o treinamento físico diminui a concentração de colesterol na parede arterial, prevenindo a aterosclerose. Baseado nos ensaios in vitro a ativação da AMPK não parece contribuir para o aumento das concentrações de óxidos de colesterol após treinamento físico / Oxysterols modulate the development of atherosclerosis by mediating cholesterol synthesis, uptake and exportation as well as inflammation and cytotoxicity in the arterial wall. Regular physical exercise prevents and regresses atherosclerosis by improving lipid metabolism, reverse cholesterol transport and antioxidant defenses. AMP-activated protein kinase (AMPK) plays an important role in the beneficial metabolic adaptations of physical exercise. In macrophages, its activation is related to the enhancement in cholesterol efflux and reduction in LDL uptake. However, it is not clear whether exercise training benefits in atherosclerosis is mediated by its action in oxysterols concentrations, and whether this can be modulated by AMPK. The aim of this study was to evaluate the role of a 6-week aerobic exercise training program in dyslipidemic mice in the arterial and plasma accumulation of cholesterol and oxysterols subspecies; expression of genes related to oxysterols metabolisms in the aortic arch, and the effect of AMPK activation in macrophage on the concentration of oxysterols and expression of genes linked to oxysterols metabolism. Sixteen-week-old male apoE knockout mice fed a chow diet were included in the protocol. Animals were trained in a treadmill running, 15 m/min, 60 min, 5 days/week, during 6 weeks. Plasma lipids and glucose were determined by enzymatic techniques and glycosometer, respectively. Cholesterol in aortic arch and oxysterols were measured by gas chromatography/mass spectrometer. The expression of genes involved in lipid metabolism was determined by RT-qPCR. Results (mean ± SD) were compared by one-way ANOVA with Newman-Keuls posttest or Student\'s t-test. Body weight and plasma total cholesterol, TG, HDL-c, glucose, and oxysterols were similar among groups. The exercise training enhanced 7beta-hydroxycholesterol (70%) and reduced cholesterol (32%) in the aortic arch. In addition, exercise increased Cyp27a1 (54%), Cd36 (75%), cat (70%), Prkaa1 (AMPKalpha1) (40%) and Prkaa2 (AMPKalpha2) (51%) mRNA. No changes were observed in the expression of Abca1, Nr1h3 (LXRalpha) and Nr1h2 (LXRbeta). In macrophages, the activation of AMPK by AICAR, reduced 7beta-hydroxycholesterol level after stimulation by HDL2. Treatment with AICAR increased Abca1 (52%) and Cd36 (220%), decreased Prkaa1 (19%) e Cyp27a1 (47%), and did not change Abcg1, Nr1h3 e Nr1h2. In conclusion, in dyslipidemic mice aerobic exercise training increases the nonenzymatic-driven oxysterol, 7beta-hydroxycholesterol, which is related to the enhanced expression of Cd36. The rapid diffusion of oxysterols, as a complementary pathway for the reverse cholesterol transport, may also be favored by the increase and reduction of Cyp27a1 and Cyp7b1 expressions, respectively, which in turns favors 27-OH C desorption from cells. Together with its direct role in improving reverse cholesterol transport as previously reported, aerobic exercise training diminishes cholesterol accumulation in the arterial wall preventing atherosclerosis. Based on in vitro assays, the AMPK activation does not seem to contribute to the effect of exercise in increasing oxysterols