The Gram-negative bacterium 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 can live on long chain fatty acids as its sole carbon source. However, the rate of fatty acid uptake was reduced after starvation (Mangroo, 1992). In this study, it was found that this starvation effect of reducing oleate uptake rate could only be observed in the initial minute post starvation. The starvation effect was not due to the depletion of its substrates ATP or reduced coenzyme A. Neither was the effect caused by a lowered acyl-CoA synthetase level. This reduction in oleate uptake rate can be reversed by incubation in oleate. It has been reported that lactate activated oleate uptake in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 (Mangroo and Gerber, 1993). In the present study, it was discovered that when [9, 10-³H]oleate was used as the radioactive tracer in uptake assay, the majority of the radioactivity effluxed from the cells. It was found that lactate did not have an effect on the overall oleate uptake. Its apparent activation on oleate uptake was due to the reduction in the efflux rate of radioactive probes, instead of increasing the uptake rate of oleate. Acyl-CoA synthetase in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 has always been described as a protein of around 45 kDa (Kameda and Nunn, 1981; Kameda 𝘦𝘵 𝘢𝘭., 1986). However, the 𝘧𝘢𝘥𝘋 gene that encodes the 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 acyl-GoA synthetase predicts a 62 kDa protein (Black 𝘦𝘵 𝘢𝘭., 1992; Fulda 𝘦𝘵 𝘢𝘭., 1994). This new form of acyl-CoA synthetase has been partially purified. This form, which has a size of around 62 kDa, can be converted to its 45 kDa counterpart in a Triton X-1 00 and temperature dependent manner. This induced processing of the 62 kDa enzyme was found to be inhibited by oleate, a natural substrate of acyl-CoA synthetase. The two forms of acyl-CoA synthetase have also been shown to be immunologically related. A much simpler purification scheme was developed for the 45 kDa acyl-CoA synthetase using the membrane bound form of acyl-CoA synthetase. The selective extraction of the 62 kDa acyl-CoA synthetase from the membrane, together with the induced processing into the 45 kDa form has provided the basis for this comparatively simple process. The availability of the expression system made the low yield associated with this simple process acceptable. Using oleate as substrate, the Kₘ and Vₘₐₓ for the 45 kDa acyl-CoA synthetase were determined to be 85 ± 18μM and 1550 ± 165 nmole/min/mg protein respectively, whereas the Kₘ and Vₘₐₓ for the 62 kDa enzyme were determined to be 38 ± 12μM and 633 ± 79 nmole/min/mg protein respectively. This suggested that the 62 kDa enzyme has higher affinity towards oleate than its 45 kDa counterpart under standard assay conditions while the ratio of Vₘₐₓ /Kₘ remained relatively constant. Evidence suggesting Triton X-1 00 activates acyl-CoA synthetase by providing a surface for catalysis was presented. This interaction between acyl-CoA synthetase and the Triton X-1 00 I oleate mixed micelle could be affected by the surface charge of the mixed micelle. / Thesis / Master of Science (MS)

Depuis une vingtaine d’années, il est possible d’allonger la durée de vie génétiquement. Nombre d’études réalisées sur des espèces allant de la levure aux primates, ont permis d’identifier des cascades de signaux intracellulaires ayant un impact sur la longévité et la qualité du vieillissement. Il est important de noter que certaines de ces interventions réduisent considérablement l’incidence de cancers et de maladies liées au vieillissement chez les mammifères. Ceci témoigne des liens existant entre vieillissement et carcinogénèse et il probable que le développement de stratégies pharmacologiques ayant pour cible le vieillissement se révèlent efficaces contre les maladies du vieillissement comme le cancer, la maladie d’Alzheimer ou de Parkinson. Nous avons criblé la banque de mutants de Saccharomyces cerevisiae pour identifier des mutations génétiques qui augmentent la durée de vie. La plupart des gènes identifiés se sont révélés conservés puisqu’ils influencent aussi la longévité chez C. elegans. La protéine de liaison à l’acyl-CoA (ACBP) est une petite protéine (10 kDa) qui se lie avec une haute affinité aux chaîne d’acyl-CoA esters (moyennes et longues) et les transporte vers les sites de consommation de l'acyl-CoA. ACBP est hautement conservée parmi les espèces eucaryotes et joue un rôle important dans la biosynthèse des lipides et le trafic vésiculaire. Chez Saccharomyces cerevisiae, la délétion d’ACBP (ACB1) entraîne une augmentation de la longévité et favorise la résistance au stress. Pour tester si l’impact d’ACBP sur la longévité s'étend aux eucaryotes supérieurs, nous avons exploré le lien entre les gènes codant pour des ACBPs chez Caenorhabditis elegans et la longévité en utilisant l’ARN interfèrent. Chez C. elegans, sept paralogues ACBP ont été identifiés, qui sont exprimés dans différents tissus. Nous avons constaté que la réduction de l'expression de maa-1 (codant une ACBP associée aux membranes) prolonge la durée de vie des vers sauvages. Nos résultats démontrent que: 1) une perte de fonction de maa-1 entraîne une résistance au superoxyde, 2) et aux événements protéotoxiques telle que l'agrégation protéique associées aux maladies neurodégénératives comme la maladie de Huntington. Enfin, nous avons montré que l'activité du facteur de transcription HIF-1 (hypoxia inducible factor-1) contribue à la longévité causée par la mutation maa-1. En effet, la délétion du gène hif-1 annule complètement l’augmentation de la longévité causée par maa-1. / Understanding the aging process, its regulation, and how to delay it has become a priority for an increasing number of scientists worldwide. The principal reason for this is that it is becoming more and more evident that anti-aging interventions may be effective against age-related diseases such as cancer, cardiovascular, and neurodegenerative diseases. Simple model organisms such as Caenorhabditis elegans and Saccharomyces cerevisiae have been instrumental to identify the principal genes implicated in aging whose role has turned out to be conserved in mammals. The project presented here has originated from a genome-wide screen performed in S. cerevisiae that has led to discover several novel life span-regulatory genes whose deletion prevents aging. One of these genes encodes for Acyl-CoA binding protein (ACBP). ACBP is a small (10 kDa) protein that binds medium- and long-chain fatty acyl-CoA esters with high affinity and transports them to acyl-CoA consuming processes. ACBP is highly conserved among eukaryotic species and plays important roles in lipid biosynthesis and vesicle trafficking. In S. cerevisiae, lack of ACBP (Acb1) extends longevity and promotes stress resistance. To test whether the life span-regulatory role of ACBP extends to higher eukaryotes, we explored the link between the C. elegans ACBP genes and longevity by RNAi screening. In C. elegans, seven ACBP paralogs have been identified, which are expressed in different tissues. We found that reducing the expression of maa-1 (encoding a membrane associated ACBP) extended the longevity of wild-type worms. Our results show that 1) a loss of function maa-1 mutant is resistant to the superoxide-generating agent paraquat and 2) reduction of maa-1 expression increases resistance to the proteotoxicity associated with the aggregation of the Huntington's disease-associated polyQ peptide. The activity of the anti-aging transcription factor HIF-1 (hypoxia inducible factor-1) contributes to the extended longevity caused by lack of maa-1. The effect of MAA-1 loss on longevity was fully reverted by the deletion of the hif-1 gene.

Abstract
 Acetyl-CoA
carboxylase
(ACC)
is
a
key
enzyme
of
fatty
acid
metabolism
with
multiple
 isozymes
often
expressed
in
different
eukaryotic
cellular
compartments.

 In
agriculture,
inhibitors
of
plastid
ACC
are
used
as
efficient
herbicides
against
grass
 weed.
However,
grass
weed
populations
resistant
to
aryloxyphenoxypropionate
(APP)
and
 cyclohexanedione
(CHD)
herbicides
represent
a
major
problem
for
sustainable
agriculture.
 Using
PCR
and
sequencing
it
was
found
out
that
five
amino
acid
substitutions
in
plastid
ACC
 were
 correlated
 with
 herbicide
 resistance
 of
 Avena
 sterilis
 ssp.
 ludoviciana
 Durieu
 populations
from
the
northern
grain-growing
region
of
Australia:
Trp-1,999-Cys,
Trp-2,027- Cys,
 Ile-2,041-Asn,
 Asp-2,078-Gly
 and
 Gly-2,096-Ala.
 We
 showed,
 using
 a
 yeast
 gene- replacement
 system,
 that
 these
 single-site
 mutations
 also
 confer
 herbicide
 resistance
 to
 wheat
plastid
ACCase:
Asp-2,078-Gly
confers
resistance
to
APPs
and
CHDs,
Trp-2,027-Cys
 and
Ile-2,041-Asn
confer
resistance
to
APPs,
and
Trp-1,999-Cys
confers
resistance
only
to
 fenoxaprop.
These
mutations
are
very
likely
to
confer
resistance
to
any
grass
weed
species
 under
selection
imposed
by
the
extensive
agricultural
use
of
the
herbicides.

...

Cinco membros da família das Acil-CoA sintetases de cadeia longa (ACSL) são responsáveis por ativar ácidos graxos, produzindo acil-CoA, e distribuí-los entre diversas vias metabólicas no interior da célula, tais como a síntese de triacilglicerol (TAG) e ?- oxidação mitocondrial. Apesar das disfunções nas ACSLs contribuirem para muitas doenças metabólicasa função de algumas isoformas de ACSL em tecidos específicos permanece ainda sem descrição na literatura. Aqui mostramos pela primeira vez a presença de mRNA da ACSL6 no músculo esquelético de seres humanos. Além disso, indivíduos obesos apresentaram menores níveis de mRNA de ACSL6 quando comparados à indivíduos magros. Após refeição hiperlipídica aguda (high fat meal, HFM, 90% de gordura) a expressão ACSL6 aumentou 2,5 vezes em relação aos níveis de jejum. Nós também verificamos as condições metabólicas que controlam a expressão ACSL6 em ratos: o jejum de 48h modulou negativamente a expressão gênica de ACSL6 e de outros genes de síntese de lipídeos tais como SREBP-1c e DGAT1, enquanto que a ingestão aguda de HFM (80% de gordura saturada , 10 mL/kg) teve o efeito oposto; Após o treinamento aeróbio (6 semanas, 5 dias /semana, uma vez por dia, 60 min a 70% da capacidade aeróbica máxima) o mRNA da ACSL6 foi reduzido em 35%. Em células primárias de músculo esquelético de ratos, a transfecção com siRNA de ACSL6 diminuiu a expressão de ACSL6, DGAT1 e SREBP-1c e o acúmulo de TAGs e gotas lipídicas. O silenciamento gênico da ACSL6 também aumentou o conteúdo dos ácidos graxos C16:0 e C18:0, AMPK-fosforilada, capacidade respiratória mitocondrial, a oxidação de palmitato e mRNA de PGC-1?, UCP2 e UCP3, mas diminuiu a produção de espécies 11 reativas de oxigênio. Em células primárias de músculo esquelético de seres humanos, a superexpressão da ACSL6 não alterou o conteúdo de TAG e da proteína DGAT1, mas aumentou as espécies lipídicas esfingomielina e fosfatidilcolinas, e reduziu a oxidação de 1-14C-palmitato e a expressão do PGC1?. Em conclusão, ACSL6 está envolvida na síntese e distribuição de acil-CoA para a síntese de lipídeos. A inibição gênica da ACSL6 melhora a capacidade de respiração mitocondrial e oxidação lipídica, através da ativação da via AMPK/PGC1?. / Five members of long-chain acyl-CoA synthetase (ACSL) family activate fatty acids providing acyl-CoA for several metabolic pathways within the cell, such as synthesis of triacylglycerol (TAG) and mitochondrial ?-oxidation, and their dysfunctions contribute to many metabolic diseases. Despite this, the existence and function of some ACSL isoforms in specific tissues remains unclear. Here we show for the first time the presence of ACSL6 mRNA and protein in skeletal muscle (SM) of humans. Obese subjects had lower levels of ACSL6 mRNA when compared to leans, and acute high fat meal (HFM, 90% fat) increased ACSL6 expression 2.5 times over fasted levels in both. We also verify the metabolic conditions that control ACSL6 expression in rats: fasting (48h) negatively modulated the ACSL6 mRNA and the expression of other genes of lipid synthesis SREBP-1c and DGAT1 in rat SM, while acute ingestion of HFM (80% saturated fat, 10 mL/Kg) had the opposite effect; After aerobic training (6 weeks, 5 days/week, once a day, 60 min at 70% of maximal aerobic capacity) ACSL6 mRNA was reduced 35%. In primary skeletal muscle cells (PSMC) of rats, ACSL6-specific siRNA oligo transfection (20 nM) decreased ACSL6, DGAT1 and SREBP-1c mRNA and the accumulation of TAGs and lipid droplets (LD). The knockdown also increased the content of C16:0 and C18:0 fatty acids, AMPK-Phosphorylated, mitochondrial content and respiratory rates, palmitate oxidation and PGC-1?, UCP2 and UCP3 mRNA, but decreased reactive oxygen species production. In PSMC of humans, ACSL6 overexpression did not change the contents of TAG or DGAT1 mRNA, but increased sphingomyelin and phosphatidylcholines and reduced 14C-palmitate oxidation and PGC1? mRNA expression. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its 13 downregulation improves mitochondrial capacity of respiration, lipid oxidation and biogenesis, which involves the activation of AMPK/PGC1-? pathway.

As dislipidemias são importantes fatores de risco para desenvolvimento de Doenças Cardiovasculares (DCV) comprovado através de grandes estudos observacionais. Estudos demonstraram que a prescrição regular de hipolipemiantes (estatinas e fibratos) pode reduzir a ocorrência de eventos cardiovasculares e diminuir a mortalidade. Objetivou-se realizar uma análise econômica e a influência de atorvastatina, sinvastatina, bezafibrato e ciprofibrato sobre a morbimortalidade cardiovascular em indivíduos que fizeram uso destes medicamentos em 2007 dispensados no Programa de Medicamentos Excepcionais do Ministério da Saúde distribuídos pela Farmácia Ambulatorial do Hospital das Clínicas da FMRP-USP. Trata-se de um estudo observacional, descritivo e de caráter transversal. A casuística foi composta por 332 (31,11%) indivíduos sorteados aleatoriamente dentre 1067 pacientes (erro padrão de 5%), de ambos os sexos, encaminhados pelo Sistema Único de Saúde (SUS) e consultórios particulares. Os indivíduos selecionados foram submetidos a uma entrevista e seus prontuários médicos analisados. Dos 310 pacientes entrevistados, 157 (51%) eram do sexo masculino. A faixa etária variou de 15 a 63 anos (X= 62,0 ± 12,23). Constatou-se 5 óbitos em 2007, sendo 100% do sexo masculino, com idade variando de 57 a 74 anos (X= 68,2 ± 6,95). 227 (73,22%) pacientes fizeram uso de estatinas, 54 (17,42%) de fibratos e 31 (10%) controles (sem uso de medicamentos). De 246 (79,35%) indivíduos analisados, a média do índice de massa corpórea (IMC) foi >28,7 Kg/m2; 121 (39%) pacientes fizeram uso de sinvastatina, 104 (34%) atorvastatina, 25 (8%) ciprofibrato, 29 (9%) bezafibrato. O perfil lipídico apresentou-se mais elevado no grupo atorvastatina e bezafibrato. Em relação aos eventos e/ou procedimentos, houve um total de 253. 132 (52,17%) pacientes apresentaram aterosclerose documentada, 60 (23,71%), angina pectoris, 28 (11,47%) insuficiência cardíaca, 6 (2,44%) infarto agudo do miocárdio, 6 (2,44%) aneurisma arterial e 4 (1,62%) acidente vascular encefálico. Quanto aos procedimentos, constatou-se a realização de 11 cateterismos cardíacos e 7 angioplastias. Quanto à análise econômica, o tratamento do grupo atorvastatina apresentou o maior custo (R$994,69 paciente/ano), já no grupo da sinvastatina (R$337,61 paciente/ano) houve maiores gastos com exames laboratoriais e complementares. Entre o grupo dos fibratos não houve diferenças importantes com relação ao custo do tratamento. Conclui-se que entre os indivíduos estudados, prevaleceu a população idosa, maior número de óbitos no sexo masculino; houve a prevalência de sobrepeso/obesidade (IMC > 25 kg/m2); aterosclerose documentada e angina pectoris foram os eventos cardiovasculares predominantes e o cateterismo cardíaco o procedimento mais realizado. O tratamento com atorvastatina foi o mais oneroso, entretanto, seus pacientes apresentaram menor ocorrência de eventos e procedimentos cardiovasculares, além do menor custo com exames laboratoriais. / Dyslipidemias are a major risk factor for the development of cardiovascular diseases. Several studies have shown that regular prescribing of lipid-lowering drugs (statins and fibrates) can reduce cardiovascular events and decrease overall mortality. The objectives of this study were to perform a economic analysis and the influence of atorvastatin, simvastatin, bezafibrate or ciprofibrate on the cardiovascular morbimortality in individuals who used these drugs during the year of 2007, dispensed by the Outpatient Pharmacy of Clinical Hospital of FMRP-USP according to the Exceptional Medicines Program of Ministry of Health. This is an observational and descriptive study of transversal character. The sample was composed of 332 (31,11%) individuals, randomly selected among 1067 patients (standard error of 5%), of both sexes, living in Ribeirão Preto-SP conveyed by the Single System of Health (SUS) and private clinics. Individuals were submitted to an interview and had their medical records examined. Among the 310 patients interviewed, 157 (51%) were males with ages ranging from 15 to 63 years old (X= 62,0 ± 12,23). Five deaths were reported in 2007, and of those patients, 100% were males, with ages ranging from 57 to 74 years old (X= 68,2 ± 6,95). 227 (73,22%) patients were using statins, 54 (17,42%) fibrates and 31 (10%) controls (no use of drugs). The average of body mass index (BMI) of 246 (79,35%) patients evaluated was above 28,7 Kg/m2; 121 (39%) patients were using simvastatin, 104 (34%) atorvastatin, 25 (8%) ciprofibrate and 29 (9%) bezafibrate. The lipid profile was more elevated in atorvastatin and bezafibrate groups. A total of 253 events and/or procedures were found. 132 (52,17%) patients had atherosclerosis documented, 60 (23,71%) angina pectoris, 28 (11,47%) heart failure, 6 (2,44%) acute myocardial infarction, 6 (2,44%) arterial aneurysm, 4 (1,62%) vascular brain accident. Regarding the procedures, 11 cardiac catheterism and 7 angioplasties were verified. Regarding the economic analysis, atorvastatin treatment group showed to be the most expensive one (R$ 994,69 patient/year). For the simvastatin group (R$337,61 patient/year), there were increased costs for lab and complementary tests, while among the group of fibrates there were no substantial differences in the cost of treatments. It is concluded that among the evaluated individuals, there was a prevalence of elderly people, deaths of male patients and overweight (BMI > 25kg/m2). The presence of atherosclerosis and angina pectoris were the predominant cardiovascular events and the cardiac catheterism procedure was the most performed. Although treatment with atorvastatin was the most expensive, patients in that treatment had a lower incidence of cardiovascular events and procedures, and lower costs with lab and complementary tests.

Orientador: Anibal Eugenio Vercesi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-09-11T21:10:31Z (GMT). No. of bitstreams: 1 Velho_JesusAntonio_D.pdf: 7243183 bytes, checksum: e41359a2677f40435f99f0f40830ee99 (MD5) Previous issue date: 2007 / Resumo: Resultados recentes do nosso grupo demonstram que mitocôndrias de camundongos hipercolesterolêmicos por deficiência do receptor de LDL (LDLR-1-) apresentam uma baixa capacidade antioxidante em função do alto consumo de NADPH para sustentar elevadas taxas de lipogênese (Oliveira et al., FASEB J. 19: 278-280; 2005). Neste trabalho, o estresse oxidativo mitocondrial dos camundongos LDLK-/- foi caracterizado, mostrando uma diminuição da razão GSH/GSSG e altos níveis de grupos carbonilas protéicos quando comparado com camundongos controles. Não foram encontradas diferenças significativas na atividade do sistema antioxidante glutationa peroxidase/redutase. Catalase exógena preveniu a oxidação espontânea de NAD(p)H endógeno em mitocôndrias isoladas de camundongos LDLR-/-, indicando que esta oxidação é mediada por 'H IND. 2' 'O IND. 2' gerado pela mitocôndria. A alta taxa de liberação de 'H IND. 2' 'O IND. 2' por mitocôndrias de camundongos LDLR-/- também foi previnida na presença de rotenona e isocitrato exógeno, os quais mantém o conteúdo endógeno de NAD(p) completamente reduzido. A nossa hipótese de que altas taxas de lipogênese diminuem a razão NADPH/NADP+ devido à redução do conteúdo de substratos ligados ao NADP é suportada pela observação de que o consumo de oxigênio mantido por substratos endógenos foi menor em mitocôndrias de camundongos LDLR-/- quando comparado com mitocôndrias controles, mas foi similar na presença de isocitrato exógeno. De fato, os níveis mitocondriais de isocitrato foram significativamente menores em camundongos LDLK-/-. Na segunda parte do trabalho, nós utilizamos estatinas (inibidores da 3- hidróxi-3-metilglutaril CoA redutase) na tentativa de corrigir o estresse oxidativo observado em camundongos LDLR-/-, mas descobrimos que o tratamento com a lovastatina piora este efeito. Mitocôndrias isoladas de figado de camundongos LDLR-/- tratados durante 15 dias com doses terapêuticas (100 mg/kg, v.o.) de lovastatina apresentaram uma maior susceptibilidade para desenvolver transição de permeabilidade mitocondrial (TPM). Em experimentosin vitro, lovastatinainduziuTPM de forma dose-dependente (10-80 'mu'M) por um mecanismo sensível a ciclosporina A (inibidor de ciclofilina), ditiotreitol (agente redutor), ADP (inibidor do translocador de nucleotídeo de adenina), catalase (redutor de 'H IND. 2' 'O IND. 2') e EGTA (quelante de cálcio). Em concordância com a inibição do inchamento mitocondrial por ditiotreitol, a lovastatina também diminuiu o conteúdo de grupos tiólicos de proteínas de membrana. Sinvastatina teve efeitos similares a lovastatina sobre as mitocôndrias; entretanto a pravastatina, uma estatina hidrofilica, apresentou menor capacidade de induzir a TPM. Estes resultados demonstram que estatinas podem atuar diretamente na mitocôndria tanto in vivo quanto in vitro induzindo transição de permeabilidade, que é um processo envolvido na morte celular / Abstract: Recent results from our group demonstrated that hypercholesterolemic LDL receptor knockout (LDLR-/-) mice mitochondria possess a lower antioxidant capacity due to a large consumption of reducing equivalents from NADPH to sustain high rates of lipogenesis (Oliveira et al., FASEB J.; 19: 278-280; 2005). In this work, LDLR-/- mice oxidative stress was further characterized by showing a lower mitochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to control mice. No differences in the activity of the antioxidant enzyme system glutathione reductase/peroxidase was observed. Exogenous catalase prevented the spontaneous oxidation of endogenous NAD(p)H in mitochondria isolated from LDLR-/- mice, indicating that this oxidation is mediated by mitochondrial generated 'H IND. 2' 'O IND. 2'. The higher rate of 'H IND. 2' 'O IND. 2' release in LDLK/- mitochondria was also prevented by the presence of exogenous isocitrate and rotenone, which maintain NADP fully reduced. Our hypothesis that high rates of lipogenesis decreases NADPH/NADP+ ratio due to a decreased content of NADP-linked substrates is supported by observations that oxygen consumption supported by endogenous substrates was lower in LDLR-/- mice than in control mitochondria, but was similar in the presence of exogenous isocitrate. Indeed, endogenous mitochondriallevels of isocitrate were significantly lower in LDLK-/- mice. ln the second part ofthis work, we used statins (3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) in an attempt to Correct the oxidative stress observed in LDLK-/- mice, but we found that the drug treatment worsened this defect. We report here that liver mitochondria isolated from hypercholesterolemic LDL receptor knockout mice treated during 15 days with therapeutic doses (100 mg/kg, p.o.) of lovastatin presented a higher susceptibility to develop membrane permeability transition (MPT). In experiments in vitro, lovastatin induced MPT in a dose-dependent manner (10-80 'mu' M) by a mechanism sensitive to cyclosporin A (cyclophilin inhibitor), dithiothreitol (reducing agent), ADP (adenine nucleotide carrier inhibitor), catalase ('H IND. 2' 'O IND. 2' reductant) and EGTA (calcium chelator). In agreement with the inhibition of the mitochondrial swelling by dithiothreitol, lovastatin also decreased the content of total mitochondrial membrane protein thiol groups. Simvastatin had similar effects on mitochondria; however, pravastatin, a hydrophilic statin, had a weaker effect in inducing MPT. These results demonstrate that statins can act directly on mitochondria either in vivo or in vitro inducing permeability transition, which is a process involved in cell death / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Selenium (Se) is an essential mineral with functions for both animals and humans. There are several regions in the world deficient in this mineral and studies have related Se supplemented with reducing cholesterol. Thus, the objective of this study was to evaluate the Se effect in different levels and sources in the diet finishing of Nellore cattle on the performance and meat quality. It was used 63 Nellore cattle (412 kg and ± 24 months of age) in a completely randomized design with two sources (sodium selenite and selenium-enriched yeast) and four supplementation levels (0; 0.3; 0.9 and 2.7 mg Se/kg DM). There were no changes in performance and carcass characteristics. The Se level reduced (P<0.01) lipid and proteins oxidation (TBARS and carbonyl) compared to the control treatment on retail display storage (0, 2, 4 and 6 days). Organic Se, regardless of level, provided Se 138% higher (P<0.0001) in meat and 22.6% higher (P<0.0001) in serum than inorganic Se. The activity of glutathione peroxides (GPx) in muscle was 288% higher for animals supplemented with selenium and consequently, the cholesterol concentration in L. dorsi was 10.2% lower (P<0.001). The serum HMG-CoA reductase concentration was 32.7% lower in animals receiving Se supplementation (organic or inorganic). In conclusion, Se supplementation in beef cattle diet is a way of naturally producing selenium-enriched meat and with better quality for human consumption. / O selênio (Se) é um mineral essencial com funções para animais e humanos. Existem várias regiões do mundo deficientes neste mineral e estudos têm relacionado a suplementação de Se com a redução do colesterol. Assim, o objetivo deste estudo foi avaliar o efeito de diferentes teores e fontes de Se na dieta de terminação de bovinos Nelore sobre o desempenho e a qualidade da carne. Foram utilizados 63 animais da raça Nelore (412 kg e ± 24 meses de idade) em delineamento inteiramente casualizado, com duas fontes (selenito de sódio e selênio levedura) e quatro teores de suplementação (0; 0,3; 0,9 e 2,7 mg Se/kg MS) em confinamento, durante 84 dias. Não houve alterações no desempenho e nas características de carcaça. O teor de Se reduziu (P <0,01) a oxidação lipídica e proteica (TBARS e carbonila) em comparação ao tratamento controle durante o armazenamento em simulação de exposição no varejo (0, 2, 4 e 6 dias). O Se orgânico, independentemente do teor, forneceu valor de Se 138% superior (P <0,0001) na carne e 22,6% superior (P <0,0001) no soro em relação ao Se inorgânico. A atividade glutationa peroxidase (GPx) no músculo foi 288% maior nos animais suplementados com selênio e, consequentemente, a concentração de colesterol na carne foi 10,2% menor (P <0,001). A concentração sérica de HMG-CoA redutase foi 32,7% menor nos animais que receberam suplementação de Se (orgânico ou inorgânico). Foi concluído que a suplementação com Se na dieta de bovinos de corte é uma forma de produzir carnes enriquecidas com selênio naturalmente e com melhor qualidade para consumo humano.

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

A deficiência da desidrogenase de acilas-CoA de cadeia média (MCAD) é um defeito da oxidação de ácidos graxos (DOAG) bioquimicamente caracterizada pelo acúmulo de acilcarnitinas de cadeia média (MCAC) em tecidos e líquidos biológicos de indivíduos afetados. Clinicamente os pacientes apresentam sintomas neurológicos como letargia e coma, geralmente desencadeados por episódios de descompensação metabólica. Outros distúrbios importantes entre os DOAG são as deficiências da proteína trifuncional mitocondrial (MTPD) e da desidrogenase de 3-hidroxiacilas-CoA de cadeia longa (LCHADD) que são caracterizadas pelo acúmulo de ácidos graxos de cadeia longa e seus derivados 3- hidroxilados (LCHFA). Geralmente, os pacientes afetados apresentam disfunção cardíaca e hepática podendo também apresentar sinais de comprometimento neurológico. Até o momento, a hipoglicemia e a toxicidade dos ácidos graxos acumulados têm sido relacionados com a fisiopatologia dessas doenças, embora os mecanismos responsáveis pelo dano tecidual não são bem conhecidos. Assim, foram investigados os efeitos dos MCAC e LCHFA sobre importantes parâmetros da função redox e homeostase energética mitocondrial em cérebro e coração de ratos, bem como a produção de superóxido e morte celular em culturas de fibroblastos da pele de pacientes afetados por essas doenças. Primeiramente, observamos que as MCAC induzem dano oxidativo lipídico e proteico, além de reduzir as defesas antioxidantes não enzimáticas em cérebro de ratos, provavelmente através da indução de radicais hidroxil e peroxil. Além disso, evidenciamos aumento dos níveis de superóxido e de morte celular em fibroblastos de pacientes afetados pela MTPD em condições padrão de cultivo indicando uma exposição crônica a níveis mais elevados de superóxido e uma vulnerabilidade à morte celular. Fibroblastos de pacientes afetados pelas MTPD e MCADD também apresentaram aumento dos níveis de superóxido quando cultivadas em condições de estresse metabólico, contudo não houve aumento de morte celular. Finalmente, a morte celular foi mais pronunciada em fibroblastos afetados pela MTPD em comparação com pacientes afetados pela MCADD, além de apresentar uma forte correlação com a produção de superóxido, sugerindo que esses eventos são provavelmente associados. Por outro lado, observamos que LCHFA prejudicam a homeostase energética em mitocôndrias de coração devido a um efeito desacoplador da fosforilação oxidativa evidenciado pelo aumento do estado 4 da respiração e diminuição da razão de controle respiratório, pela redução do potencial de membrana, do conteúdo de NAD(P)H e da produção de H2O2. O ácido 3- hidroxitetradecanóico (3 HTA) também induziu inchaço mitocondrial provavelmente como consequência da abertura do poro de transição de permeabilidade mitocondrial (mPTP) uma vez que a ciclosporina A (CsA), um inibidor clássico da abertura do poro, impediu esse efeito em mitocôndrias cardíacas carregadas com Ca2+. LCHFA também dissipadaram o potencial de membrana, diminuíram o coneúdo de NAD(P)H e de ATP na presença ou ausência de Ca2+ em mitocondriais de córtex cerebral. Além disso, na presença de Ca2+, 3 HTA induziu inchamento mitocondrial e liberação de citocromo c, que ativa rotas apoptóticas. 3 HTA também afetou a homeostase de Ca2+ evidenciado por uma reduzida capacidade de retenção de Ca2+ pela mitocôndria e induziu a produção de H2O2 na presença de Ca2+. Essas alterações foram prevenidas pelo vermelho de rutênio (RR), um bloqueador da captação mitocondrial de Ca2+, e por CsA mais ADP, inibidores da abertura do mPTP, o que implica o seu envolvimento nesses efeitos. Em contraste, LCHFA não causaram dano oxidativo nem alteraram as defesas antioxidantes em coração de ratos jovens. Em conjunto, nós demonstramos que MCAC e LCHFA que se acumulam em pacientes afetados pelas deficiências da MCAD e MTP/LCHAD, respectivamente, são potencialmente tóxicos para as funções mitocondriais essenciais em cérebro e coração de ratos. Além disso, demonstramos que fibroblastos de pacientes com MTPD cultivados em condições padrão estão sob estresse oxidativo, que é acentuado quando os fibroblastos são submetidos a condições de estresse metabólico. Presume-se que esses mecanismos fisiopatológicos podem ser responsáveis, pelo menos em parte, pelo dano tecidual característico desses pacientes. / The medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder (FAOD) biochemically characterized by accumulation of medium-chain acylcarnitines (MCAC) in tissues and biological fluids of affected individuals. The clinical presentation includes neurological symptoms as lethargy and coma, generally followed by episodes of metabolic decompensation. Other important FAOD are the mitochondrial trifunctional protein (MTPD) and the long-chain 3- hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies that are characterized by accumulation of long-chain fatty acids and their 3-hydroxylated (LCHFA) derivatives. Usually, affected patients present cardiac and hepatic dysfunction and may present signs of neurological impairment. So far, hypoglycemia and the toxicity of accumulating fatty acids have been related with the pathophysiology in these disorders, although the mechanisms responsible for tissue damage are poorly known. Thus, we investigated the effects of MCAC and LCHFA on important parameters of mitochondrial redox and energetic homeostasis in brain and heart of rats, as well as superoxide production and cell death in skin fibroblasts from patients affected by these diseases. First, we observed that MCAC induced lipid and protein oxidative damage, and reduced the antioxidant non-enzymatic defenses in rat brain, probably through induction of hydroxyl and peroxyl radicals. Furthermore, we evidenced increased superoxide levels and cell death in skin fibroblasts from MTP deficient patients under standard growing conditions indicating a chronic exposure to higher superoxide levels and a vulnerability to cell death. In addition, cells from MCADD and MTPD patients cultured under metabolic stress conditions presented increased levels of superoxide, although cell death was not increased. Finally, cell death was more pronounced in fibroblasts from MTP compared to MCAD deficient patients, and presented a strong correlation with superoxide production, suggesting that these events are probably associated. On the other hand, we observed that LCHFA provoked an impairment of heart mitochondrial energetic homeostasis, by behaving as uncouplers of oxidative phosphorylation, evidenced by increase of state 4 respiration and decrease of the respiratory control ratio, by reducing the membrane potential, the NAD(P)H content and H2O2 production. 3-Hydroxytetradecanoic acid (3 HTA) also induced mitochondrial swelling probably as a consequence of the mitochondrial permeability transition pore (mPTP) opening once cyclosporin A (CsA), a classical inhibitor, prevented this effect in heart mitochondria loaded with Ca2+. LCHFA also dissipated membrane potential, diminished NAD(P)H content and decreased ATP content in the presence or absence of Ca2+ in cerebral cortex mitochondrial preparations. Moreover, 3 HTA induced mitochondrial swelling and cytochrome c release in the presence of Ca2+, which activates apoptotic cascades. 3 HTA also affected Ca2+ homeostasis evidenced by a diminished mitochondrial Ca2+ retention capacity and induced hydrogen peroxide production in the presence of Ca2+. These alterations where prevented by ruthenium red (RR), a blocker of mitochondrial Ca2+ uptake, and by CsA plus ADP, inhibitors of the mPTP, implying its involvement in these effects. In contrast, LCHFA did not cause oxidative damage nor altered the antioxidant defenses in heart of young rats. Taken together, we demonstrated that MCAC and LCHFA found accumulated in patients affected by MCAD and MTP/LCHAD deficiencies, respectively, are potentially toxic to essential mitochondrial functions in rat brain and heart. We also found that fibroblasts from MTP deficient patients cultured under basal growing conditions are under oxidative stress that is accentuated when cultured metabolic stress conditions are used. It is presumed that these pathomechanisms may be responsible, at least in part, for the tissue damage characteristic of these patients.

Cinco membros da família das Acil-CoA sintetases de cadeia longa (ACSL) são responsáveis por ativar ácidos graxos, produzindo acil-CoA, e distribuí-los entre diversas vias metabólicas no interior da célula, tais como a síntese de triacilglicerol (TAG) e ?- oxidação mitocondrial. Apesar das disfunções nas ACSLs contribuirem para muitas doenças metabólicasa função de algumas isoformas de ACSL em tecidos específicos permanece ainda sem descrição na literatura. Aqui mostramos pela primeira vez a presença de mRNA da ACSL6 no músculo esquelético de seres humanos. Além disso, indivíduos obesos apresentaram menores níveis de mRNA de ACSL6 quando comparados à indivíduos magros. Após refeição hiperlipídica aguda (high fat meal, HFM, 90% de gordura) a expressão ACSL6 aumentou 2,5 vezes em relação aos níveis de jejum. Nós também verificamos as condições metabólicas que controlam a expressão ACSL6 em ratos: o jejum de 48h modulou negativamente a expressão gênica de ACSL6 e de outros genes de síntese de lipídeos tais como SREBP-1c e DGAT1, enquanto que a ingestão aguda de HFM (80% de gordura saturada , 10 mL/kg) teve o efeito oposto; Após o treinamento aeróbio (6 semanas, 5 dias /semana, uma vez por dia, 60 min a 70% da capacidade aeróbica máxima) o mRNA da ACSL6 foi reduzido em 35%. Em células primárias de músculo esquelético de ratos, a transfecção com siRNA de ACSL6 diminuiu a expressão de ACSL6, DGAT1 e SREBP-1c e o acúmulo de TAGs e gotas lipídicas. O silenciamento gênico da ACSL6 também aumentou o conteúdo dos ácidos graxos C16:0 e C18:0, AMPK-fosforilada, capacidade respiratória mitocondrial, a oxidação de palmitato e mRNA de PGC-1?, UCP2 e UCP3, mas diminuiu a produção de espécies 11 reativas de oxigênio. Em células primárias de músculo esquelético de seres humanos, a superexpressão da ACSL6 não alterou o conteúdo de TAG e da proteína DGAT1, mas aumentou as espécies lipídicas esfingomielina e fosfatidilcolinas, e reduziu a oxidação de 1-14C-palmitato e a expressão do PGC1?. Em conclusão, ACSL6 está envolvida na síntese e distribuição de acil-CoA para a síntese de lipídeos. A inibição gênica da ACSL6 melhora a capacidade de respiração mitocondrial e oxidação lipídica, através da ativação da via AMPK/PGC1?. / Five members of long-chain acyl-CoA synthetase (ACSL) family activate fatty acids providing acyl-CoA for several metabolic pathways within the cell, such as synthesis of triacylglycerol (TAG) and mitochondrial ?-oxidation, and their dysfunctions contribute to many metabolic diseases. Despite this, the existence and function of some ACSL isoforms in specific tissues remains unclear. Here we show for the first time the presence of ACSL6 mRNA and protein in skeletal muscle (SM) of humans. Obese subjects had lower levels of ACSL6 mRNA when compared to leans, and acute high fat meal (HFM, 90% fat) increased ACSL6 expression 2.5 times over fasted levels in both. We also verify the metabolic conditions that control ACSL6 expression in rats: fasting (48h) negatively modulated the ACSL6 mRNA and the expression of other genes of lipid synthesis SREBP-1c and DGAT1 in rat SM, while acute ingestion of HFM (80% saturated fat, 10 mL/Kg) had the opposite effect; After aerobic training (6 weeks, 5 days/week, once a day, 60 min at 70% of maximal aerobic capacity) ACSL6 mRNA was reduced 35%. In primary skeletal muscle cells (PSMC) of rats, ACSL6-specific siRNA oligo transfection (20 nM) decreased ACSL6, DGAT1 and SREBP-1c mRNA and the accumulation of TAGs and lipid droplets (LD). The knockdown also increased the content of C16:0 and C18:0 fatty acids, AMPK-Phosphorylated, mitochondrial content and respiratory rates, palmitate oxidation and PGC-1?, UCP2 and UCP3 mRNA, but decreased reactive oxygen species production. In PSMC of humans, ACSL6 overexpression did not change the contents of TAG or DGAT1 mRNA, but increased sphingomyelin and phosphatidylcholines and reduced 14C-palmitate oxidation and PGC1? mRNA expression. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its 13 downregulation improves mitochondrial capacity of respiration, lipid oxidation and biogenesis, which involves the activation of AMPK/PGC1-? pathway.