L’entérotoxine stable à la chaleur STb est produite par les Escherichia coli entérotoxinogènes (ETEC). Son rôle dans la diarrhée post-sevrage porcine est établi. L’internalisation de STb a été observée dans des cellules épithéliales intestinales humaines et de rat. Cependant, le mécanisme d’internalisation n’est pas totalement compris, particulièrement dans le jéjunum porcin, la cible in vivo de STb. Par la cytométrie en flux, nous avons examiné l’internalisation de STb couplée à un marqueur fluorescent dans les cellules épithéliales intestinales porcines IPEC-J2 et les fibroblastes murins NIH3T3. Nos résultats révèlent que l’internalisation de STb est températureindépendante dans les IPEC-J2 tandis qu’elle est température-dépendante dans les NIH3T3, où la réorganisation de l’actine est aussi nécessaire. Toutefois, les niveaux de sulfatide, le récepteur de STb, sont semblables à la surface des deux lignées. Le sulfatide est internalisé à 37°C de façon similaire entre les deux types cellulaires. La rupture des lipid rafts, les microdomaines membranaires contenant le sulfatide, par la méthyl-βcyclodextrine ou la génistéine, n’affecte pas l’internalisation de STb dans les deux lignées. Notre étude indique que le mécanisme d’internalisation de STb est dépendant du type cellulaire. L’activité de la cellule hôte peut être requise ou non. Le récepteur de STb, le sulfatide, n’est pas directement impliqué dans ces mécanismes. L’internalisation activité cellulaire-dépendante suggère une endocytose, nécessitant la réorganisation de l’actine mais pas les lipid rafts. L’internalisation de STb est donc un processus complexe dépendant du type cellulaire, qu’il apparait plus relevant d’étudier dans des modèles cellulaires représentatifs des conditions in vivo. / Heat-stable enterotoxin b (STb) is one of the toxins produced by enterotoxigenic Escherichia coli (ETEC) and its role in swine post-weaning diarrhea is well established. Internalization of STb in intestinal human and rat epithelial cells has been shown by previous studies. However, the uptake mechanism is still not fully understood, especially in porcine jejunum epithelium, the in vivo STb target. Using flow cytometry, we studied internalization of fluorescently-labelled STb in porcine epithelial intestinal IPEC-J2 and murine fibroblast NIH3T3 cell lines. Our results revealed that STb is internalized in both cell lines. Toxin uptake is not dependent on the temperature in IPEC-J2 cells, whereas it is in NIH3T3 fibroblasts. Actin reorganization is only required for STb internalization in NIH3T3 cells. However, membrane sulfatide, the toxin receptor, is similarly present in both cell lines and similarly internalized with time at 37°C. Disruption of lipid rafts, known to contain sulfatide, with inhibitors (methyl-βcyclodextrin or genistein), did not affect toxin uptake in both cell lines. Altogether, these data indicate that STb internalization mechanisms are cell-type dependent. Moreover, uptake can depend on host cell activity or not. Sulfatide, the toxin receptor, is not directly involved in these mechanisms. Uptake independent on cell activity occurs in porcine intestinal epithelium. The cell activity-dependent uptake suggests an endocytosis, which requires actin rearrangement and is not mediated by lipid rafts. STb internalization is therefore a complex process varying upon cell type, which should preferentially be studied in cellular models representative of in vivo conditions, such as porcine cell lines.

Prostate cancer is the most common malignancy afflicting Canadian men in 2011. Physicians use digital rectal exams (DRE), blood tests for prostate specific antigen (PSA) and transrectal ultrasound (TRUS)-guided biopsies for the initial diagnosis of prostate cancer. None of these tests detail the spatial extent of prostate cancer - information critical for using new therapies that can target cancerous prostate. With an MRI technique called proton magnetic resonance spectroscopic imaging (1H-MRSI), biochemical analysis of the entire prostate can be done without the need for biopsy, providing detailed information beyond the non-specific changes in hardness felt by an experienced urologist in a DRE, the presence of PSA in blood, or the “blind-guidance” of TRUS-guided biopsy. A hindrance to acquiring high quality 1H-MRSI data comes from signal originating from fatty tissue surrounding prostate that tends to mask or distort signal from within the prostate, thus reducing the overall clinical usefulness of 1H-MRSI data. This thesis has three major areas of focus: 1) The development of an optimized 1H-MRSI technique, called conformal voxel magnetic resonance spectroscopy (CV-MRS), to deal the with removal of unwanted lipid contaminating artifacts at short and long echo times. 2) An in vivo human study to test the CV-MRS technique, including healthy volunteers and cancer patients scheduled for radical prostatectomy or radiation therapy. 3) A study to determine the efficacy of using the 1H-MRSI data for optimized radiation treatment planning using modern delivery techniques like intensity modulated radiation treatment. Data collected from the study using the optimized CV-MRS method show significantly reduced lipid contamination resulting in high quality spectra throughout the prostate. Combining the CV-MRS technique with spectral-spatial excitation further reduced lipid contamination and opened up the possibility of detecting metabolites with short T2 relaxation times. Results from the in vivo study were verified with post-histopathological data. Lastly, 1H-MRSI data was incorporated into the radiation treatment planning software and used to asses tumour control by escalating the radiation to prostate lesions that were identified by 1H-MRSI. In summary, this thesis demonstrates the clinical feasibility of using advanced spectroscopic imaging techniques for improved diagnosis and treatment of prostate cancer.

The human genome project has allowed for the rapid identification of a large number of protein families based on similarities in their genetic sequences. The acyl-glycerol phosphate acyltransferase (AGPAT) family of enzymes have been largely identified through sequence homology, with eleven isoforms identified in both mice and humans. Interestingly, very little work has been done on the characterization of AGPAT isoform 4. In the present study, I report the functional characterization of AGPAT4 as an acyl-CoA: lysocardiolipin acyltransferase (ALCAT), which we have renamed ALCAT2. Although ALCAT2 is present in most tissues, it is abundant in multiple brain regions including olfactory bulbs, hippocampus, cerebellum, cortex, and brain stem, and is detectable in both primary neurons and glial cells. In assays performed in vitro, ALCAT2 significantly increased the incorporation of [14C]oleoyl-CoA into phosphatidylinositol and CL using either lysophosphatidylinositol, or monolysocardiolipin or dilysocardiolipin as acyl acceptors, respectively. ALCAT2 did not display significant acyltransferase activity with lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, or lysophosphatidylglycerol acyl acceptors. Overexpressing ALCAT2 in HEK-293 cells increased the total CL content, but did not significantly affect levels of other glycerophospholipids including phosphatidylinositol. Analysis of the fatty acyl profile of CL from ALCAT2-overexpressing cells indicated increased total saturated fatty acids, particularly stearate, palmitate, and myristate, and increased levels of n-3 polyunsaturated fatty acids α-linolenic acid (18:3n-3), eicosatrienoic acid (20:3n-3), and eicosapentanoic acid (20:5n-3). In accordance with its observed role in cardiolipin remodeling, ALCAT2 localized predominately to the mitochondria. ALCAT2 was also regulated during embryogenesis, and in varying metabolic states. In summary, ALCAT2 is a new enzyme in CL remodeling with a potential role in mitochondrial function.

Prostate cancer is the most common malignancy afflicting Canadian men in 2011. Physicians use digital rectal exams (DRE), blood tests for prostate specific antigen (PSA) and transrectal ultrasound (TRUS)-guided biopsies for the initial diagnosis of prostate cancer. None of these tests detail the spatial extent of prostate cancer - information critical for using new therapies that can target cancerous prostate. With an MRI technique called proton magnetic resonance spectroscopic imaging (1H-MRSI), biochemical analysis of the entire prostate can be done without the need for biopsy, providing detailed information beyond the non-specific changes in hardness felt by an experienced urologist in a DRE, the presence of PSA in blood, or the “blind-guidance” of TRUS-guided biopsy. A hindrance to acquiring high quality 1H-MRSI data comes from signal originating from fatty tissue surrounding prostate that tends to mask or distort signal from within the prostate, thus reducing the overall clinical usefulness of 1H-MRSI data. This thesis has three major areas of focus: 1) The development of an optimized 1H-MRSI technique, called conformal voxel magnetic resonance spectroscopy (CV-MRS), to deal the with removal of unwanted lipid contaminating artifacts at short and long echo times. 2) An in vivo human study to test the CV-MRS technique, including healthy volunteers and cancer patients scheduled for radical prostatectomy or radiation therapy. 3) A study to determine the efficacy of using the 1H-MRSI data for optimized radiation treatment planning using modern delivery techniques like intensity modulated radiation treatment. Data collected from the study using the optimized CV-MRS method show significantly reduced lipid contamination resulting in high quality spectra throughout the prostate. Combining the CV-MRS technique with spectral-spatial excitation further reduced lipid contamination and opened up the possibility of detecting metabolites with short T2 relaxation times. Results from the in vivo study were verified with post-histopathological data. Lastly, 1H-MRSI data was incorporated into the radiation treatment planning software and used to asses tumour control by escalating the radiation to prostate lesions that were identified by 1H-MRSI. In summary, this thesis demonstrates the clinical feasibility of using advanced spectroscopic imaging techniques for improved diagnosis and treatment of prostate cancer.

MARCKS-Protein ist in den Signalübertragungsweg der Zelle involviert. Durch einen Adsorptions-/Desorptionszyklus mit der Zellmembran reguliert es die Konzentration bestimmter Botenmoleküle. Im Rahmen dieser Arbeit wurde untersucht, inwieweit strukturelle Änderungen der Membran, verursacht durch die Membran-Protein-Wechselwirkung, mit einem Reaktions-Diffusions-System korrelieren. Die elektrostatische Wechselwirkung von MARCKS-Protein mit negativ geladenen Membranlipiden geschieht an der inneren Seite der Zellmembran. Als Modellsystem lässt sich dies mit einer monomolekularen Lipidschicht an der Wasser-Luft-Grenzfläche realisieren. Anhand von oberflächensensitiven Messungen konnte gezeigt werden, dass die Wechselwirkung von MARCKS mit negativ geladenen Membranlipiden und damit die Adsorption an der Membran, zu einer Änderung der Membrantopologie führt. Damit verbundenen ist auch der partielle Einbau von MARCKS in die Membran, was zu einem größeren molekularen Flächenbedarf führt. Dieser korreliert mit dem Anstieg des lateralen Drucks der Lipidmonoschicht bei konstanter Fläche. Die Desorption von MARCKS kann durch die Wechselwirkung mit PKC induziert werden, detektierbar durch die Reduktion des lateralen Drucks. Bei Vorhandensein eines Reservoirs an MARCKS und PKC oszilliert der laterale Druck, was als zyklische Adsorption und Desorption von MARCKS an bzw. von der Lipidschicht interpretiert wird. Anhand der experimentellen Ergebnisse wurde ein mathematisches Modell entwickelt, dass dieses oszillierende Verhalten als ein Reaktions-Diffusions-System erklärt.

Understanding the function of membrane proteins is crucial to elucidate the molecular mechanisms by which transmembrane signaling based physiological processes,i. e., the interactions of extracellular ligands with membrane-bound receptors, are regulated. In this work, synthetic transmembrane segments derived from the visual photoreceptor rhodopsin, the full length system rhodopsin and mutants of opsin are used to study physical processes that underlie the function of this prototypical class-A G-protein coupled Receptor. The dependency of membrane protein hydration and protein-lipid interactions on side chain charge neutralization is addressed by fluorescence spectroscopy on synthetic transmembrane segments in detergent and lipidic environment constituting transmembrane segments of rhodopsin in the membrane. Results from spectroscopic studies allow us to construct a structural and thermodynamical model of coupled protonation of the conserved ERY motif in transmembrane helix 3 of rhodopsin and of helix restructuring in the micro-domain formed at the protein/lipid water phase boundary. Furthermore, synthesized peptides and full length systems were studied by time resolved FTIR-Fluorescence Cross Correlation Hydration Modulation, a technique specifically developed for the purpose of this study, to achieve a full prospect of time-resolved hydration effects on lipidic and proteinogenic groups, as well as their interactions. Multi-spectral experiments and time-dependent analyses based on 2D correlation where established to analyze large data sets obtained from time-resolved FTIR difference spectra and simultaneous static fluorescence recordings. The data reveal that lipids play a mediating role in transmitting hydration to the subsequent membrane protein response followed by water penetration into the receptor structure or into the sub-headgroup region in single membrane-spanning peptides carrying the conserved proton uptake site (monitored by the fluorescence emission of hydrophobic buried tryptophan). Our results support the assumption of the critical role of the lipid/water interface in membrane protein function and they prove in particular the important influence of electrostatics, i. e., side chain charges at the phase boundary, and hydration on that function. / Für die Aufklärung der molekularen Wirkungsweise von physiologischen, auf Signaltransduktion, d. h. dem Zusammenspiel von extrazellulären Reizen und membrangebundenen Rezeptoren, beruhenden Prozessen ist das Verständnis der Funktion von Membranproteinen unerlässlich. In dieser Arbeit werden von Rhodopsin abgleitete, synthetische transmembrane Segmentpeptide, Opsin-Mutanten und der vollständige Photorezeptor Rhodopsin untersucht, um die physikalischen Prozesse zu beleuchten, die der Funktionen dieses prototypischen Klasse-A G-Protein gekoppelten Rezeptors zugrunde liegen. Die Abhängigkeit der Membranprotein-Hydratation und der Lipid-Protein-Wechselwirkung von der Ladung einer Aminosäuren-Seitenkette wird erforscht. Hierzu werden synthetische, transmembrane Segmentpeptide in Lipid und Detergenz, als Modell transmembraner Segmente von Rhodopsin in der Membran mittels Fluoreszenzspektroskopie untersucht. Aus den erhaltenen Ergebnissen wird ein thermodynamisches und strukturelles Modell hergeleitet, welches die Kopplung der Protonierung des hochkonservierten ERY-Motivs in Transmembranhelix 3 von Rhodopsin an die Restrukturierung der Helix in der Mikroumgebung der Lipid-Wasser-Phasengrenze erklärt. Des Weiteren werden sowohl die Segementpeptide als auch die vollständigen Systeme Opsin und Rhodopsin mittels zeitaufgelöster FTIR-Fluoreszenz-Kreuzkorrelations-Hydratations-Modulation untersucht. Diese Technik wurde eigens zur Aufklärung von zeitabhängigen Hydratationseffekten auf Lipide und Proteine oder Peptide entwickelt. Dabei werden zeitaufgelöste FTIR Differenz-Spektren und gleichzeitig statische Fluoreszenzsignale aufgenommen und diese zeitabhängigen multispektralen Datensätze mittels 2D Korrelation analysiert. Die Auswertung der Experimente enthüllt einen sequentiellen Hydratationsprozess. Dieser beginnt mit der Bildung von Wasserstoffbrückenbindungen an der Carbonylgruppe des Lipids, gefolgt von Strukturänderungen der Transmembranproteine und abgeschlossen durch das Eindringen von Wasser in das Proteininnere. Letzteres wird nachgewiesen durch die Fluoreszenz von Tryptophan im hydrophoben Peptid- oder Proteininneren. Die Ergebnisse dieser Arbeit unterstreichen die Annahme, dass Lipid-Protein-Wechselwirkungen eine entscheidende Rolle in der Funktion von Membranproteinen spielen und das insbesondere Elektrostatik, in Form von Ladungen an der Phasengrenze, und die Hydratisierung einen kritischen Einfluss auf diese Funktion haben.

Aquaculture represents a sustainable alternative to natural fisheries for provision of high quality, animal protein. Crustaceans make a significant contribution to global aquaculture production, of which decapods are the most economically important group. Among freshwater crayfish, the genus Cherax includes several species that have emerged as important culture species. A suite of favourable biological attributes, including fast growth and an omnivorous feeding habit, have contributed to establishment of successful culture of Cherax quadricarinatus (redclaw) in many countries. Aspects of redclaw production, however, remain relatively undeveloped, in particular feed formulation. To better understand the digestive processes and nutritional requirements of redclaw, this study examined the relationship between diet composition and digestive enzyme activity, growth performance and diet digestibility coefficients. The extent to which redclaw can efficiently utilise complex polysaccharides, such as cellulose, has been speculated on by authors who reported endogenous cellulase activity in this species. I evaluated the use of insoluble α-cellulose by redclaw, demonstrated that high dietary levels (30%) can significantly reduce the specific activity of selected digestive enzymes (amylase and cellulase), while also lowering apparent digestibility coefficients. Inclusion of α-cellulose above 12% also significantly reduced survival rate, specific growth rate and feeding efficiency in this organism which corresponds with low tolerance for insoluble fibre by other decapods. Even though redclaw possess endogenous cellulases, they appear to have only a limited capacity to utilise insoluble fibre in their diets. Further, I assessed the impact of different nutrient profiles on digestive enzyme activity, growth and tail muscle composition in redclaw. Purified diets containing varying levels of dietary protein significantly affected activity of digestive enzymes (protease, amylase and cellulase) and the composition of the tail muscle tissue. Redclaw have a relatively low protein requirement, which was reflected here, as little significant difference was observed in growth rates and the feed conversion ratio was only significantly affected by the lowest protein diet. Manipulation of the non-protein energy component in purified diets (protein to lipid ratio) had no effect on growth performance indices in redclaw. Digestive enzyme activity (protease) was however, strongly influenced by both the amount of protein and lipid in the diet and a significant correlation was observed between protease activity and growth performance indices. The findings here, provide preliminary data for consideration of digestive enzymes such as proteases as potential growth indicators for freshwater crayfish. These enzymes are already recognised as reliable biological indicators for comparison of digestive efficiency and potential growth rate in fish. The relationship between diet composition and digestive enzyme expression observed here, stress the need for further empirical evaluation of specific ingredients in artificial diets for redclaw. A range of single cell, plant and animal-based, agricultural products were assessed for their potential use in diets formulated for redclaw. Analysis of dietary supplements revealed that apparent digestibility of crude protein was generally higher for diets containing plant-based ingredients. A similar outcome was observed for digestibility coefficients of test ingredients. Ingredient type also had a significant effect on digestive enzyme activity. Importantly, a significant correlation was observed for enzyme activity and apparent digestibility coefficients. It appears that redclaw have the capacity to utilise nutrients from a broad range of dietary ingredients successfully including animal, single cell and in particular, plant matter in their diet. Taken together, the results presented here demonstrate that digestive enzyme activities in redclaw are significantly influenced by diet composition. I show clearly that the ability of redclaw to utilise various nutrients (measured as digestibility coefficients) is highly correlated with digestive enzyme activity. Finally, protease activity demonstrated a potential for use as an indicator of redclaw growth performance. The data presented here will contribute to development of better and cheaper feed formulations for use in redclaw aquaculture and have broader applications to freshwater crustacean culture. In particular, the potential for use of plant-based ingredients in aqua-feeds for redclaw will contribute to a more economically and environmentally sustainable redclaw culture.

[Truncated abstract] The low density lipoprotein receptor-related protein (LRP), a member of the low-density lipoprotein (LDL) receptor gene family is involved in numerous biological processes including lipoprotein metabolism. This thesis concerns investigations into some aspects of LRP metabolism/regulation and possible roles in coronary artery disease (CAD). Specific aims were: to investigate the association between polymorphisms in the LRP gene and in its associated protein, the lipoprotein receptor-associated protein (RAP), with the risk of CAD; to extensively examine the influence of the LRP exon 22 C200T polymorphism on lipid metabolism; to develop and characterise assays for the mRNA expression of LRP and 2 other genes relevant to lipid metabolism, the LDL receptor (LDLR), and HMG CoA reductase (HMGCR); and finally, to apply the latter techniques to studies on the influence of genetic variation in LRP, and dietary and drug interventions, on LRP, LDLR and HMGCR mRNA expression in nucleated blood cells from healthy human subjects. Six hundred CAD subjects and 700 similarly aged controls were genotyped for 8 LRP gene polymorphisms as well as for the RAP V311M polymorphism. ... In the final phase of my studies, I examined the influence of 4 weeks therapy with a cholesterol lowering drug, an HMGCR inhibitor, atorvastatin (20mg daily), on the mRNA expression of LDLR, LRP and HMGCR in human nucleated blood cells. Twelve normal Caucasian male subjects aged 49 ? 5 (SD) years were studied. Plasma total cholesterol and LDL-C decreased by averages of 29 % and 41 % after the 4 week period. This was accompanied by an elevation in LDLR mRNA expression by approximately 30 35 %. In contrast, there was no significant effect on LRP and HMGCR mRNA expression. In conclusion, the original findings in this thesis included: demonstration of a strong influence of the LRP exon 22 C200T polymorphism on coronary artery disease and LDLR expression, but without a clear effect on fasting or postprandial lipid levels; data on the biological variation in LDLR and LRP gene expression in nucleated blood cells from normal subjects; the influence of an oral fat load on the expression viii of these genes, finding that LDLR was significantly depressed; and finally, the observation that statin therapy upregulated LDLR in nucleated blood cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Oxidative stress is a process characterized by the antioxidant defense system decrease and/or an excessive reactive species (RS) production. RS are substances capable of attacking proteins, lipids and DNA. The oxidative damage caused to lipids is known as lipid peroxidation, which leads to the increase of malondialdehyde (MDA) levels. Oxidative stress is involved in the pathogenesis of many chronic diseases, including diabetes, cancer, Parkinson s disease and damaged tissue in exposed to chemical agents, such as neurotoxicity, hematotoxicity and nefrotoxicity. It is known that there is a close relation between organic solvents, present in paints, and oxidative stress. Thus, the body has an elaborate antioxidant defense system, the exogenous antioxidants, such as lipid soluble vitamins, and the endogenous system, such as antioxidant enzymes. In this study a method has been validated and optimized for simultaneous quantification of retinol, α-tocopherol, lycopene and β- carotene using high performance liquid chromatography (HPLC-UV/fluorescence). The analytical parameters of validation analyzed were linearity, precision, accuracy, recovery and limits of detection (LOD) and quantification (LOQ). For all the vitamins analyzed, the linear regression coefficients were > 0.99, CV% < 5%;% bias < ± 6% recovery > 92% and the LOD and LOQ values obtained were satisfactory for routine clinical application for all analytes. The validated method was applied to a group of occupationally exposed to paints (n = 45) and a non-exposed group (control group, n = 30). The results indicated that all vitamins, except vitamin E, were significantly lower in the exposed group. Moreover, the possible correlation between endogenous and exogenous antioxidants and lipid damage was evaluated. Quantifications were done to assess endogenous antioxidants, reduced glutathione (GSH) in erythrocytes, the enzymes superoxide dismutase (SOD) and catalase (CAT) in whole blood through spectroscopic methods, and malondialdehyde (MDA) levels in plasma through HPLC-VIS in both study groups, exposed (n=42) and controls (n=28). The biological monitoring was performed by measurement of blood toluene, since in previous studies, it was suggested that this solvent would be the major inducer of lipid peroxidation. Despite the low levels of toluene found, exposed workers presented higher levels of MDA and the antioxidant enzymes (SOD and CAT) activities were significantly elevated when compared with the control group; and this increase was accompanied by depletion of GSH levels. Also, several correlations were observed between MDA and the enzymatic (SOD and CAT) and non-enzymatic antioxidants (GSH), and with lipid-soluble vitamins as well, except vitamin E. Through statistical tests the antioxidants which have a greater influence on the levels of MDA were evaluated. Among the antioxidants tested, GSH and carotenoids (mainly β- carotene) were suggested as main responsible for the reduction of lipid peroxidation. Thus, it can be suggested that high intakes of exogenous antioxidants, such as carotenoids,, tend to decrease lipid damage in occupationally exposed individuals to solvents constituent of paints. / O estresse oxidativo é um processo caracterizado pela diminuição do sistema de defesa antioxidante e/ou por uma produção excessiva de espécies reativas (ERs). As ERs são substâncias capazes de lesar proteínas, lipídios e DNA. Quando os lipídios são atingidos ocorre um processo chamado de peroxidação lipídica que leva ao aumento nos níveis de malondialdeído (MDA). O estresse oxidativo está envolvido com a patogênese de muitas doenças crônicas, como diabetes, câncer, doença de Parkinson e em danos teciduais em expostos a agentes químicos, como neurotoxicidade, hematotoxicidade e nefrotoxicidade. Sabe-se que existe uma estreita relação entre os solventes orgânicos, presentes em tintas, e o estresse oxidativo. Diante disso, o organismo dispõe de um elaborado sistema de defesa antioxidante, os antioxidantes exógenos, como as vitaminas lipossolúveis e o sistema endógeno como enzimas antioxidantes. Dessa forma, nesse estudo foi primeiramente otimizada e validada metodologia para simultânea quantificação de antioxidantes exógenos: retinol, α-tocoferol, licopeno e β-caroteno, utilizando cromatografia líquida de alta eficiência (CLAE-VIS/fluorescência). Os parâmetros analíticos de validação analisados foram linearidade, precisão, exatidão, recuperação e limites de detecção (LD) e quantificação (LQ). Para todas as vitaminas analisadas, os coeficientes de regressão linear foram > 0,99; CV% < 5%; bias% < ± 6%; recuperação > 92% e os valores de LD e LQ obtidos foram satisfatórios para aplicação na rotina clínica. O método validado foi aplicado em um grupo de expostos ocupacionalmente a tintas (n=45) e um grupo de não expostos (controle, n=30). Os resultados indicaram que todas as vitaminas, exceto a vitamina E, foram significativamente menores no grupo exposto. Além disso, avaliou-se o possível efeito protetivo de antioxidantes exógenos e endógenos sobre o dano lipídico. Foram realizadas as dosagens dos antioxidantes endógenos, glutationa reduzida (GSH) em eritrócitos, das enzimas superóxido dismutase (SOD) e catalase (CAT) em sangue total por métodos espectrofotométricos e os níveis de malondialdeído (MDA) em plasma por CLAE-VIS nos dois grupos de estudo, expostos (n=42) e controles (n=28). A monitorização biológica foi realizada através da dosagem de tolueno sanguíneo, uma vez que, em trabalhos prévios, foi sugerido que este solvente seria o principal indutor de peroxidação lipídica. Apesar dos baixos níveis de tolueno sanguíneo encontrados, os trabalhadores expostos apresentaram níveis de MDA e atividade das enzimas antioxidantes (SOD e CAT) significativamente elevados, quando comparados com o grupo controle e esse aumento foi acompanhado de depleção nos níveis de GSH. Ainda, foram observadas várias correlações entre os níveis de MDA e os antioxidantes endógenos enzimáticos (SOD e CAT) e não enzimático (GSH) e ainda com as vitaminas lipossolúveis, exceto vitamina E. Através de testes estatísticos foram avaliados quais antioxidantes teriam uma maior influência nos níveis de MDA. Dentre os antioxidantes analisados, a GSH e os carotenóides (principalmente o β- caroteno) foram sugeridos como principais responsáveis pela redução da peroxidação lipídica. Com isso, pode-se sugerir que aumento nos níveis de carotenóides, via dieta, tendem a diminuir o dano lipídico em indivíduos ocupacionalmente expostos a solventes constituintes de tintas.

Made available in DSpace on 2016-06-02T19:22:53Z (GMT). No. of bitstreams: 1 3145.pdf: 4807955 bytes, checksum: e7ba1c165199af2b5606877c0fadf24b (MD5) Previous issue date: 2010-03-12 / Universidade Federal de Sao Carlos / The aim of the present study was to investigate the effects of resistance training on skeletal muscle lipid content, liver lipid content, heart lipid content, fat depots and lipid profile in ovariectomized rats. Wistar adult female rats were grouped into: sedentary (Sed-intact); ovariectomized sedentary (Sed-Ovx); strength trained (ChronicEx-intact) and ovariectomized strength trained (ChronicEx-Ovx) (n= 10 per group). A 12-week strength training period that consisted in climbing a 1.1-m vertical ladder with weights attached to rats tail was used. The sessions were performed once every 3 days with 4-9 climbs and 8-12 dynamic movements per climb. Ovariectomy increased liver lipid content, fat depots, heart and muscle lipid content. There was an increase in atherogenic index and negative change in lipid profile due to ovariectomy. Resistance training decreased lipid content in liver, soleus, tibialis anterior, fat depots (mesenteric and retroperitoneal) and lipid profile, independently of ovarian hormone status. These results indicate the potential benefits of resistance training as an alternative strategy to control the effects of ovariectomy on fat depot, lipid profile, and tissue lipid content. / O objetivo do estudo foi investigar os efeitos do treinamento de força sobre o conteúdo lipídico no músculo esquelético, fígado, coração, depósitos de gorduras e perfil lipídico em ratas ovariectomizadas. Ratas fêmeas adultas foram divididas em quarto grupos: Sedentário (Sed-Intacto); Sedentário ovariectomizado (Sed-Ovx); Treinado intacto (CrônicoEx-Intacto); Treinado ovariectomizado (CrônicoEx-Ovx) (n = 10 por grupo). Foi realizado um período de 12 semanas de treinamento de força que consistia em subidas de uma escada vertical de 1,1 metros, com peso atado no rabo. As sessões foram realizadas uma vez a cada três dias com 4- 9 subidas e 8-12 movimentos por subida. Foram analisados os conteúdos de lipídios no músculo esquelético, fígado, coração, depósitos de gorduras (urogenital, mesentérico e retroperitoneal) e perfil lipídico. Ovariectomia aumentou o conteúdo lipídico no fígado, músculos esqueléticos, coração e depósitos de gordura. Foi observado um aumento no índice aterogênico e mudanças negativas no perfil lipídico devido à ovariectomia. O treinamento de força diminuiu o conteúdo de lipídio hepático, nos músculos esqueléticos sóleo, tibial anterior, depósitos de gordura (mesentérico e retroperitoneal) e perfil lipídico independente do estado hormonal ovariano. Esses resultados indicam os benefícios potenciais do treinamento de força com uma estratégia alternativa para controlar os efeitos da ovariectomia sobre os depósitos de gordura, perfil lipídico e conteúdo tecidual de lipídio.