Dissertations / Theses on the topic 'Lipid metabolism disorders'

Secretin, the first hormone commencing the field of endocrinology, has been studied for its pleiotropic role in the body inclusive of its neuroactive and body water homeostatic and gastrointestinal functions. Yet, the metabolic effect of secretin remains elusive and is being proposed recently for a revisit. Recent discovery from our lab showed an anorectic response for secretin, while its role in lipid homeostasis remains largely unexplored. Exerting functions such as exocrine pancreatic secretion and gastric motility inhibition, intestinal fatty acid induced release of secretin was recently shown to be mediated by CD36. Fasting related increase in plasma secretin concentration has been proposed to be involved in lipolysis but evidences regarding lipolytic actions of secretin remain contradictory. Recent report has suggested that secretin stimulates both lipolysis and lipogenesis in adipose cells. Thus, we hypothesize that secretin modulates lipid homeostasis, which was examined under two opposite, energy deficient and energy excess, conditions. Under energy deficient/starved state, secretin level in circulation and secretin receptor level in epididymal adipose tissue were found to be upregulated. Using secretin receptor knockout (SCTR-/-) and secretin knockout (SCT-/-) mice as controls, it was found that secretin stimulated a dose- and time-dependent lipolysis in vitro and acute lipolysis in vivo. H-89, a protein kinase A (PKA) inhibitor, attenuated the lipolytic effects of secretin in vitro, while secretin induced an increase in cAMP dependent PKA activity in vivo. Using western blot analysis, secretin was found to phosphorylate hormone sensitive lipase (HSL) at serine residue 660. Additionally, immunofluorescent studies revealed that secretin stimulated translocation of HSL from cytosol to surface of lipid droplet subsequently leading to lipolysis. Under excess energy condition, when SCTR-/- mice and its littermates SCTR+/+ mice were subjected to high fat diet (HFD) feeding for 3 months, it was found that SCTR-/- mice gained lesser weight. Nuclear magnetic resonance imaging revealed that SCTR-/- mice exhibited lower body fat content. Additionally, HFD-associated hyperleptinaemia was alleviated in SCTR-/- mice along with metabolic syndrome as they performed better in insulin and glucose tolerance tests. Continuous monitoring by indirect calorimetry revealed similar food intake, energy expenditure and locomotor activity between SCTR-/- and SCTR+/+ mice. Interestingly, intestinal fatty acid absorption, measured by a noninvasive method, was impaired in HFD-fed SCTR-/- mice. While postprandial triglyceride release was reduced in SCTR-/- mice, it also had a significant reduction in transcript and protein levels of CD36 and its downstream mediator MTTP. Secretin, when incubated with isolated enterocytes, upregulated the expression of CD36. In summary, during starvation, secretin stimulates lipolysis through a HSL and PKA mediated pathway. When fed a HFD, SCTR-/- mice is resistant to diet induced obesity due to impaired intestinal lipid absorption. A novel short positive feedback pathway between CD36 and secretin, functioning to maximize lipid absorption, is also being proposed. Thus for the first time, two independent role of secretin in lipolysis and in intestinal lipid absorption were discovered along with their mechanistic insights. This study paves way for developing new therapeutic strategies against metabolic disorders associated with lipid metabolism.<br>published_or_final_version<br>Biological Sciences<br>Doctoral<br>Doctor of Philosophy

Adipose tissue plays a central role in the regulation of lipid storage and mobilization. A tight control between adipose tissue lipid storage and mobilization functions must be exerted to prevent an overload of lipids at other organs such as the heart, liver and skeletal muscles, and favor the risk of developing metabolic disorders, such as Type 2 diabetes and cardiovascular diseases (CVD). There is strong evidence from animal studies that low oxygen levels (hypoxia) are noted in adipose tissue as the mass of the organ excessively expands and, in turn, exacerbates some adipose tissue functions. Whether hypoxia exposure, which could be derived from reduced environmental oxygen availability, disease or a combination of both, affects adipose tissue lipid storage and mobilization functions in humans is not well known. Using in vitro and in vivo approaches, this thesis aimed at characterizing the effects of hypoxia on human adipose tissue lipid storage and lipid mobilization functions. Study I investigated how hypoxia can modulate human adipose functions such as lipid storage and lipid mobilization in vitro. Study II examined whether acute intermittent hypoxia, which simulates obstructive sleep apnea, affects adipose tissue lipid storage/mobilization functions and triglyceride levels in healthy young men in postprandial state. Study III tested the effect of an acute 6-hour continuous exposure to hypoxia (fraction of inspired oxygen (FIO2) = 0.12)) on plasma triglyceride levels in healthy young men in the fasting state. Study I indicates that both acute (24h) and chronic (14d) hypoxia (3%, and 10% O2) modulate human adipose tissue lipid storage and mobilization functions in a different manner. Study II demonstrates that acute exposure to intermittent hypoxia (6h) is sufficient to increase plasma non-esterified fatty acids (NEFA) levels, as well as insulin levels, but does not alter circulating triglyceride or subcutaneous adipose tissue lipid storage and/or mobilization capacity ex vivo in healthy men. Study III shows that acute exposure to normobaric hypoxia increases circulating NEFA and glycerol concentrations but did not translate in altering circulating triglycerides in fasting healthy men. In conclusion, our observations suggest that an exposure to reduced oxygen levels impairs human adipose tissue storage and/or mobilization functions, a phenomenon known in the development of metabolic disorders, such as Type 2 diabetes and CVD.

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids<br>Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon

Osteoporosis remain major clinical challenges. RNA interference (RNAi) provides a promising approach for promoting osteoblastic bone formation to settle the challenges. However, the major bottleneck for translating RNAi with efficacy and safety to clinical bone anabolic strategy is lack of osteoblast-specific delivery systems for osteogenic siRNAs. Previously, we developed a targeting system involving DOTAP-based cationic liposomes attached to oligopeptides (AspSerSer)6, (also known as (DSS)6), which had good affinity for bone formation surface. Using this system, osteogenic Pleckstrin Homology Domain Containing, Family O Member 1 (Plekho1) siRNA could be specifically delivered to bone formation surface at tissue level and promoted bone formation in osteopenic rodents. However, concerns still exist regarding indirect osteoblast-specific delivery, detrimental retention in hepatocytes, mononuclear phagocyte system (MPS)-induced dose reduction and inefficient nanoparticle extravasation. Aptamers, selected by cell-based Systematic evolution of ligands by exponential enrichment (cell-SELEX), are single-stranded DNA (ssDNA) or RNA which binds to target cells specifically by distinct tertiary structures. By performing positive selection with osteoblasts and negative selection with hepatocytes and peripheral blood mononuclear cells (PBMCs), we aimed to screen an aptamer that could achieve direct osteoblast-specific delivery and minimal hepatocyte and PBMCs accumulation of Plekho1 siRNAs. In addition, lipid nanoparticles (LNPs) have been widely used as nanomaterials encapsulating siRNA due to their small particle size below 90 nm. Polyethylene glycol¡(PEG) as the mostly used hydrophilic polymer, could efficiently prevent LNPs from MPS uptake. So, LNPs with PEG shielding could serve as siRNA carriers to realize efficient extravasation from fenestrated capillaries to osteoblasts and help reduce MPS uptake of the siRNAs. Recently, we screened an aptamer (CH6) by cell-SELEX specifically targeting both rat and human osteoblasts and developed the aptamer-functionalized LNPs encapsulating osteogenic Plekho1 siRNA, i.e., CH6-LNPs-siRNA. Our results demonstrated that CH6-LNPs-siRNA had an average particle size below 90 nm and no significant cytotoxicity in vitro. CH6 aptamer facilitated osteoblast-selective uptake of Plekho1 siRNA and gene silencing in vitro. In this study, we further found that CH6 aptamer facilitated the bone-specific distribution of siRNA by biophotonic imaging and quantitative analysis. Immunohistochemistry results showed that CH6 achieved in vivo osteoblast-specific delivery of Plekho1 siRNA. Dose-response experiment indicated that CH6-LNPs-siRNA achieved almost 80% gene knockdown at the siRNA dose of 1.0 mg/kg and maintained 12 days for over 50% gene silencing. microCT, bone histomorphometry and mechanical testing confirmed that CH6 facilitated bone formation, leading to improved bone micro-architecture, increased bone mass and enhanced mechanical properties in osteoporotic rodents. Furthermore, CH6-LNPs-siRNA achieved better bone anabolic action when compared to the previously developed (AspSerSer)6-liposome-siRNA. There was no obvious toxicity in rats injected with CH6-LNPs-siRNA. All these results indicated that osteoblast-specific aptamer-functionalized LNPs could act as a novel RNAi-based bone anabolic strategy and advance selectivity of targeted delivery for osteogenic siRNAs from tissue level toward cellular level. In addition, the generation of ssDNA from double-stranded PCR products is an essential step in selection of aptamers in SELEX. We found that the size separation derived from unequal primers with chemical modification could be a satisfactory alternative to the classic magnetic separation.

2013/2014<br>Il ferro è un elemento fondamentale per molti processi di cellule e tessuti ma è potenzialmente tossico e un eccesso può danneggiare diversi componenti cellulari. A livello fisiologico il ferro circolante è regolato da segnali da pathway che lo consumano e da cellule che lo forniscono. Il principale ormone regolatore epcidina agisce insieme al recettore cellulare ferroportina nel controllare l’assorbimento con la dieta, l’immagazzinamento e la distribuzione del ferro nel flusso sanguigno. Disturbi genetici che colpiscono i componenti del pathway così altamente regolato possono causare serie malattie nell’uomo come l’emocromatosi ereditaria e l’anemia sideropenia refrattaria al ferro (IRIDA) principalmente causate da mutazioni note nei geni HFE e TMPRSS6. Gli studi descritti in questa tesi hanno lo scopo di evidenziare varianti nuove e causative che hanno un ruolo nella regolazione del pathway di ecpidina-ferroportina per spiegare l’effetto delle variazioni di ferro e le basi molecolari dell’insorgenza dei disturbi genetici del ferro nell’uomo. Studi di associazione sull’intero genoma (GWAS) sono stati condotti sui valori quantitativi di epcidina, parametri del ferro e tratti eritroidi misurati nell’ampia popolazione della Val Borbera che include 1785 individui genotipizzati da un isolato genetico italiano. Il principale risultato mostra che l’associazione di HFE e TMPRRS6 ai tratti eritroidi dipendono in maggior parte dal totale di ferro disponibile e non da un effetto diretto di HFE e TMPRSS6. I livelli di epcidina sono stati associati alle variazioni in HFE e TMPRSS6 e una prima ampia meta-analisi condotta su circa 6,000 individui VBI e olandesi ha evidenziato 2 nuovi loci associati significativamente (p<5x10-8) all’epcidina: il primo sul cromosoma 10 vicino al gene FOXI2 e il secondo sul cromosoma 2 nel gene EML6 e vicino a SPTBN1 (alias ELF). SPTBN1 è un gene interessante in quanto essenziale nel signaling TGF-β mediante le proteine SMAD, una delle quali svolge un ruolo anche nella regolazione della trascrizione dell’epcidina. Per identificare ulteriori loci che hanno un ruolo nell’omeostasi del ferro è stata condotta un’ampia meta-analisi sui marcatori clinici dello stato del ferro su 48,000 individui di origine europea in collaborazione con il consorzio australiano per lo studio genetico del ferro (GIS): lo studio mostra associazioni più significative ed effetto pleiotropico dei geni noti del ferro HFE, TF, TFR2 and TMPRSS6 e cinque nuovi geni associati a livello Bonferroni (ABO, ARNTL, FADS2, NAT2, TEX14). In particolare la trasferrina è associata a NAT2 precedentemente associato a disturbi dei lipidi e a rischio cardiovascolare e FADS2 le cui variazioni hanno un effetto su diversi fenotipi come gli acidi grassi, il glucosio nel sangue e gli enzimi del fegato. I risultati mostrano una forte correlazione tra omeostasi del ferro e metabolismo dei lipidi nell’uomo e quindi il ferro potrebbe avere un ruolo nell’insorgenza dei disturbi cardiovascolari. I risultati confermano che l’isolato della Val Borbera ha costituito un modello della popolazione generale adatto a studi genetici su malattie comuni. Per aumentare la possiblità di trovare varianti rare e causative sfruttando i vantaggi e le caratteristiche delle popolazioni isolate, la coorte della Val Borbera e gli altri isolati genetici italiani si sono riuniti in un progetto che utilizza le tecniche innovative di sequenziamento dell’intero genoma allo scopo di creare un pannello di riferimento ricco di sequenze italiane rare e di altà qualità per ulteriori studi genetici sul ferro, epcidina e altri tratti di rischio.<br>Iron is a key element for cellular and tissue processes. It is also potentially toxic and excess iron can damage various cellular components. At physiological levels circulating iron is regulated by signals from pathways that use iron and from cells that supply iron. The main iron-regulatory hormone hepcidin and its receptor iron channel ferroportin play a critical role controlling the dietary absorption, storage, and tissue distribution of iron through the bloodstream. Genetic disorders that affect the components of the tightly regulated hepcidin-ferroportin pathway could cause severe pathologies in humans as hereditary hemocromatosis and iron-refractory iron-deficiency anemia or IRIDA mainly caused respectively by mutation in two known loci, HFE and TMPRSS6. The studies described in this thesis aimed at highlighting novel and causative variants in loci that have a role in the regulation of hepcidin-iron pathway to explain the effect of unbalanced iron in humans and the molecular basis of the onset of genetic iron disorders. First, genome-wide association studies (GWAS) have been carried out on quantitative hepcidin, iron parameters and erythrocyte traits measured in the population of Val Borbera (VBI) that includes 1785 genotyped individuals from an Italian genetic isolate. The main result showed that association of HFE and TMPRSS6 to erythroid traits is mostly dependent on the amount of iron available and not a direct effect of HFE and TMPRSS6 variants. Hepcidin levels have been associated to HFE and TMPRSS6 variations and a first large meta-analysis, performed on 6,000 VBI and Dutch individuals, revealed two novel loci associated to hepcidin at genome-wide significance (p<5x10-8): the first on chromosome 10, near the gene FOXI2 and the second signal on chromosome 2 in the EML6 gene and near SPTBN1 (alias ELF). SPTBN1 is an interesting gene as it is essential in TGF-β signaling by SMAD proteins and one of the SMADs is involved in hepcidin transcription regulation. To identify additional loci affecting iron homeostasis, a large international meta-analysis on the serum biomarkers commonly used to determine the clinical iron status has been carried out in 48,000 European ancestry individuals in collaboration with Australian Genetic Iron Status (GIS) Consortium: the study showed more significant associations and pleiotropic effect for known loci as HFE, TF, TFR2 and TMPRSS6 and five novel associated loci at significant levels (ABO, ARNTL, FADS2, NAT2, TEX14). In particular, transferrin is associated to NAT2, previously associated to lipids affections and cardiovascular risk, and to FADS2 that affects several phenotypes as lipids fatty acids, fasting glucose and liver enzyme. The results highlighted a strong correlation between iron homeostasis and lipid metabolism in humans that could have implication on the onset of cardiovascular disorders. The Val Borbera genetic isolate has represented a suitable model for genetic study on common disease. To increase the power of detection of rare and causative variants and to take advantage of the characteristics of genetic isolates, VBI and other Italian isolated populations are now involved in an innovative whole-genome sequencing (WGS) project with the aim to create an Italian specific panel enriched in lower-frequency high-quality variants to be used in further genetic analysis on iron parameters, hepcidin and other traits.<br>XXVII Ciclo<br>1981

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-13Bitstream added on 2014-06-13T20:27:54Z : No. of bitstreams: 1 siloto_ev_dr_botfmvz.pdf: 494737 bytes, checksum: 2225a359b9e80a74e8528c9b0ea4cfd8 (MD5)<br>O presente estudo avaliou o efeito da suplementação do cromo e dois níveis de energia sobre o desempenho e a qualidade de ovos de poedeiras leves. Foram utilizadas 192 poedeiras da linhagem Bovans com 47 semanas de idade. As aves foram distribuídas em um delineamento inteiramente casualizado, em esquema fatorial 4x2, com quatro níveis de suplementação de cromo (0, 200, 400 e 800 μg Cr/kg) e dois níveis energéticos (2.780 e 2.900 kcal EM/kg de ração). Cada tratamento continha seis repetições de quatro aves cada. O cromo utilizado foi o derivado de levedura. O experimento teve a duração de 112 dias, divididos em 4 períodos de 28 dias. Os parâmetros de desempenho não foram influenciados pelos níveis de cromo e de energia da dieta. Houve aumento linear no percentual de gema com o aumento da suplementação de cromo em dietas contendo 2.780 kcal/kg. As aves alimentadas com dietas contendo 2.780 kcal de EM/kg e isentas de suplementação de cromo, apresentaram redução no percentual de gema. Quando as aves foram suplementadas com 800 μg Cr/kg de dieta, foi observada uma maior deposição de gema. A suplementação de 800 μg Cr/kg na dieta com 2.780 kcal EM/kg provocou aumento no percentual de gema sem afetar o desempenho das poedeiras<br>This study aimed to evaluate the performance and egg quality in laying hens receiving diets supplemented with crescent levels of Chromium yeast and two different dietary energy levels. One hundred and ninety-two 47-week-old Bovans laying hens were distributed in a completely randomized design with 4x2 factorial arrangement: 4 levels of chromium yeast supplementation (0, 200, 400, 800 μg/kg feed) and two energy levels (2,780 and 2,900 kcal ME / kg of feed). Each treatment consisted of 6 replications of 4 birds each. The experiment lasted 112 days (four 28-day periods). The following performance parameters were evaluated: feed intake, egg weight, egg production, percentage of whole eggs, egg mass, feed: gain ratio (kg feed/dozen eggs and kg feed/kg eggs). The egg quality was accessed through the weight of total egg, shell, yolk and albumen and their proportions, Haugh unit, the specific gravity, and the shell thickness. The performance parameters were not influenced by the levels of chromium and dietary energy. There was a linear increase in the percentage of yolk with increasing chromium supplementation in diets containing 2.780kcal/kg. Laying hens fed with diets containing 2.780kcal/kg ME and without chromium supplementation showed reduced percentage of yolk. When birds were supplemented with 800 mg Cr / kg diet a greater deposition of yolk was observed. Dietary supplementation of 800 mg Cr / kg with 2.780kcal of ME/kg induced an increase in the percentage of yolk without affecting performance of laying hens

A pre-operative energy restrictive diet is common practice prior to bariatric surgery in the United Kingdom. A review of current practice found variability in form, duration and application of the diet used, with little evidence for efficacy beyond patients successfully undergoing surgery. Imperative for best practice was to provide evidence of the efficacy of dietary approaches both in terms of the original aim of the diet to reduce liver size and improve access to enable laparoscopic surgery, but also to investigate the wider effects of the diet on this population. A clinical trial was designed to compare two very low energy dietary approaches applied over two weeks before bariatric surgery, using a) a food-based diet, which is standard practice at the Royal Derby Hospital, and comparing this to b) an alternative meal replacement approach with products supplied by LighterLife. The diets were designed to offer similar energy intakes, 800kcal/d, but with varying macronutrient composition and format. Both diets achieved comparable body weight loss (median -3.4%) and excess body mass index loss (median -1.79kg/m2). The trial found similar results from both diets for the original aim of the diet, which is to enable surgery. Surgeons’ visual assessment of the liver at the time of surgery for operative difficulty was associated to histologically assessed levels of steatosis, with lower levels of steatosis associated to less operative difficulty. Patient evaluation of the diets revealed no significant difference in outcomes between diets, and overall satisfaction of the diets was very high, with 92% and 85% reporting that they were satisfied or very satisfied with their diet respectively. Both diets produced a favourable change in circulating lipids, inflammatory markers and glucose (pooled median LDL -0.4mmol/L, Glucose-0.2mmol/L, CRP -3.6mg/L, IL6 -0.4pg/mL, ELF -0.8). Findings including histology and gene expression data suggest a dynamic lipid environment in both liver and omental adipose tissue post-diet, concurrent with insulin sensitivity, but suggest a less dynamic environment in subcutaneous adipose tissue. Overall the diets could not be separated in terms of effects on circulating biomarkers, Non-Alcoholic Fatty Liver disease outcomes, operative difficulty, gene expression levels, or patient acceptability. Negative associations with diabetes status were found for steatosis and inflammation post-diet and warrant further investigation. Also, childhood weight status was found to be linked to the level of improvement in insulin sensitivity and requires further work. Limitations suggest additional work should incorporate reporting or recording of physical activity levels, pre-diet assessment of liver condition, diabetes duration and habitual dietary intake. Work should also confirm findings and corroborate suppositions regarding physiological changes observed from mRNA transcript expression levels, into protein and onto functional levels. This work informs the format and application of a short-term pre-operative diet in severe obesity. It has established that pre-bariatric surgery patients were satisfied with either format of VLED when applied in clinical practice. It has also extended the existing evidence base on the effects of a short-term VLED, specifically the potential beneficial effects on progressive features of NAFLD, and adipose tissue inflammatory status, which could be of value in the management of the disease.

Made available in DSpace on 2014-08-13T14:50:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-22Bitstream added on 2014-08-13T18:00:39Z : No. of bitstreams: 1 000756842.pdf: 789344 bytes, checksum: 061035505a8bd708cd9dbac41aad0d39 (MD5)<br>A obesidade, doença crônica metabólica caracterizada pelo acúmulo excessivo de tecido adiposo em relação à massa magra tecidual, é considerada uma epidemia global e um importante problema de saúde pública, que afeta tanto países desenvolvidos quanto subdesenvolvidos. O adipócito recebe influência de diversas substâncias e secreta inúmeros peptídeos, como leptina, angiotensina I e II, TGF-β, entre outros, que atuam diretamente ou indiretamente no sistema cardiovascular. Assim, o tecido adiposo não é simplesmente um reservatório de energia, mas um ativo órgão endócrino, parácrino e autócrino com múltiplas funções, capaz de sintetizar e liberar mediadores que participam de diversos processos biológicos, incluindo os que ocorrem no coração. O coração é composto por miócitos, nervos, vasos e matriz extracelular. O principal componente da matriz é o colágeno, com predomínio dos tipos I e III, sendo que, o tipo I é o mais abundante, correspondendo á aproximadamente 80% do colágeno total miocárdico. O colágeno é produzido, principalmente, pelos fibroblastos e degradado pelas metaloproteinases (MMPs). O colágeno, em situação estável, contribui para a manutenção da arquitetura e função cardíaca; entretanto, em resposta a estímulos desencadeados por agentes neuro-hormonais e/ou mecânicos, pode sofrer alterações; esta mudança pode ser resultante do aumento da síntese e/ou diminuição da degradação ou vice-versa. Em pesquisa recente realizada em nosso laboratório foi encontrado diminuição dos níveis protéicos de colágeno tipo I miocárdico em ratos Wistar obesos, por dieta hiperlipídica insaturada por 30 semanas, em relação ao grupo controle. Em razão, dos resultados encontrados em nosso laboratório e da literatura mostrar que a leptina aumenta a atividade das MMP-2 e a síntese da expressão gênica da MMP-9, a proposta deste estudo foi testar a hipótese ...<br>Obesity is a chronic metabolic disorder characterized by excessive adipose tissue accumulation in relation to lean tissue. Currently, it is a global epidemic and a major public health problem that affects both developed as well as undeveloped countries. The adipocyte receives influence of several substances and secretes numerous peptides, such as leptin, angiotensin I and II, TGF-β, among others, that act directly or indirectly on the cardiovascular system. Thus, adipose tissue is not simply an energy reservoir, but an active endocrine, paracrine and autocrine organ with multiple functions, able to synthesize and release mediators that participate in many biological processes, including those that occur in the heart. The heart is composed of myocytes, nerves, vessels and extracellular matrix. The main component of matrix is collagen, predominantly type I and III, being type I the most abundant, corresponding to approximately 80% of total myocardial collagen. Collagen is mainly produced by fibroblasts and degraded by metalloproteinases (MMPs). Collagen, in a stable condition, contributes to the maintenance of cardiac architecture and function, however, in response to stimuli triggered by neuro-hormonal and/or mechanical agents, it may change, and this change can be due to increased synthesis and/or decreased degradation, or vice versa. In recent research conducted in our laboratory, we found decreased protein levels of myocardial type I collagen in obese Wistar rats by unsaturated high-fat diet for 30 weeks. Due to the results found in our laboratory and because the literature shows that leptin increases MMP-2 activity and MMP-9 gene expression, the purpose of this study was to test the hypothesis that the reduction of myocardial type I collagen is associated with increased MMPs 2 and 9 activities in obese rats by unsaturated high-fat diet. Thirty-day-old male Wistar rats were randomized into to two groups: control ...

Orientador: Antonio Carlos Cicogna<br>Banca: Marcos Ferreira Minicucci<br>Banca: André Soares Leopoldo<br>Resumo: A obesidade, doença crônica metabólica caracterizada pelo acúmulo excessivo de tecido adiposo em relação à massa magra tecidual, é considerada uma epidemia global e um importante problema de saúde pública, que afeta tanto países desenvolvidos quanto subdesenvolvidos. O adipócito recebe influência de diversas substâncias e secreta inúmeros peptídeos, como leptina, angiotensina I e II, TGF-β, entre outros, que atuam diretamente ou indiretamente no sistema cardiovascular. Assim, o tecido adiposo não é simplesmente um reservatório de energia, mas um ativo órgão endócrino, parácrino e autócrino com múltiplas funções, capaz de sintetizar e liberar mediadores que participam de diversos processos biológicos, incluindo os que ocorrem no coração. O coração é composto por miócitos, nervos, vasos e matriz extracelular. O principal componente da matriz é o colágeno, com predomínio dos tipos I e III, sendo que, o tipo I é o mais abundante, correspondendo á aproximadamente 80% do colágeno total miocárdico. O colágeno é produzido, principalmente, pelos fibroblastos e degradado pelas metaloproteinases (MMPs). O colágeno, em situação estável, contribui para a manutenção da arquitetura e função cardíaca; entretanto, em resposta a estímulos desencadeados por agentes neuro-hormonais e/ou mecânicos, pode sofrer alterações; esta mudança pode ser resultante do aumento da síntese e/ou diminuição da degradação ou vice-versa. Em pesquisa recente realizada em nosso laboratório foi encontrado diminuição dos níveis protéicos de colágeno tipo I miocárdico em ratos Wistar obesos, por dieta hiperlipídica insaturada por 30 semanas, em relação ao grupo controle. Em razão, dos resultados encontrados em nosso laboratório e da literatura mostrar que a leptina aumenta a atividade das MMP-2 e a síntese da expressão gênica da MMP-9, a proposta deste estudo foi testar a hipótese ...<br>Abstract: Obesity is a chronic metabolic disorder characterized by excessive adipose tissue accumulation in relation to lean tissue. Currently, it is a global epidemic and a major public health problem that affects both developed as well as undeveloped countries. The adipocyte receives influence of several substances and secretes numerous peptides, such as leptin, angiotensin I and II, TGF-β, among others, that act directly or indirectly on the cardiovascular system. Thus, adipose tissue is not simply an energy reservoir, but an active endocrine, paracrine and autocrine organ with multiple functions, able to synthesize and release mediators that participate in many biological processes, including those that occur in the heart. The heart is composed of myocytes, nerves, vessels and extracellular matrix. The main component of matrix is collagen, predominantly type I and III, being type I the most abundant, corresponding to approximately 80% of total myocardial collagen. Collagen is mainly produced by fibroblasts and degraded by metalloproteinases (MMPs). Collagen, in a stable condition, contributes to the maintenance of cardiac architecture and function, however, in response to stimuli triggered by neuro-hormonal and/or mechanical agents, it may change, and this change can be due to increased synthesis and/or decreased degradation, or vice versa. In recent research conducted in our laboratory, we found decreased protein levels of myocardial type I collagen in obese Wistar rats by unsaturated high-fat diet for 30 weeks. Due to the results found in our laboratory and because the literature shows that leptin increases MMP-2 activity and MMP-9 gene expression, the purpose of this study was to test the hypothesis that the reduction of myocardial type I collagen is associated with increased MMPs 2 and 9 activities in obese rats by unsaturated high-fat diet. Thirty-day-old male Wistar rats were randomized into to two groups: control ...<br>Mestre

Altered glucose metabolism is the underlying factor in many metabolic disorders, including diabetes. A novel protein recently linked to diabetes through animal and clinical studies is Sarcolemmal Membrane Associated Protein (SLMAP) but its role in metabolism remains undefined. The data here reveals a novel role for SLMAP isoform1 in glucose metabolism within the myocardium. Neonatal cardiomyocytes (NCMs) harvested from hearts of transgenic mice expressing SLMAP1, presented with increased glucose uptake, glycolytic rate, as well as glucose transporter 4 (GLUT4) expressions with minimal impact on lipid metabolism. SLMAP1 expression markedly increased the machinery required for endosomal trafficking of GLUT4 to the membrane within NCMs, accounting for the observed effects on glucose metabolism. The data here indicates SLMAP1 as a unique regulator of glucose metabolism through endosomal regulation of GLUT4 trafficking and suggests it may uniquely serve as a target to limit cardiovascular disease in metabolic disorders such as diabetes.

INTRODUÇÃO: A esteato-hepatite não alcóolica (NASH) é considerada problema de saúde pública, dada sua crescente incidência e seu possível papel na carcinogênese hepato-celular. Terapias atuais envolvem alterações de dieta e estilo de vida, mas têm seu resultado prejudicado pela baixa aderência dos pacientes. Abordagens farmacológicas ainda são precárias. Uma grande dificuldade no manejo de NASH reside no limitado entendimento de sua fisiopatologia, que parece envolver complexas alterações metabólicas e inflamatórias. Ácidos graxos poli-insaturados ômega-3 (AGPIs n-3) são reconhecidos por suas propriedades moduladoras do metabolismo lipídico e da inflamação, e estão diminuídos em pacientes com NASH. O uso clínico de AGPIs n-3 tem mostrado benefício no controle da esteatose e na produção de marcadores da resposta metabólica e inflamatória em NASH, embora com algumas observações contraditórias. A compreensão de mecanismos moleculares modulados por AGPIs n-3 em NASH podem ser úteis para identificar alvos moleculares que auxiliem no desenho de intervenção farmacológica efetiva. Nesse sentido, ciências ômicas são particularmente úteis para a compreensão de mecanismos moleculares com alto valor translacional para a prática clínica e podem contribuir para a identificação desses alvos. OBJETIVO: O presente estudo avaliou a resposta proteômica hepática e lipidômica plasmática de pacientes com NASH perante o tratamento com AGPIs n-3. MÉTODO: As avaliações proteômicas e lipidômicas foram desenvolvidas por espectometria de massas e/ou cromatografia gasosa em amostras de biópsias hepáticas e plasma coletadas de pacientes envolvidos em estudo preliminar, realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. O referido estudo envolveu pacientes adultos, de ambos os sexos e com diagnóstico de NASH tratados diariamente, durante 6 meses, com 3 cápsulas contendo mistura de óleo de linhaça e óleo de peixe [0,315 g AGPIs: sendo 0,065 g de ácido eicosapentaenoico (EPA), 0,050 g de docosahexaenoico (DHA) e 0,2 g alfa linolênico (ALA) por cápsula]. Pacientes, após o tratamento com AGPIs n-3, que apresentaram altas concentrações plasmáticas de ALA e/ou DHA e/ou baixas de ácido araquidônico (AA) mostraram melhora parcial das alterações de histologia hepática. No presente estudo, avaliamos as vias proteômicas e marcadores lipidômicos resultantes do tratamento com AGPIs n-3. Isto foi feito por meio da comparação, antes (grupo AT) e depois do tratamento (grupo DT), de pools de tecido hepático (análise por interactoma) e amostras de plasma (OPLS-DA). RESULTADOS: Foram identificadas proteínas hepáticas, exclusivamente e/ou alteradamente expressas, no grupo DT, relacionadas com vias de matriz celular, metabolismo lipídico, de estresse oxidativo, e de retículo endoplasmático e respiração celular. Com excessão da via de matriz celular, a análise do interactoma revelou alteração funcional significativa das vias moduladas por essas proteínas. Em conjunto, essas alterações foram sugestivas de diminuição de lipotoxicidade, estresse oxidativo e respiração anaeróbia, e aumento de respiração aeróbia após tratamento com AGPIs n-3. Estas modificações são marcadores potenciais de melhora de função de retículo endoplasmático e mitocondrial. Em adição, após o tratamento com AGPIs n-3, o perfil lipidômico plasmático mostrou-se alterado com significativo aumento de glicerofosfolípides, ALA e EPA, e diminuição de ácido araquidônico (n-6) e da razão AGPIs n-6/n-3. Estes dados são concordantes com potencial melhora das funções de retículo endoplasmático e mitocondriais. CONCLUSÃO: O tratamento com AGPIs n-3 em pacientes com NASH influenciou favoravelmente o perfil proteômico hepático e lipidômico sistêmico. Em conjunto, essas alterações sugerem melhora da função de retículo endoplasmático e mitocondrial, com potencial impacto na homeostase celular, por meio da modulação de diferentes vias biológicas<br>INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is considered a public health problem, given its increasing incidence and its possible role in hepatocellular carcinogenesis. Current therapies involve diet and lifestyle changes, but its applicability suffers from low patients adherence. Pharmacological approaches are still missing. A main difficulty in the NASH management lies in the limited understanding of its pathophysiology, which seems to involve complex metabolic and inflammatory disturbances. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are recognized for its modulatory properties on lipid metabolism and inflammation and are decreased in patients with NASH. The clinical use of these PUFAs has shown benefit in controlling steatosis and the production of metabolic and inflammatory response markers in NASH, despite some conflicting reports. Understanding mechanisms modulated by n-3 PUFAs in NASH may be useful for identifying molecular targets that could assist in the design of effective pharmacologic interventions. In this sense, omics sciences are particularly useful for understanding molecular mechanisms with high translational value to clinical practice and may contribute to the identification of these targets. AIM: This study evaluated the liver proteomic and plasma lipidomics responses of patients with NASH towards treatment with n-3 PUFAs. METHODS: The proteomic and lipidomic evaluations were studied by mass spectrometry and / or gas chromatography in samples from liver biopsies and plasma collected from patients enrolled in a preliminary clinical trial of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. This study involved adult patients of both sexes diagnosed with NASH treated daily for 6 months, with 3 capsules containing a mixture of linseed and fish oils [0.315 g PUFAs: 0.065 g eicosapentaenoic acid (EPA) , 0.050 g docosahexaenoic (DHA) and 0.2 g alpha linolenic acid (ALA) per capsule]. Patients, after treatment with n-3 PUFAs, with higher concentrations of ALA and DHA and lower arachidonic acid (AA) showed improvement of liver histology alterations. In the present study we evaluated the proteomics pathways and lipidomics markers resulted from treatment with PUFAs n-3. This was performed by comparing, before (BT group) and after (AT group) treatment, liver tissue pools (analysis interactome) and plasma samples (OPLS-DA). RESULTS: It was identified, in a way exclusive and altered, the expressed liver proteins in AT group, related to pathways of cellular matrix, lipid metabolism, oxidative and endoplasmic reticulum stress and cellular respiration. With the exception of cell matrix, the analysis of the interactome revealed substantial functional alterations of the pathways modulated by these proteins. Together, these changes were suggestive of decreased lipotoxicity, oxidative stress and anaerobic respiration and increased aerobic respiration following treatment with PUFAs n-3. These modifications are potential markers of endoplasmic reticulum and mitochondrial functions improvement. In addition, after treatment with n-3 PUFAs, the lipidomics profile was modified, with significant increase in glycerophospholipids, ALA and EPA and decrease of arachidonic acid (AA) and n-6/n-3 AGPIs ratio. These findings are concordant with potential improvement of reticulum endoplasmic and mitochondrial functions. CONCLUSION: In patients with NASH the treatment with n-3 PUFAs favorably influenced hepatic proteomic and systemic lipidomics profiles. Together, these changes suggest improved endoplasmic reticulum and mitochondrial functions, with potential impact on cellular homeostasis through the modulation of different biological pathways

Tese de Doutoramento em Ciência e Tecnologia Animal<br>The daily intake of conjugated linoleic acid (CLA) isomers by humans, through diet and supplementation, and the controversial effects of these compounds in human health, were the main motivation for the elaboration of this thesis. Firstly, the present work intended to estimate the daily CLA ingestion by the Portuguese population. Secondly, the biological effects of CLA were exploited using two distinct animal models, normal-weight (Wistar rat) and genetically fat (obese Zucker rat), in combination with saturated fat based diets. The estimative of total CLA intake for the Portuguese population was 73.70 mg/day. The cis(c)9,trans(t)11 and t7,c9 were the most prevalent CLA isomers, with, respectively, 76.10 and 12.56% of the total CLA intake value. Concerning the animal trials, CLA in conjugation with saturated fats revealed beneficial but also deleterious biological effects. In the normal-weight Wistar rat fed a palm oil based diet, the administration of c9,t11 CLA isomer increased the serum triacylglycerols and the size of adipocytes from epididymal and retroperitoneal fat depots. In addition, a CLA mixture of c9,t11 and t10,c12 isomers increased the glycerol membrane permeability of kidney proximal tubules, which may indicate an improvement of glycerol reabsorption pathway. In the obese Zucker rat, CLA (as a mixture) induced changes in fatty acid profile of liver, muscle and adipose depots. CLA supplemented with a vegetable saturated fat diet seemed to promote a more beneficial adipokine serum profile and an alleviation of hepatic steatosis. In contrast, adverse effects of CLA were observed with hypercholesterolaemia promotion. Regardless CLA, the ovine fat diets worsened the insulin resistance and increased the pro-inflammatory serum cytokines. In the liver, different levels of cell death and apoptotic pathways were modulated by CLA, depending on the type of saturated fat present in the diet. The most striking result of this study was that CLA was not able to promote fat loss in both experimental models. Moreover, new mechanisms of CLA action were disclosed in this work, which reinforce the need to further investigate this compound.<br>RESUMO - Efeitos biológicos do ácido linoleico conjugado e de gorduras saturadas na composição da gordura corporal, na obesidade e patologias associadas: estudos experimentais em ratos Wistar e Zucker obesos - A ingestão diária de isómeros do ácido linoleico conjugado (CLA), através da dieta e da sua suplementação, bem como, os efeitos controversos destes compostos na saúde humana, constituíram a principal motivação para a elaboração desta tese. Numa primeira fase, o trabalho pretendeu estimar a ingestão diária de CLA pela população Portuguesa. Posteriormente, foram avaliados os efeitos biológicos do CLA quando suplementado a dietas à base de gordura saturada. Para tal, recorreu-se a dois modelos animais distintos, o rato Wistar e o rato Zucker (geneticamente obeso). A ingestão média total de CLA pela população Portuguesa foi estimada em 73.70 mg/dia. Os isómeros do CLA mais representativos foram o cis(c)9,trans(t)11 e o t7,c9, correspondendo, respectivamente, a 76.10 e 12.56% do total de CLA ingerido. Quanto aos estudos in vivo, o CLA revelou efeitos biológicos tanto benéficos como prejudiciais. No modelo Wistar alimentado com dieta à base de óleo de palma, a administração do isómero do CLA c9,t11 elevou os níveis de triacilgliceróis no soro, bem como, o tamanho dos adipócitos das gorduras epididimal e retroperitoneal. Adicionalmente, a mistura de isómeros do CLA (c9,t11 e t10,c12) aumentou a permeabilidade membranar ao glicerol no túbulo proximal do rim, sugerindo uma melhoria no processo de reabsorção desta molécula. No modelo Zucker, o CLA (como mistura de isómeros) induziu alterações no perfil dos ácidos gordos do fígado, músculo e gorduras epididimal e retroperitoneal. O CLA administrado com óleo de palma promoveu um perfil de adipocinas no soro mais benéfico e melhorou a esteatose hepática. Em contraste, a suplementação com CLA apresentou um efeito hipercolesterolémico. Independentemente do CLA, a dieta rica em gordura de ovinos agravou a resistência à insulina e aumentou as adipocinas pró-inflamatórias no soro. No fígado, diferentes níveis de morte celular e vias apoptóticas foram modeladas pelo CLA em função do tipo de gordura presente na dieta. É de salientar que não se observaram efeitos anti-adipogénicos do CLA em nenhum dos dois modelos animais. Por último, esta tese contribuiu para a descoberta de novos mecanismos de acção dos isómeros do CLA, reforçando a necessidade de continuar a estudar estes compostos.<br>This work was funded by Fundação para a Ciência e a Tecnologia (FCT) through the individual fellowship SFRH/BD/22566/2006 and co-financed by the grant POCTI/44750/2002

Los pacientes con dislipemia son tratados con estatinas para reducir sus concentraciones de lípidos, y disminuir el riesgo cardiovascular. El grado de respuesta a estos fármacos es heterogéneo, y en él podrían influir ciertos genes. Este trabajo trata de valorar la influencia de seis variantes genéticas sobre la eficacia de las estatinas en pacientes con dislipemia, tanto de manera independiente como junto a una serie de variables de control. Las magnitudes lipídicas evaluadas fueron el colesterol total (CT) y el excluido de HDL (noHDL), y las variables de control fueron: edad, dosis media diaria de estatina, cambio en el índice de masa corporal, cambio en el hábito tabáquico, cambio en las horas de ejercicio practicadas semanalmente y cambio en el consumo de gramos de alcohol diarios. La eficacia del tratamiento se valoró mediante tres indicadores: disminución relativa de CT y no HDL según la concentración al final (%C) y según la concentración media durante el tratamiento (%CM), y grado de consecución de objetivos terapéuticos. Este estudio se llevó a cabo en una población NAIF (n=100), pacientes en los que se inicia el tratamiento en la primera visita, y posteriormente en una población NO NAIF (n=57), pacientes ya tratados en la primera visita a quienes se les modifica el tratamiento. Las variantes genéticas estudiadas fueron: APOE c.526C>T (APOE2), APOE c.388T>C (APOE4), SLCO1B1 c.521T>C, CYP3A4 c.-392G>A, HMGCR c.1564-106A>G y LPA c.3947+467T>C. El estudio estadístico se basó en modelos de regresión múltiple. Para la población NAIF, la variante HMGCR c.1564-106A>G resultó influir sobre el %C y el %CM tanto para CT como para noHDL. Aportó al modelo basal un porcentaje de explicación adicional para %C de CT y noHDL de un 9,5% y 8,2%, respectivamente, y 6,2% y 3,5% al del %CM. También resultó influyente para los NO NAIF, suponiendo una explicación adicional de aproximadamente un 8% para los cuatro casos. Cabe destacar que en ambas poblaciones el efecto de la presencia de la variante resultó contrario: perjudicial para los NAIF y beneficioso para los NO NAIF. El gen HMGCR codifica para la enzima sobre la que ejercen su acción las estatinas. La variante estudiada está implicada en la regulación del splicing alternativo del exón 13, que modifica los centros activos sobre los que puede actuar la estatina. Este splicing alternativo también se ve modulado por la concentración de lípidos circulantes, pero esto ocurre únicamente en los no portadores de la variante. En esta tesis se postula que la disminución del CT y el noHDL se producirían de manera gradual y constante en el caso de los portadores de la variante, o según una función de tipo sigmoideo en los no portadores. Además, para los NO NAIF, la variante SLCO1B1 c.521T>C resultó influyente y perjudicial para el %C y %CM del CT, aportando un 7,1% y 5,9% de explicación al modelo basal respectivamente. Esto podría deberse a la relación entre la variante y los efectos adversos de las estatinas, que suele llevar aparejada una mala adhesión al tratamiento. Respecto al grado de consecución de los objetivos terapéuticos, las variantes influyentes fueron HMGCR c.1564-106A>G para el CT para los NAIF, y SLCO1B1 c.521T>C para CT y noHDL de los NO NAIF. La presencia de las variantes supuso mayor dificultad para alcanzar los objetivos. Se puede concluir que la variante estudiada del gen HMGCR tiene cierta influencia sobre la eficacia de las estatinas, aunque su efecto depende del tipo de población estudiada. La presencia de la variante del gen SLCO1B1 también podría presentar cierto efecto perjudicial, pero en ambos casos sería recomendable ampliar el estudio con un mayor número de pacientes.<br>Patients with dislipemia are often treated with statins to reduce lipids and cardiovascular risk. It is known that the efficacy of statins is variable between patients, so a genetic influence is suspected. This work tries to assess the influence of six genetic variants on the efficacy of statins employing three indicators: percentage reduction of total cholesterol (CT) and noHDL cholesterol (noHDL) according to final concentration (%C) and to mean concentration (%CM), as well as achievement of therapeutic objectives. The study was first developed in a population of patients who were not treated at the first visit (NAIF) and then it was repeated with patients treated in the first visit whose treatment was changed (NO NAIF). The genetic variants were: APOE c.526C>T (APOE2), APOE c.388T>C (APOE4), SLCO1B1 c.521T>C, CYP3A4 c.-392G>A, HMGCR c.1564-106A>G y LPA c.3947+467T>C. The statistical analysis employs multiple regression models to define the percentage of explanation added by the variant to a basal model constructed with the significant control variables. The most influential variant was HMGCR c.1564-106A>G which added an explanation of 9,5%, 8,5%, 6,2% and 3,5% to the indicators %C CT and noHDL, and %CM CT and noHDL in NAIF population. For NO NAIF it added an explanation of over 8% in the same cases. The presence of the variant showed an opposite effect in both populations: harmful for NAIF and beneficial for NO NAIF. This variant is related to an alternative splicing of the exon 13, which is also regulated by lipids concentrations, but only in patients without the variant. This work postulates that the reduction of CT and noHDL is gradual for variant carriers, but follows a sigmoid function for non-carriers. For NO NAIF the SLCO1B1 c.521T>C variant was harmful for %C and %CM of CT, adding explanations of 7,1% and 5,9%. This could be due to the relationship between this variant and the adverse effects of statins, which usually means worse treatment adherence. About therapeutic objectives, the variant HMGCR c.1564-106A>G was influent for CT of NAIF and SLCO1B1 c.521T>C for CT and noHDL of NO NAIF, hindering the achievement of therapeutic objectives in both cases.

Il sistema immunitario è costituito da una varietà di cellule, molecole e processi biologici che interagiscono per prevenire le invasioni microbiche, riconoscere le molecole estranee ed eliminare le fonti esistenti di lesioni cellulari, ripristinando le normali funzioni tissutali una volta risolto il problema. L'immunità innata è la prima linea di difesa contro le invasioni di agenti patogeni. Nelle vacche da latte, il suo funzionamento subisce gravi alterazioni durante il periodo di transizione (TP). In questa fase è stata segnalata una compromissione delle funzioni delle cellule polimorfonucleate (PMN) correlate alla produzione di metaboliti reattivi dell'ossigeno (ROM), all’attività della mieloperossidasi (MPO), alla chemiotassi e alla fagocitosi. I PMN bovini hanno un alterata espressione dei geni codificanti per tali funzioni tra -1 e 2 settimane dal parto, rispetto al livello rilevato 4 settimane dopo il parto per gli stessi geni. La causa esatta delle disfunzioni immunitarie che si verificano nel periparto non è mai stata chiaramente identificata. In esse possono contribuire diversi fattori, principalmente imputati alle alterazioni metaboliche tipiche del periparto (cambiamenti nell’assetto ormonale, limitazione della risposta immunitaria materna al fine di mantenere la gravidanza, alterazioni nel bilancio energetico e stato di stress ossidativo). Tuttavia, la durata e l’entità delle disfunzioni immunitarie può aumentare qualora subentri uno stato di squilibrio fisiologico (PI). In tali condizioni, le alterazioni metaboliche del periparto sfuggono al controllo dei meccanismi omeostatici e omeoretici, ed una infiammazione sistemica è la conseguenza frequente di questo squilibrio. Lo stato infiammatorio sistemico è scatenato da un aumento dei livelli di citochine proinfiammatorie (PIC), che è collegato ad un aumento della temperatura corporea al parto, e che tipicamente inficia le funzionalità epatiche, modificando le priorità anaboliche dell'organo in fase di inizio lattazione. A seguito di tale slittamento, il fegato produce più α-globuline, note come proteine positive di fase acuta (+APP), cioè aptoglobina, ceruloplasmina e siero amiloide alfa (SAA). Al contrario, riduce la sintesi di albumina, retinol binding protein (RBP), paraoxonasi (PON) e lipoproteine, note come proteine negative di fase acuta (-APP), e sequestra minerali, quali zinco e ferro, dal flusso ematico. L'infiammazione porta all'attivazione dei PMN, mentre la ridotta competenza immunitaria comunemente riportata in TP è stata associata ad un effetto opposto sui leucociti. Pertanto, questi dovrebbero essere considerati come due fenomeni distinti, ma lo stato di PI potrebbe essere considerato un denominatore comune, direttamente correlato al rischio di patologie in avvio di lattazione. Le strategie nutrizionali per ottimizzare l'immunità delle vacche da latte durante il TP dovrebbero quindi essere focalizzate sulla riduzione del grado di PI correlato al parto. Tra tali strategie nutrizionali, dovrebbe essere presa in considerazione la corretta gestione delle fonti energetiche per adattarle alle variazioni dei fabbisogni. Inoltre, il profilo degli acidi grassi delle fonti lipidiche può contribuire nel modificare le funzioni immunitarie. Infine, la somministrazione di prodotti supplementari con attività antiossidanti o antinfiammatorie, così come di specie donatrici di gruppi metilici, potrebbero essere strategie utili a favorire la funzionalità immunitaria delle bovine durante il TP. In una prospettiva più ampia, sebbene strategie nutrizionali e supplementi possano talora mitigare le alterazioni immunitarie, possiamo concludere che l'adozione di pratiche volte a minimizzare il PI durante il periodo di transizione sia la strategia più efficace per prevenire le disfunzioni. Al fine di chiarire il legame tra le alterazioni che si verificano nel periparto e le disfunzioni immunitarie delle bovine da latte sono stati condotti tre esperimenti. Bovine di razza frisona sono state alloggiate in poste individuali a stabulazione fissa e monitorate regolarmente per le condizioni corporee (BCS), il peso (BW), l'assunzione di alimenti (DMI), la produzione di latte (MY) e il tempo di ruminazione. Campioni di sangue sono stati raccolti regolarmente per valutare un ampio profilo ematochimico e per testare le funzioni dei globuli bianchi mediante stimolazioni ex-vivo. Inoltre, la diapedesi dei PMN è stata testata in vivo mediante test della carragenina e sono stati raccolti campioni di rumine a 30 giorni dal parto (DFC). Il primo esperimento era volto a chiarire le cause dei cambiamenti metabolici che si verificano al momento della messa in asciutta, ed il contributo del livello produttivo in tali alterazioni. Infatti, i profondi cambiamenti nell’alimentazione, gli adattamenti gastrointestinali, del metabolismo e dei parametri immunitari che si verificano nelle bovine alla messa in asciutta sono note scatenare il rilascio di cortisolo, indurre segnali di infiammazione sistemica ed alterare il bilancio redox. Produzioni di latte elevate al momento della messa in asciutta hanno un ruolo nell'aggravare tali condizioni. Nel nostro studio, un gruppo di 13 bovine è stato asciugato a 55 giorni dalla data prevista per il parto. Gli animali sono stati divisi in due gruppi in base alla produzione media dell'ultima settimana di lattazione, assumendo un cut-off di 15 kg * d-1: bassa (LM; 6 animali) e alta produzione (HM; 7 animali). I dati sono stati sottoposti ad ANOVA utilizzando un modello per misure ripetute, assumendo il livello produttivo al termine della lattazione, il tempo e la loro interazione come effetti fissi. L'aumento delle quantità di fibra nella razione di asciutta ha ridotto la DMI e aumentato il tempo di ruminazione. La migrazione dei leucociti nella ghiandola mammaria per contribuire alla fase di involuzione ha ridotto la loro abbondanza nel sangue e aumentato la loro attività. Tale attivazione dei leucociti nella mammella ha aumentato l'abbondanza di specie reattive dell’azoto nel plasma e innescato un'infiammazione sistemica in tutti gli animali (aumento delle +APP e riduzione delle -APP). Tale infiammazione ha compromesso le funzioni epatiche (aumento delle concentrazioni di gamma-glutamil transferasi -GGT- bilirubina e fosfatasi alcalina -ALP-). Sia la produzione di specie dell’azoto che lo stato infiammatorio sistemico hanno contribuito all'esaurimento degli antiossidanti circolanti (gruppi tiolici -SHp-, tocoferolo, β-carotene, potere antiossidante ferrico riducente -FRAP- e capacità antiossidante contro specie reattive dell'ossigeno -ORAC-). Gli animali con una produzione più elevata alla messa in asciutta hanno mostrato la peggiore condizione, probabilmente per i più profondi cambiamenti metabolici che hanno affrontato dopo l'interruzione delle mungiture, e per la fase involutiva verosimilmente più dispendiosa. Questo studio evidenzia la messa in asciutta come una fase critica per gestire la salute delle vacche da latte, e suggerisce un potenziale legame della messa in asciutta con le alterazioni delle funzioni immunitarie che si verificano nel periparto. Nel secondo esperimento si sono cercati di identificare i cambiamenti del sistema immunitario che precedono l'insorgenza della chetosi, al fine di chiarire il loro ruolo nella comparsa della malattia. Pertanto, 13 bovine sono state monitorate tra -48 e 35 DFC e suddivise in due gruppi sulla base dei loro livelli plasmatici di beta idrossibutirrato (BHB): inferiore (CTR, 7 animali) o superiore a 1,4 mMol / L (KET; 6 animali). I dati sono stati sottoposti ad ANOVA utilizzando un modello per misure ripetute, assumendo lo stato di salute, il tempo e la loro interazione come effetti fissi. Le vacche KET hanno avuto una maggiore attivazione del sistema immunitario prima del parto (maggiori concentrazioni plasmatiche di PIC, MPO e specie ossidanti e maggiori produzione di interferone gamma in risposta alla stimolazione con Mycobacterium avium) alterazioni della funzionalità epatica (più alta concentrazione sanguigna di GGT) e minori minerali plasmatici. Elevati livelli plasmatici di NEFA, BHB e glucosio nelle vacche KET suggeriscono uno stato di insulinoresistenza e una marcata mobilizzazione del grasso corporeo durante il periodo di asciutta. Tali andamenti dei parametri relativi al metabolismo energetico durante l’asciutta sono stati associati alla riduzione della DMI al momento del parto e al peggioramento del bilancio energetico negativo ad avvio lattazione. Ciò ha causato a sua volta una riduzione di MY e accresciuto ulteriormente la mobilizzazione dei grassi in avvio di lattazione. Compromissione della funzionalità epatica e attivazione dei leucociti durante il periodo di asciutta hanno determinato una marcata risposta infiammatoria di fase acuta nelle vacche KET dopo il parto (maggiori concentrazioni di +APP minori concentrazioni di RBP), ed ulteriormente compromesso la funzionalità epatica (maggiori concentrazioni di glutammato-ossalacetato transaminasi -AST-GOT- e bilirubina). I leucociti delle vacche KET hanno mostrato ridotte funzioni infiammatorie dopo stimolazione ex-vivo con lipopolisaccaridi batterici (minore produzione di PIC e maggiore produzione di lattato). Queste alterazioni potrebbero essere guidate dall'azione combinata dei metaboliti legati alla mobilizzazione dei lipidi e delle azioni antinfiammatorie volte a prevenire un'infiammazione eccessiva. Ciò suggerisce che le alterazioni dei parametri immunitari osservate prima del parto siano altamente correlate con la probabilità di sviluppare chetosi in avvio di lattazione. Nel terzo esperimento è stato somministrato un prodotto immunostimolante dalla comprovata efficacia nel migliorare le funzioni leucocitarie degli animali immunodepressi e nel ridurre l'incidenza delle malattie infettive delle bovine ad inizio lattazione. La sua modalità di azione non è mai stata chiarita, e un’indagine approfondita sul suo effetto metabolico potrebbe evidenziarne l’efficacia anche nei confronti dei disordini metabolici del periodo di transizione. Pertanto, un gruppo di10 bovine è stato monitorato da -62 a 42 DFC. Il gruppo trattato (TRT, 5 animali) ha ricevuto 32,5 g di Omnigen-AF® (Phibro Animal Health Corporation) due volte al giorno (65 g d-1), mentre il gruppo di controllo (CTR, 5 animali) non ha ricevuto alcun supplemento. I dati sono stati sottoposti ad ANOVA utilizzando un modello per misure ripetute, assumendo il trattamento, il tempo e la loro interazione come effetti fissi. La somministrazione dell’immunostimolante alla messa in asciutta non ha influenzato BW, BCS, MY, composizione del latte e del fluido ruminale e nemmeno modificato la concentrazione di neutrofili del sangue. Tuttavia, ha aumentato il tempo di ruminazione e migliorato il metabolismo energetico dopo il parto (concentrazioni di NEFA e BHB inferiori). Le bovine TRT avevano maggiori concentrazioni ematiche di linfociti e i loro leucociti avevano una maggiore efficienza nel rispondere alla stimolazione con lipopolisaccaridi batterici (produzione di lattato inferiore e minore consumo di glucosio). Nonostante questi effetti positivi sulle cellule immunitarie, l'immunostimolante non ha influenzato le concentrazioni di +APP dopo il parto. Inoltre, l’immunostimolante ha ridotto le concentrazioni di albumina, PON e antiossidanti dopo il parto, suggerendo la compromissione di alcune funzioni epatiche negli animali trattati. Tuttavia, la mancanza di qualsiasi effetto sui biomarcatori di funzionalità (bilirubina) e danno epatico (GGT, AST-GOT, ALP) smentisce una reale compromissione delle attività epatiche a seguito del trattamento. Gli effetti positivi nel favorire il recupero delle funzioni del rumine, riducendo la mobilizzazione dei grassi corporei dopo il parto, suggeriscono che l'immunostimolante sia una strategia efficace nella prevenzione dei disturbi metabolici del periodo di transizione.<br>Immune system is made of a variety of cells, molecules and biological processes that interacts to prevent microbial invasions, recognize foreign molecules and eliminate existing sources of cellular injuries to restore tissues to their normal functions once problem has been solved. Innate immunity is the primary defense line against pathogens invasions. Its functioning typically undergoes severe alterations during transition period (TP) of dairy cows. An impairment of polymorphonuclear cells (PMN) functions related to reactive oxygen metabolites (ROM) production, myeloperoxidase (MPO) activity, chemotaxis and phagocytosis has been reported in this phase. Bovine PMN have an altered abundance in mRNA transcripts encoding for such functions between -1 and 2 weeks from calving, in comparison to the level found at 4 weeks after calving for the same genes. The exact cause of immune dysfunctions occurring in peripartum has never been clearly identified. Reduced immune competence could arise from the interaction of different factors affected from the typical peripartum trends (i.e. changes in endocrine asset, limitations of maternal immune responses against the allogeneic conceptus, alterations in energy balance and oxidative stress status). Nevertheless, its duration could be modified when peripartal changes exceed the control of homeorhetic and homeostatic mechanisms, leading to the physiological imbalance (PI) condition. Such a condition could also trigger the inflammatory-like status. It consists in a prepartal raise of pro-inflammatory cytokines (PICs) levels, that is linked to a raise in body temperature at calving, and that typically affects liver metabolism, implying severe losses in hepatic functions and a shift of anabolic priority of the organ in early lactation. The liver produces more α-globulins, known as positive acute phase proteins (APP), i.e. haptoglobin, ceruloplasmin and serum amyloid alpha (SAA). Conversely, it reduces the synthesis of albumin, retinol binding protein, paraoxonase (PON) and lipoproteins, known as negative APP and sequesters minerals, as zinc and iron, from blood flow. Inflammation lead to the activation of PMN, while the reduced immune competence commonly reported in TP has been associated to an opposite effect on leukocytes. Thus, these should be considered as two distinct phenomena, but they could arise from a common cause with a different magnitude and duration. Nutritional strategies to optimize dairy cow’s immunity during TP should be focused on reducing the PI degree related to calving, as this condition could be referred as a common denominator between immune dysfunction and diseases causes. Among such nutritional strategies, the correct management of energy sources to fit with altered requirements should be considered. Furthermore, fatty acids profile of lipid sources administered could also modify immune functions. Finally, the administration of supplementary products exerting antioxidant or anti-inflammatory activities, as well as methyl donors species, could be beneficial for dairy cows immunity in TP. In a wider perspective, although feed additives and nutritional strategy could be effective in mitigate immune alterations, we can conclude that adoption of proper management practices aimed to avoid PI condition in peripartal period of dairy cows could be the most effective strategy to prevent dysfunctions. Three experiments have been designed to elucidate the linkage between sudden changes occurring in peripartum and immune alterations in dairy cows. Throughout such experiments Holstein dairy cows were housed in tied stalls and monitored regularly for body condition score (BCS), body weight (BW), dry matter intake (DMI), milk yield (MY) and rumination time. Blood samples were collected regularly to assess a wide hematochemical profile and to test white blood cell functions through ex-vivo challenges. Furthermore, PMN diapedesis has been tested in-vivo through a carrageenan-skin test and rumen samples were collected at 30 days from calving (DFC). The first experiment was aimed in investigate the main causes of metabolic changes occurring at dry-off and the contribution of MY in such alterations. In fact, dry-off is related to deep changes in feeding behavior, gastro intestinal adaptations, metabolism and immune parameters in high-yielding cow’s career. Indeed, the release of cortisol, signals of systemic inflammation and altered redox balance have been reported immediately after milking interruption, and high MY have a role in aggravating such conditions. In our study, a group of 13 Holstein dairy cows were dried off at 55 days from expected calving day, and regularly monitored from -7 to 34 days from dry-off (DFD). Animals were retrospectively divided in two groups according to their average MY in the last week of lactation, assuming a cut-off of 15 kg*d-1: low MY (6 cows) and high MY (7 cows). Data were submitted to ANOVA using a mixed model for repeated measures including MY at dry-off, time and their interaction as fixed effects. Increased fiber amounts of dry ration reduced DMI and increased rumination time. Leukocytes migration into mammary gland to contribute in the involution phase decreased their abundance in blood at dry-off, and their activity. Such activation of leukocytes at mammary site increased the abundance of nitrogen species in plasma and triggered a systemic inflammation in all the animals, as reflected from increased concentrations of positive and reduced concentrations of negative APPs. Such inflammation impaired liver functions, as suggested from the increased gamma-glutamyl transferase (GGT), bilirubin and alkaline phosphatase (ALP) concentrations. Both the production of nitrogen species and the systemic inflammatory status contributed in the depletion of antioxidant system in blood (thiol groups -SHp-, tocopherol, β-carotene, ferric reducing antioxidant power -FRAP- and oxygen reactive antioxidant capacity -ORAC-). Animals with higher MY at dry-off showed the worst condition, likely for the deeper metabolic changes they faced at milking interruption, and to the greater amount of mammary parenchyma to be reabsorbed. This study highlights the dry-off as a thorny point to manage dairy cows’ health and depose for a relationship between dry-off and immune alteration that typically occurs at calving. The second experiment was aimed in investigate changes occurring in the immune system prior to ketosis onset to elucidate their role in disease occurrence. Thus, a group of 13 Holstein dairy cows were monitored from -48 to 35 DFC and retrospectively divided into 2 groups basing on their plasma BHB levels: lower (CTR; 7 cows) or higher than 1.4 mMol/L (KET; 6 cows). Data were submitted to ANOVA using a mixed model for repeated measures including health status, time and their interaction as fixed effects. KET cows had a greater activation of the immune system prior to calving (higher plasma concentrations of PICs, myeloperoxidase and oxidant species, and greater interferon gamma responses to Mycobacterium avium) impaired liver functions (higher blood concentration of GGT) and lower plasma minerals. High plasma NEFA, BHB and glucose levels in KET cows suggest an insulin resistance status and a marked mobilization of body fat occurring during dry period. They were also associated to reduced DMI around calving and worse negative energy balance in early lactation. This caused in turn reduced MY and increased fat mobilization in early lactation. Impairment of liver function and activation of leukocytes during the dry period accentuated the acute phase response in KET cows after calving (greater concentrations of positive APPs and lower concentration of retinol binding protein), further impairing liver function (higher blood concentrations of glutamate-oxaloacetate transaminase -AST-GOT- and bilirubin). Leukocytes of KET cows had reduced inflammatory functions after an ex vivo stimulation assay (lower production of PICs and greater production of lactate). These alterations on WBC could be driven by the combined action of metabolites related to the mobilization of lipids and of anti-inflammatory actions aimed to prevent over exuberant inflammation. This suggests that prepartal trends of immune parameters be highly related with the likelihood of developing diseases in early lactation. The third experiment consisted in the administration of Omnigen-AF (OAF), an immune stimulant that is effective in increasing leukocytes functions in immunosuppressed animals and in reducing incidence of infectious diseases in early lactating dairy cows. Its mode of action has never been elucidated, and a wider perspective of its metabolic effect could highlight its effectiveness in facing metabolic disorders of transition period also. Thus, a group of 10 Holstein dairy cows were divided into 2 groups: treated group (TRT; 5 cows) received 32.5 g of Omnigen-AF® (Phibro Animal Health Corporation) twice a day (65 g d-1) as top-dress on the morning and afternoon feeds, while control group (CTR; 5 cows) did not receive any supplementation. From -62 to 42 DFC animals were monitored regularly. Data were submitted to ANOVA using a mixed model for repeated measures including treatment, time and their interaction as fixed effects. Administration of OAF at dry-off did not affect BW, BCS, milk yield, milk and rumen fluid composition, and neither affected blood neutrophils concentrations. Nevertheless, it increased rumination time and improved the energy metabolism after calving (lower NEFA and BHB concentrations). TRT cows had an increased lymphocytes abundance at blood level, and their leukocytes had greater efficiency in facing biological stressors during the peripartum (lower lactate production and lower glucose consumption after a challenge with bacterial lipopolysaccharides). Despite these positive effects on immune cells, OAF did not affect the positive APPs concentrations after calving. A reduced abundance of albumin, PON and antioxidants also occurred with OAF after calving, suggesting some impairment of hepatic functions to occur. Nevertheless, the lack of any effect on main biomarkers related to liver function (bilirubin) and liver damage (GGT, AST-GOT, ALP) dismisses a real impairment of liver activities to occur with OAF. Positive effects in favoring the recovery of rumen functions, reducing mobilization of body fats after calving suggest OAF to be an effective strategy in preventing metabolic disorders of transition period.

Il sistema immunitario è costituito da una varietà di cellule, molecole e processi biologici che interagiscono per prevenire le invasioni microbiche, riconoscere le molecole estranee ed eliminare le fonti esistenti di lesioni cellulari, ripristinando le normali funzioni tissutali una volta risolto il problema. L'immunità innata è la prima linea di difesa contro le invasioni di agenti patogeni. Nelle vacche da latte, il suo funzionamento subisce gravi alterazioni durante il periodo di transizione (TP). In questa fase è stata segnalata una compromissione delle funzioni delle cellule polimorfonucleate (PMN) correlate alla produzione di metaboliti reattivi dell'ossigeno (ROM), all’attività della mieloperossidasi (MPO), alla chemiotassi e alla fagocitosi. I PMN bovini hanno un alterata espressione dei geni codificanti per tali funzioni tra -1 e 2 settimane dal parto, rispetto al livello rilevato 4 settimane dopo il parto per gli stessi geni. La causa esatta delle disfunzioni immunitarie che si verificano nel periparto non è mai stata chiaramente identificata. In esse possono contribuire diversi fattori, principalmente imputati alle alterazioni metaboliche tipiche del periparto (cambiamenti nell’assetto ormonale, limitazione della risposta immunitaria materna al fine di mantenere la gravidanza, alterazioni nel bilancio energetico e stato di stress ossidativo). Tuttavia, la durata e l’entità delle disfunzioni immunitarie può aumentare qualora subentri uno stato di squilibrio fisiologico (PI). In tali condizioni, le alterazioni metaboliche del periparto sfuggono al controllo dei meccanismi omeostatici e omeoretici, ed una infiammazione sistemica è la conseguenza frequente di questo squilibrio. Lo stato infiammatorio sistemico è scatenato da un aumento dei livelli di citochine proinfiammatorie (PIC), che è collegato ad un aumento della temperatura corporea al parto, e che tipicamente inficia le funzionalità epatiche, modificando le priorità anaboliche dell'organo in fase di inizio lattazione. A seguito di tale slittamento, il fegato produce più α-globuline, note come proteine positive di fase acuta (+APP), cioè aptoglobina, ceruloplasmina e siero amiloide alfa (SAA). Al contrario, riduce la sintesi di albumina, retinol binding protein (RBP), paraoxonasi (PON) e lipoproteine, note come proteine negative di fase acuta (-APP), e sequestra minerali, quali zinco e ferro, dal flusso ematico. L'infiammazione porta all'attivazione dei PMN, mentre la ridotta competenza immunitaria comunemente riportata in TP è stata associata ad un effetto opposto sui leucociti. Pertanto, questi dovrebbero essere considerati come due fenomeni distinti, ma lo stato di PI potrebbe essere considerato un denominatore comune, direttamente correlato al rischio di patologie in avvio di lattazione. Le strategie nutrizionali per ottimizzare l'immunità delle vacche da latte durante il TP dovrebbero quindi essere focalizzate sulla riduzione del grado di PI correlato al parto. Tra tali strategie nutrizionali, dovrebbe essere presa in considerazione la corretta gestione delle fonti energetiche per adattarle alle variazioni dei fabbisogni. Inoltre, il profilo degli acidi grassi delle fonti lipidiche può contribuire nel modificare le funzioni immunitarie. Infine, la somministrazione di prodotti supplementari con attività antiossidanti o antinfiammatorie, così come di specie donatrici di gruppi metilici, potrebbero essere strategie utili a favorire la funzionalità immunitaria delle bovine durante il TP. In una prospettiva più ampia, sebbene strategie nutrizionali e supplementi possano talora mitigare le alterazioni immunitarie, possiamo concludere che l'adozione di pratiche volte a minimizzare il PI durante il periodo di transizione sia la strategia più efficace per prevenire le disfunzioni. Al fine di chiarire il legame tra le alterazioni che si verificano nel periparto e le disfunzioni immunitarie delle bovine da latte sono stati condotti tre esperimenti. Bovine di razza frisona sono state alloggiate in poste individuali a stabulazione fissa e monitorate regolarmente per le condizioni corporee (BCS), il peso (BW), l'assunzione di alimenti (DMI), la produzione di latte (MY) e il tempo di ruminazione. Campioni di sangue sono stati raccolti regolarmente per valutare un ampio profilo ematochimico e per testare le funzioni dei globuli bianchi mediante stimolazioni ex-vivo. Inoltre, la diapedesi dei PMN è stata testata in vivo mediante test della carragenina e sono stati raccolti campioni di rumine a 30 giorni dal parto (DFC). Il primo esperimento era volto a chiarire le cause dei cambiamenti metabolici che si verificano al momento della messa in asciutta, ed il contributo del livello produttivo in tali alterazioni. Infatti, i profondi cambiamenti nell’alimentazione, gli adattamenti gastrointestinali, del metabolismo e dei parametri immunitari che si verificano nelle bovine alla messa in asciutta sono note scatenare il rilascio di cortisolo, indurre segnali di infiammazione sistemica ed alterare il bilancio redox. Produzioni di latte elevate al momento della messa in asciutta hanno un ruolo nell'aggravare tali condizioni. Nel nostro studio, un gruppo di 13 bovine è stato asciugato a 55 giorni dalla data prevista per il parto. Gli animali sono stati divisi in due gruppi in base alla produzione media dell'ultima settimana di lattazione, assumendo un cut-off di 15 kg * d-1: bassa (LM; 6 animali) e alta produzione (HM; 7 animali). I dati sono stati sottoposti ad ANOVA utilizzando un modello per misure ripetute, assumendo il livello produttivo al termine della lattazione, il tempo e la loro interazione come effetti fissi. L'aumento delle quantità di fibra nella razione di asciutta ha ridotto la DMI e aumentato il tempo di ruminazione. La migrazione dei leucociti nella ghiandola mammaria per contribuire alla fase di involuzione ha ridotto la loro abbondanza nel sangue e aumentato la loro attività. Tale attivazione dei leucociti nella mammella ha aumentato l'abbondanza di specie reattive dell’azoto nel plasma e innescato un'infiammazione sistemica in tutti gli animali (aumento delle +APP e riduzione delle -APP). Tale infiammazione ha compromesso le funzioni epatiche (aumento delle concentrazioni di gamma-glutamil transferasi -GGT- bilirubina e fosfatasi alcalina -ALP-). Sia la produzione di specie dell’azoto che lo stato infiammatorio sistemico hanno contribuito all'esaurimento degli antiossidanti circolanti (gruppi tiolici -SHp-, tocoferolo, β-carotene, potere antiossidante ferrico riducente -FRAP- e capacità antiossidante contro specie reattive dell'ossigeno -ORAC-). Gli animali con una produzione più elevata alla messa in asciutta hanno mostrato la peggiore condizione, probabilmente per i più profondi cambiamenti metabolici che hanno affrontato dopo l'interruzione delle mungiture, e per la fase involutiva verosimilmente più dispendiosa. Questo studio evidenzia la messa in asciutta come una fase critica per gestire la salute delle vacche da latte, e suggerisce un potenziale legame della messa in asciutta con le alterazioni delle funzioni immunitarie che si verificano nel periparto. Nel secondo esperimento si sono cercati di identificare i cambiamenti del sistema immunitario che precedono l'insorgenza della chetosi, al fine di chiarire il loro ruolo nella comparsa della malattia. Pertanto, 13 bovine sono state monitorate tra -48 e 35 DFC e suddivise in due gruppi sulla base dei loro livelli plasmatici di beta idrossibutirrato (BHB): inferiore (CTR, 7 animali) o superiore a 1,4 mMol / L (KET; 6 animali). I dati sono stati sottoposti ad ANOVA utilizzando un modello per misure ripetute, assumendo lo stato di salute, il tempo e la loro interazione come effetti fissi. Le vacche KET hanno avuto una maggiore attivazione del sistema immunitario prima del parto (maggiori concentrazioni plasmatiche di PIC, MPO e specie ossidanti e maggiori produzione di interferone gamma in risposta alla stimolazione con Mycobacterium avium) alterazioni della funzionalità epatica (più alta concentrazione sanguigna di GGT) e minori minerali plasmatici. Elevati livelli plasmatici di NEFA, BHB e glucosio nelle vacche KET suggeriscono uno stato di insulinoresistenza e una marcata mobilizzazione del grasso corporeo durante il periodo di asciutta. Tali andamenti dei parametri relativi al metabolismo energetico durante l’asciutta sono stati associati alla riduzione della DMI al momento del parto e al peggioramento del bilancio energetico negativo ad avvio lattazione. Ciò ha causato a sua volta una riduzione di MY e accresciuto ulteriormente la mobilizzazione dei grassi in avvio di lattazione. Compromissione della funzionalità epatica e attivazione dei leucociti durante il periodo di asciutta hanno determinato una marcata risposta infiammatoria di fase acuta nelle vacche KET dopo il parto (maggiori concentrazioni di +APP minori concentrazioni di RBP), ed ulteriormente compromesso la funzionalità epatica (maggiori concentrazioni di glutammato-ossalacetato transaminasi -AST-GOT- e bilirubina). I leucociti delle vacche KET hanno mostrato ridotte funzioni infiammatorie dopo stimolazione ex-vivo con lipopolisaccaridi batterici (minore produzione di PIC e maggiore produzione di lattato). Queste alterazioni potrebbero essere guidate dall'azione combinata dei metaboliti legati alla mobilizzazione dei lipidi e delle azioni antinfiammatorie volte a prevenire un'infiammazione eccessiva. Ciò suggerisce che le alterazioni dei parametri immunitari osservate prima del parto siano altamente correlate con la probabilità di sviluppare chetosi in avvio di lattazione. Nel terzo esperimento è stato somministrato un prodotto immunostimolante dalla comprovata efficacia nel migliorare le funzioni leucocitarie degli animali immunodepressi e nel ridurre l'incidenza delle malattie infettive delle bovine ad inizio lattazione. La sua modalità di azione non è mai stata chiarita, e un’indagine approfondita sul suo effetto metabolico potrebbe evidenziarne l’efficacia anche nei confronti dei disordini metabolici del periodo di transizione. Pertanto, un gruppo di10 bovine è stato monitorato da -62 a 42 DFC. Il gruppo trattato (TRT, 5 animali) ha ricevuto 32,5 g di Omnigen-AF® (Phibro Animal Health Corporation) due volte al giorno (65 g d-1), mentre il gruppo di controllo (CTR, 5 animali) non ha ricevuto alcun supplemento. I dati sono stati sottoposti ad ANOVA utilizzando un modello per misure ripetute, assumendo il trattamento, il tempo e la loro interazione come effetti fissi. La somministrazione dell’immunostimolante alla messa in asciutta non ha influenzato BW, BCS, MY, composizione del latte e del fluido ruminale e nemmeno modificato la concentrazione di neutrofili del sangue. Tuttavia, ha aumentato il tempo di ruminazione e migliorato il metabolismo energetico dopo il parto (concentrazioni di NEFA e BHB inferiori). Le bovine TRT avevano maggiori concentrazioni ematiche di linfociti e i loro leucociti avevano una maggiore efficienza nel rispondere alla stimolazione con lipopolisaccaridi batterici (produzione di lattato inferiore e minore consumo di glucosio). Nonostante questi effetti positivi sulle cellule immunitarie, l'immunostimolante non ha influenzato le concentrazioni di +APP dopo il parto. Inoltre, l’immunostimolante ha ridotto le concentrazioni di albumina, PON e antiossidanti dopo il parto, suggerendo la compromissione di alcune funzioni epatiche negli animali trattati. Tuttavia, la mancanza di qualsiasi effetto sui biomarcatori di funzionalità (bilirubina) e danno epatico (GGT, AST-GOT, ALP) smentisce una reale compromissione delle attività epatiche a seguito del trattamento. Gli effetti positivi nel favorire il recupero delle funzioni del rumine, riducendo la mobilizzazione dei grassi corporei dopo il parto, suggeriscono che l'immunostimolante sia una strategia efficace nella prevenzione dei disturbi metabolici del periodo di transizione.<br>Immune system is made of a variety of cells, molecules and biological processes that interacts to prevent microbial invasions, recognize foreign molecules and eliminate existing sources of cellular injuries to restore tissues to their normal functions once problem has been solved. Innate immunity is the primary defense line against pathogens invasions. Its functioning typically undergoes severe alterations during transition period (TP) of dairy cows. An impairment of polymorphonuclear cells (PMN) functions related to reactive oxygen metabolites (ROM) production, myeloperoxidase (MPO) activity, chemotaxis and phagocytosis has been reported in this phase. Bovine PMN have an altered abundance in mRNA transcripts encoding for such functions between -1 and 2 weeks from calving, in comparison to the level found at 4 weeks after calving for the same genes. The exact cause of immune dysfunctions occurring in peripartum has never been clearly identified. Reduced immune competence could arise from the interaction of different factors affected from the typical peripartum trends (i.e. changes in endocrine asset, limitations of maternal immune responses against the allogeneic conceptus, alterations in energy balance and oxidative stress status). Nevertheless, its duration could be modified when peripartal changes exceed the control of homeorhetic and homeostatic mechanisms, leading to the physiological imbalance (PI) condition. Such a condition could also trigger the inflammatory-like status. It consists in a prepartal raise of pro-inflammatory cytokines (PICs) levels, that is linked to a raise in body temperature at calving, and that typically affects liver metabolism, implying severe losses in hepatic functions and a shift of anabolic priority of the organ in early lactation. The liver produces more α-globulins, known as positive acute phase proteins (APP), i.e. haptoglobin, ceruloplasmin and serum amyloid alpha (SAA). Conversely, it reduces the synthesis of albumin, retinol binding protein, paraoxonase (PON) and lipoproteins, known as negative APP and sequesters minerals, as zinc and iron, from blood flow. Inflammation lead to the activation of PMN, while the reduced immune competence commonly reported in TP has been associated to an opposite effect on leukocytes. Thus, these should be considered as two distinct phenomena, but they could arise from a common cause with a different magnitude and duration. Nutritional strategies to optimize dairy cow’s immunity during TP should be focused on reducing the PI degree related to calving, as this condition could be referred as a common denominator between immune dysfunction and diseases causes. Among such nutritional strategies, the correct management of energy sources to fit with altered requirements should be considered. Furthermore, fatty acids profile of lipid sources administered could also modify immune functions. Finally, the administration of supplementary products exerting antioxidant or anti-inflammatory activities, as well as methyl donors species, could be beneficial for dairy cows immunity in TP. In a wider perspective, although feed additives and nutritional strategy could be effective in mitigate immune alterations, we can conclude that adoption of proper management practices aimed to avoid PI condition in peripartal period of dairy cows could be the most effective strategy to prevent dysfunctions. Three experiments have been designed to elucidate the linkage between sudden changes occurring in peripartum and immune alterations in dairy cows. Throughout such experiments Holstein dairy cows were housed in tied stalls and monitored regularly for body condition score (BCS), body weight (BW), dry matter intake (DMI), milk yield (MY) and rumination time. Blood samples were collected regularly to assess a wide hematochemical profile and to test white blood cell functions through ex-vivo challenges. Furthermore, PMN diapedesis has been tested in-vivo through a carrageenan-skin test and rumen samples were collected at 30 days from calving (DFC). The first experiment was aimed in investigate the main causes of metabolic changes occurring at dry-off and the contribution of MY in such alterations. In fact, dry-off is related to deep changes in feeding behavior, gastro intestinal adaptations, metabolism and immune parameters in high-yielding cow’s career. Indeed, the release of cortisol, signals of systemic inflammation and altered redox balance have been reported immediately after milking interruption, and high MY have a role in aggravating such conditions. In our study, a group of 13 Holstein dairy cows were dried off at 55 days from expected calving day, and regularly monitored from -7 to 34 days from dry-off (DFD). Animals were retrospectively divided in two groups according to their average MY in the last week of lactation, assuming a cut-off of 15 kg*d-1: low MY (6 cows) and high MY (7 cows). Data were submitted to ANOVA using a mixed model for repeated measures including MY at dry-off, time and their interaction as fixed effects. Increased fiber amounts of dry ration reduced DMI and increased rumination time. Leukocytes migration into mammary gland to contribute in the involution phase decreased their abundance in blood at dry-off, and their activity. Such activation of leukocytes at mammary site increased the abundance of nitrogen species in plasma and triggered a systemic inflammation in all the animals, as reflected from increased concentrations of positive and reduced concentrations of negative APPs. Such inflammation impaired liver functions, as suggested from the increased gamma-glutamyl transferase (GGT), bilirubin and alkaline phosphatase (ALP) concentrations. Both the production of nitrogen species and the systemic inflammatory status contributed in the depletion of antioxidant system in blood (thiol groups -SHp-, tocopherol, β-carotene, ferric reducing antioxidant power -FRAP- and oxygen reactive antioxidant capacity -ORAC-). Animals with higher MY at dry-off showed the worst condition, likely for the deeper metabolic changes they faced at milking interruption, and to the greater amount of mammary parenchyma to be reabsorbed. This study highlights the dry-off as a thorny point to manage dairy cows’ health and depose for a relationship between dry-off and immune alteration that typically occurs at calving. The second experiment was aimed in investigate changes occurring in the immune system prior to ketosis onset to elucidate their role in disease occurrence. Thus, a group of 13 Holstein dairy cows were monitored from -48 to 35 DFC and retrospectively divided into 2 groups basing on their plasma BHB levels: lower (CTR; 7 cows) or higher than 1.4 mMol/L (KET; 6 cows). Data were submitted to ANOVA using a mixed model for repeated measures including health status, time and their interaction as fixed effects. KET cows had a greater activation of the immune system prior to calving (higher plasma concentrations of PICs, myeloperoxidase and oxidant species, and greater interferon gamma responses to Mycobacterium avium) impaired liver functions (higher blood concentration of GGT) and lower plasma minerals. High plasma NEFA, BHB and glucose levels in KET cows suggest an insulin resistance status and a marked mobilization of body fat occurring during dry period. They were also associated to reduced DMI around calving and worse negative energy balance in early lactation. This caused in turn reduced MY and increased fat mobilization in early lactation. Impairment of liver function and activation of leukocytes during the dry period accentuated the acute phase response in KET cows after calving (greater concentrations of positive APPs and lower concentration of retinol binding protein), further impairing liver function (higher blood concentrations of glutamate-oxaloacetate transaminase -AST-GOT- and bilirubin). Leukocytes of KET cows had reduced inflammatory functions after an ex vivo stimulation assay (lower production of PICs and greater production of lactate). These alterations on WBC could be driven by the combined action of metabolites related to the mobilization of lipids and of anti-inflammatory actions aimed to prevent over exuberant inflammation. This suggests that prepartal trends of immune parameters be highly related with the likelihood of developing diseases in early lactation. The third experiment consisted in the administration of Omnigen-AF (OAF), an immune stimulant that is effective in increasing leukocytes functions in immunosuppressed animals and in reducing incidence of infectious diseases in early lactating dairy cows. Its mode of action has never been elucidated, and a wider perspective of its metabolic effect could highlight its effectiveness in facing metabolic disorders of transition period also. Thus, a group of 10 Holstein dairy cows were divided into 2 groups: treated group (TRT; 5 cows) received 32.5 g of Omnigen-AF® (Phibro Animal Health Corporation) twice a day (65 g d-1) as top-dress on the morning and afternoon feeds, while control group (CTR; 5 cows) did not receive any supplementation. From -62 to 42 DFC animals were monitored regularly. Data were submitted to ANOVA using a mixed model for repeated measures including treatment, time and their interaction as fixed effects. Administration of OAF at dry-off did not affect BW, BCS, milk yield, milk and rumen fluid composition, and neither affected blood neutrophils concentrations. Nevertheless, it increased rumination time and improved the energy metabolism after calving (lower NEFA and BHB concentrations). TRT cows had an increased lymphocytes abundance at blood level, and their leukocytes had greater efficiency in facing biological stressors during the peripartum (lower lactate production and lower glucose consumption after a challenge with bacterial lipopolysaccharides). Despite these positive effects on immune cells, OAF did not affect the positive APPs concentrations after calving. A reduced abundance of albumin, PON and antioxidants also occurred with OAF after calving, suggesting some impairment of hepatic functions to occur. Nevertheless, the lack of any effect on main biomarkers related to liver function (bilirubin) and liver damage (GGT, AST-GOT, ALP) dismisses a real impairment of liver activities to occur with OAF. Positive effects in favoring the recovery of rumen functions, reducing mobilization of body fats after calving suggest OAF to be an effective strategy in preventing metabolic disorders of transition period.

No presente trabalho analisou-se um total de 101 indivíduos, sendo 26 não infectados e 75 infectados pelo HIV e classificados de acordo com o Walter Reed Army Institute (28 pacientes WR 1, 31 pacientes WR 2 e 16 pacientes WR 3/4). 05 indivíduos infectados pelo HIV apresentaram, nos estágios iniciais, uma diminuição progressiva do peso corporal, dos níveis séricos de uréia, albumina, colesterol total, HOL colesterol e LOL colesterol. Já os níveis séricos de proteínas totais, globulinas, IgG, IgA, &#945;1 glicoproteína ácida, haptoglobina e as atividades enzimáticas da AST e da LD apresentaram elevação nos indivíduos infectados e em conseqüência da evolução da infecção. Os triglicérides demonstraram apenas tendência para aumento dos níveis séricos nos indivíduos estadiados como WR3/4. Os níveis de ferro sérico encontraram-se diminuídos nos indivíduos estadiados como WR 3/4, enquanto que a concentração de transferrina apresentou-se diminuída apenas no Grupo WR 2. Houve uma tendência para a elevação progressiva dos níveis médios de ferritina com a evolução da doença. Nenhuma alteração foi verificada nos níveis de proteína \"C\" reativa. A EC-SOO apresentou diminuição dos níveis de atividade nos indivíduos infectados pelo HIV, enquanto que em células mononucleares a SOD apresentou atividade diminuída nos indivíduos estadiados como WR 3/4. A GSH-Px não apresentou alteração de sua atividade em decorrência da infecção pelo HIV. Os níveis plasmáticos do &#945;-tocoferol e do ascorbato apresentaram tendência para diminuição, enquanto o &#946;-caroteno não apresentou alteração nos grupos estudados. Estes resultados sugerem que a haptoglobina, as globulinas e a IgA podem ser utilizadas para a avaliação da evolução da infecção pelo HIV. Por outro lado, os níveis dos constituintes do sistema de defesa antioxidante analisados indicam que os indivíduos soropositivos encontram-se menos protegidos contra a ação de espécies reativas de oxigênio, o que favoreceria a presença de um estresse oxidativo e a replicação viral.<br>A total number of 101 individuals, including 26 controls and 75 patients classified according to the Walter Reed Army Institute (28 WR 1, 31 WR 2 and 16 WR 3/4) was studied. HIV infected individuals presented, during the early stages, a progressive reduction of body weigth, as well as urea, albumin, total cholesterol, HDL cholesterol and LDL cholesterol in blood serum. However, increased serum levels of total protein, globulin, IgG, IgA, &#945;1 acid glycoprotein, haptoglobin, AST and LD were observed in HIV infected individuals during the evolution of infection. Decreased serum iron and a trend for increasing triglyceride was shown only for those individuals classified as WR 3/4. Transferrin was diminished only in the WR 2 group. A trend for enhancing serum ferritin following the progession of HIV infection was also observed. No alteration was observed on the levels of reactive \"C\" protein. Decreased EC-SOD activities were observed in HIV infected individuals as compared to controls, whereas in mononuclear cells the SOD activity was diminished only in WR 3/4 patients. HIV infection did not alter GSH-Px activity. A trend for decreasing &#945;-tocopherol and ascorbate plasma levels was shown during the evolution of HIV infected patients, while no difference was observed for &#946;-carotene levels in the studied groups. The above results suggest that haptoglobin, globulins and IgA can be used to assess the evolution of the HIV infection. Moreover, the decreased levels of the antioxidant defense system components observed in HIV infected patients may indicate that they are under an oxidative stress that could favor HIV replication.

L'obesitat mòrbida (índex de massa corporal superior a 40 kg/m2) és una malaltia amb una elevada prevalença a la societat occidental, que es troba associada amb nombroses comorbilitats (diabetes, dislipèmia, fetge gras, apnea obstructiva del son, malalties cardiovasculars, determinats tipus de càncer, etc). Són nombrosos els tractaments que s'han proposat per combatre l'obesitat mòrbida, des de modificacions en l'estil de vida (dieta i exercici físic), passant per tractaments farmacològics o la cirurgia bariàtrica en les seves diferents modalitats. De tots ells, només la darrera ha resultat un mètode efectiu per tractar la malaltia en aquest tipus de pacients, alhora que s'ha vist que també millora moltes de les comorbilitats que l'acompanyen. L’objectiu del treball és estudiar des d'un punt de vista antropomètric, bioquímic i sempre que sigui possible genètic, les alteracions del metabolisme lipídic i glucidic en l'obès mòrbid i la seva evolució fins un any després d'un bypass gàstric en Y-de-Roux, dut a terme a l'Hospital Universitari de la Vall d'Hebron, centrant-nos en l'anàlisi del plasma, el teixit adipós blanc subcutani i visceral i el fetge. Amb aquest estudi hem pogut concloure que: 1. El teixit adipós dels obesos es troba hipertrofiat i inflamat. El dèficit de capacitat d'emmagatzematge del teixit adipós, reflectit per la dislipèmia aterogènica que pateix aquest tipus de pacient, faria que els lípids s'acumulessin en altres teixits, promovent d'aquesta manera l'aparició de les comorbilitats metabòliques que acompanyen l'obesitat. 2. L'esteatosi hepàtica present en els obesos es podria veure afavorida per l'expressió anòmala d’LPL en aquest teixit. En l'activació d'aquesta expressió hi podria intervenir TNF-alfa. 3. El bypass gàstric en Y-de-Roux és una tècnica de cirurgia bariàtrica útil per aconseguir reduccions de pes sostingudes en el temps en aquest tipus de pacients. Aquesta reducció es deu principalment a la pèrdua de greix corporal. 4. La reducció de l'excés de pes comporta la millora de les comorbilitats que acompanyen l'obesitat mòrbida: a. La millora de la insulinoresistència s'aconsegueix molt abans d'obtenir la pèrdua de l'excés de pes desitjada. b. El perfil lipídic dels individus es normalitza en la majoria dels casos, de manera que la dislipèmia remet i el risc cardiovascular que comporta disminueix. Aquesta millora es podria deure, en part, a la normalització gairebé total de la funcionalitat del teixit adipós i el fetge.<br>Morbid obesity (BMI greater than 40 kg/m2) is a disease with a high prevalence in Western society, and is associated with numerous comorbidities (diabetes, hyperlipidemia, fatty liver disease, obstructive sleep apnea, cardiovascular diseases, certain cancers, etc.) Many treatment have been proposed to combat morbid obesity, from lifestyle changes (diet and physical exercise) or drug treatments to bariatric surgery. Of these, only the latter has been proved as an effective way to treat the disease in these patients. Moreover, it also improves many of the comorbidities accompaning morbid obesity. We want to study from an anthropometric, biochemical and whenever it has been possible genetic point of view the alterations in lipid and carbohydrate metabolism in the morbidly obese and their evolution at one year follow-up period after an Roux-en-Y gastric bypass conducted at the Hospital Vall d'Hebron, focusing on the analysis of plasma, subcutaneous and visceral white adipose tissue and liver. In this study we have concluded that: 1. Adipose tissue of obese is hypertrophied and inflamed. The shortage of adipose tissue storage capacity, reflected by the atherogenic dyslipidemia observed in this type of subjects, would make that lipids accumulate in other tissues, thus promoting the emergence of metabolic comorbidities that accompany the obesity. 2. The present hepatic steatosis in obese could be favored by the abnormal expression of LPL in this tissue. In the activation of this expression could mediate TNF-alpha. 3. Roux-en-Y gastric bypass is a surgery technique useful for obtain sustained weight reductions over time in morbidly obese subjects. Principally, this reduction is due to the loss of body fat. 4. Reducing excess weight leads to an improvement in comorbidities: a. The improvement in insulinoresistance is achieved much earlier than the excess weight loss desired. b. The lipid profile of individuals is normalized in most cases, so dyslipidemia and cardiovascular risk involved decrease. This improvement could be due in part to the almost complete normalization of the adipose tissue and liver functions.

Newborn screening programs measure analyte levels in neonatal blood spots to identify individuals at high risk of disease. Carnitine and acylcarnitine levels are primary markers used in the detection of fatty acid oxidation disorders. These analytes may be influenced by certain pre/perinatal or newborn screening related factors. The primary objective of this study was to explore the association between these characteristics and levels of blood carnitines and acylcarnitines in the newborn population. The study was composed of two parts: a systematic review and a clinical database analysis of existing newborn screening data. The systematic review results suggested considerable variability across studies in the presence and directionality of associations between analyte levels and birth weight, gestational age, age at time of blood spot collection, type of sample, and storage time. Sex was not significantly associated with carnitine or acylcarnitine levels in neonatal blood. We identified a need to more fully investigate a potential interaction between gestational age and birth weight in regard to analyte levels. The secondary data analyses indicated a statistically significant relationship between analyte levels and all perinatal / infant and newborn screening related factors of interest, but effect sizes were generally small. The interaction between gestational age and birth weight was significant in all models; when further explored through graphical analysis with conditional means, extremely premature neonates stood out as having distinct analyte patterns in relation to birth weight. Variation in the ratio of total acylcarnitine to free carnitine was better accounted for by the perinatal and newborn factors than was variation in any individual carnitine or acylcarnitine, indicating that proportions of carnitine and acylcarnitines may be more important in understanding an individual’s metabolic functioning than individual analyte levels. A low proportion of variation was explained in all multivariate models, supporting the use of universal algorithms in newborn screening and suggesting the need for further large scale empirical research targeted at previously unaccounted for perinatal factors such as birth stress.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2007<br>Cerebrovascular disease and coronary heart disease (CHD) are of the most important causes of morbidity and mortality amongst South Africans. The risk factor prevalence for stroke and CHD becomes altered by changes in lifestyle, including diet. In general it is suggested that lifestyle management should be the first choice when having to treat patients with increased cardiovascular risk. The prudent low-fat, high-fibre diet is regarded as an apparently healthy diet. It is suspected that this diet is effective for the control of known coronary risk factors as well as raised clotting factors. Research studies have shown the addition of dietary fibre to the diet as a promising therapeutic agent for the limited control of known coronary risk factors. The physiological effects of dietary fibre in humans are significantly influenced by the degree to which fibre is fermented in the colon. Fermentation results in the production of short-chain fatty acids (SCFAs); acetate, propionate and butyrate. The aim of this study was to examine the possible effects of different combinations of short-chain fatty acids on some metabolic risk markers. In this study a group of westernised African male volunteers was recruited and randomly assigned to three groups. Group one received a placebo. Group two received a supplement containing 50% acetate and 50% propionate. Group three received a SCFA supplement in the ratio of 70% acetate, 15% propionate and 15% butyrate. Supplementation was sustained for a period of six weeks. Blood samples were drawn during the different visits. At baseline the study group represented a group of black African men without any apparent metabolic or physical abnormalities. All measured variables fell within the normal range. In the placebo group, there was a statistically significant decrease in plasma fibrinogen levels from baseline to the end of supplementation. In the acetatepropionate supplement study group a statistically significant decrease in factor VIII (from 91.1 ± 11.2 to 90.9 ± 8.3%, respectively), and ATIII (from 114.3 ± 13.1 to 108.34 ± 9.5%), as well as a statistically significant decrease in low-density lipoprotein cholesterol (LDL-C) from 3.10 ± 0.79 to 2.64 ± 0.73 mmol/L. The significant increase in %HDL-C from 26.3 ± 6.5 to 30.2 ± 9.3% should also be noted. Both triglycerides (8%) and plasma fibrinogen (2%) showed a statistically significant increase. However, these changes are of no clinical significance. For the high-acetate supplement study group (with the addition of butyrate), a statistically significant decrease in factor VII (from 102.5 ± 13.7 to 101.1 ± 6.4%), VIII (from 92.6 ± 12.8 to 87.6 ± 6.0%), ATIII (from 109.2 ± 16.0 to 103.0 ± 9.9%) as well as fibrin monomer concentration (from 13.9 ± 2.2 to 12.1 ± 3.6 mg/L), were measured. Fibrin network compaction increased significantly from 14.2 ± 4.6 to 13.7 ± 4.0%. Other changes include a statistically significant increase in the serum-TC of 4.2%. From the results it is evident that the acetate-propionate supplement, with exclusion of butyrate, has a beneficial effect on metabolic parameters when compared to a highacetate- propionate supplement. The results do provide evidence of a possible therapeutic application for the propionate-acetate containing supplement. The specific mechanism should, however, still be investigated. It can be concluded from this study that acetate, propionate and butyrate each have different effects on human metabolism. It is evident that the use of a mixture of acetate and propionate may have a beneficial effect on patients at risk of developing CVD. Further studies that investigate the optimum ratio of these two products may lead to the development of a naturally derived therapeutic product for the prevention or treatment of CVD in black African men, as well as the population at large.

THESIS SUBMITED IN FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF TECHNOLOGY OF BIOMEDICAL TECHNOLOGTY IN THE FACULTY OF HEALTH AND WELLNESS SCIENCES AT THE CAPE PENINSULA UNIVERSITY OF TECHNOLOGY SUPERVISORS: PROF T.E. MATSHA PROF R.T. ERASMUS DR A. ZEMLIN SUBMITED DECEMBER 2013<br>Introduction: Cardiovascular diseases (CVD) have become the leading cause of morbidity and mortality amongst the global population. Originally thought to be a health burden of high income countries, the prevalence is rapidly increasing in developing countries. For example, in 2008, an estimated 17.3 million died from CVD, and 80% of these (13.8 mil) were from low to middle income countries. Epidemiological data on CVD in Africa is scanty and of poor quality and national vital registration is available in only 5% of Africa’s 53 countries. Furthermore, data on CVD risk amongst the South African population and specifically the mixed ancestry community is poorly described. The increasing global population of people with CVD has been largely attributed to increasing rates of determinants and risk factors which include obesity, metabolic syndrome (MetS), type 2 diabetes mellitus (DM) and chronic kidney diseases (CKD). The prevalence of DM in South Africa is known to be on the rise with more affected communities being South African Asians followed by coloureds. Aims and objectives: The aim of this study was to determine the CVD risk profile of the Bellville South community during a baseline and three year follow-up study, by assessment of known risk factors, MetS, type 2 DM, obesity and CKD. Methods: Participants for this study were drawn from an urban community of the Bellville South suburb of Cape Town. At baseline (January 2008 and March 2009) 946 individuals aged 16 to 95 participated. All participants received a standardized interview and physical examination during which anthropometric measurements were performed three times and their average used for analysis: weight (kg), height (cm), waist (cm) and hip (cm) circumferences. Body Mass Index (BMI) was calculated as weight per square metre (kg/m2). A blood sample was obtained from all participants after an overnight fast for the determination of biochemical profiles: glucose, glycated haemoglobin, creatinine, total cholesterol, high density lipoprotein cholesterol (HDL-C), triglycerides and low density lipoprotein cholesterol (LDL-C) which was calculated using Friedewald’s formula. Kidney function test was assessed through estimated glomerular filtration rate (eGFR) using the cockcroft-Gault and MDRD equations. Blood pressure was measured according to the World Health Organisation (WHO) guidelines. Participants with no history of doctor diagnosed DM underwent a 75 g oral glucose tolerance test as recommended by the WHO. Metabolic syndrome was determined using JIS, NCEP ATPIII and IDF criteria. The follow-up examination was conducted in 2011 (3 years from vii baseline) using similar procedures. A total of 198 participants formed the follow-up cohort whose measurements were compared to those of the baseline. Finally, the prediction and processes/progression of the risk factors were determined. Results: At both baseline and follow-up studies, females had a higher BMI compared to their male counterparts. The crude prevalence of type 2 DM, including the previously diagnosed type 2 DM was 28.59% (age-adjusted = 33.5%, 95%CI: 30.01 – 36.92), and that of undiagnosed type 2 DM was 17.8% (age-adjusted = 12.4%, 95%CI: 9.8 – 14.8). The overall prevalence of CKD was 28.7% (269) and was higher in females (31.4%) compared to 20.2% in males. MetS was present in 46.5% of the participants. Gender-specific prediction for CVD risk calculated using the 30-year CVD interactive risk calculator showed that high CVD risk was present in normoglycaemic and younger subjects (under 35 years). At follow-up, the cumulative incidence of progression in glucose tolerance status was: 16.2% (32 participants including 11 with new-onset diabetes), and increased in a stepwise fashion with the number of components of MetS. Between baseline and 3-year evaluation glomerular filtration rate (eGFR) increased by 8.7 ml/min (95% confidence interval: 6.9-10.7), reflecting variables trajectories across baseline strata of kidney functions. Conclusion: Given the findings of this study and the estimated increases in the determinants and risk factors of CVD in the mixed ancestry population of South Africa this trend may continue to worsen if current trajectories do not change.

A dissertation submitted to the Faculty of medicine of the university of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Masters of science Johannesburg 1991<br>Carnitine plays a role in the transport of activated long chain fatty acyl groups from the site of activation to the site of beta-oxidation in the mitochondria. Endogenous synthesis of carnitine from the amino acids methionine and lysine, takes place mainly in the liver. From there free and acyl carnitine are released into the blood and transported to other tissues.A few studies have indicated that poor nutritional status may lead to carnitine deficiency.[Abbreviated Abstract. Open document to view full version]<br>GR2017

Trabalho final do 6º ano médico com vista à atribuição do grau de mestre (área científica de cirurgia plástica) no âmbito do ciclo de estudos de Mestrado Integrado em Medicina.<br>Introdução A doença de Madelung (DM) é uma entidade rara, caracterizada pelo aparecimento de volumosas massas de tecido adiposo, que afectam várias áreas corporais, mais frequentemente a região cervical, cintura escapular e região dorsal. Background Madelung disease (MD) is a rare entity characterized by multiple voluminous lipomas affecting several body areas, most frequently the neck, trunk, limbs and head region. Methods We made a search through the database PubMed on October 2014, selecting articles published on the last five years and their respective bibliographies with the keywords Madelung disease and lipomatosis. Afterwards, we analysed a series of 62 patients with MD operated on the last 11 years at the Plastic Surgery department, at Centro Hospitalar e Universitário de Coimbra. Results Of the 62 patients reviewed, 59 were male and 3 female. Their ages ranged from 34 to 76 years old with a mean age of 54,2 years. Aesthetic deformities and functional limitations, namely difficulties with dressing, were the most frequent. According to Enzi classification, 59 patients fit the type 1 phenotype, and three patients type 2. The lipomas were more frequently observed at the neck region (n=79 cases), followed by: the thorax (n=10, five of them with gynecomastia); abdominal (n=13); brachial (n=7); dorsal (n=7, four of them interscapular); axillar, suprapubic, lumbar and chin (n=3); deltoid and facial (n=2); occipital, supraclavicular, suprasternal and inguinal (n=1). A history of alcoholism was found in 91,9% (n=57), with 11,3% (n=7) having some sort of hepatic disease. We found dyslipidaemia in 19,1% (n=12) and hyperuricemia in 7,9% (n=5). The diagnosis of hypertension was made in 17,5% (n=11) of the patients. From the 132 surgical interventions made, 119 were dermolipectomies (with 13 abdominoplasties), eight liposuctions and 5 mastectomies. We registered complications in three interventions: hematoma (n=2) and a suture dehiscence (n=1). The relapse rate was 18,2% (n=24), most of them on the cervical region. Conclusion MD is a rare entity but responsible for severe deformities, with great aesthetic and functional impact. Although the medical literature only describes about 200 to 300 cases, this disease seems to be quite frequent at the Plastic Surgery department in Centro Hospitalar e Universitário de Coimbra. According to the literature, this disease often affects white middle-aged men from the Mediterranean population, with an estimated incidence of 1:25.000; men twenty times fold as women. The series supports the association with alcoholism, present in 91,9% of patients, although the exact etiology for the disease is unknown. The most frequently affected areas are the neck, shoulder, dorsal region and proximal segments of the limbs, however lipomas can appear in many other locations, such as the abdomen, retroauricular, submandibular, or mammary regions. As the etiology of the disease is not clear, surgery is currently the only effective therapy. In our opinion, dermolipectomy has great aesthetic advantages. Furthermore, it is technically more effective, and so liposuction should be restricted to selected cases, with very diffuse lipomatosis. Hematomas are the most frequent complication. Relapses are common, (18,2% interventions), even with alcohol abstinence. Materiais e métodos A revisão bibliográfica foi realizada em Outubro de 2014, através da base da PubMed, filtrando artigos publicados nos últimos 5 anos e respectivas bibliografias, seleccionados com as palavras-chave madelung disease e lipomatosis. Posteriormente, fizemos a análise de uma casuística de 62 doentes portadores de DM operados no Serviço de Cirurgia Plástica do Centro Hospitalar e Universitário de Coimbra, nos últimos 11 anos. Resultados Dos 62 doentes com DM, 59 eram do sexo masculino e três do sexo feminino. As idades variaram entre os 34 e os 76 anos (média = 54,2 anos). Os motivos de ida à consulta mais frequentes foram deformidades estéticas e limitações funcionais, nomeadamente dificuldades com o vestuário. De acordo com a classificação de Enzi, 59 doentes enquadravam-se no tipo 1 de DM e três doentes no tipo 2. A localização mais frequentemente operada foi a região cervical (n=79 casos), seguida das regiões: torácica (n=10, cinco dos quais por ginecomastia); abdominal (n=13); braquial (n=7); dorsal (n=7, dos quais quatro interescapulares); axilar, suprapúbica, lombar e mento (n=3); deltóideia e facial (n=2); occipital, supraclavicular, supraesternal e inguinal (n=1). Em 91,9% (n=57) dos doentes havia antecedentes de alcoolismo, estando algum tipo de doença hepática presente em 11,3% (n=7). Analiticamente, 19,1% (n=12) tinham dislipidémia e 7,9% (n=5) hiperuricemia. 17,5% (n=11) tinham o diagnóstico de hipertensão arterial. Das 132 intervenções cirúrgicas, efectuaram-se em 119 casos dermolipectomias (13 abdominoplastias), lipoaspiração em 8 casos e mastectomia em 5 casos. Houve registo de complicações em três operações: hematoma (n=2) e deiscência de suturas (n=1). A taxa de recidiva foi de 18,2% (n=24) nas 132 cirurgias, correspondendo a maioria (17 casos), a localizações a nível da região cervical. Conclusão A DM é uma doença rara, mas responsável por deformidades severas, com grande impacto estético e funcional. Embora estejam descritos na literatura médica apenas 200 a 300 casos, esta doença parece ser relativamente frequente no Serviço de Cirurgia Plástica do Centro Hospitalar e Universitário de Coimbra (CHUC). De acordo com a literatura, é uma doença mais frequente em países mediterrânicos com uma incidência estimada de 1:25.000, afectando 20 vezes mais os homens que as mulheres. É mais prevalente em indivíduos de meia-idade, entre a quinta e sexta década de vida. A experiência do serviço suporta a associação com o alcoolismo, presente em 91,9% dos doentes, embora a etiologia exacta ainda seja desconhecida. As localizações mais afectadas são o pescoço, os ombros, a região dorsal e os segmentos proximais de ambos os membros, mas os lipomas podem aparecer em outras localizações como a região retroauricular, submandibular, a região mamária e o abdómen. Como é uma doença de etiologia desconhecida, a cirurgia é ainda a única terapêutica eficaz. Em nossa opinião, a dermolipectomia tem grandes vantagens estéticas, e tecnicamente é mais eficaz, devendo-se reservar a lipoaspiração a casos seleccionados, de lipomatose muito difusa. O hematoma pós-operatório é a complicação principal. A recidiva é frequente (cerca 18,2% das intervenções), mesmo nos doentes que cessam o consumo de álcool.

Cyclopropenoid fatty acids (CPFA), which are a group of fatty acids produced by plants of the order Malvales, are known to induce adverse physiological effects when administered to a variety of animal species. A structurally strained cyclopropene ring is present in all CPFA and is believed responsible for the toxic action of these fatty acids. Dietary consumption of CPFA by mammals, poultry and fish has resulted in toxic responses including hepatic damage, impaired reproductive capabilities and sizeable alterations in lipid metabolism. Furthermore, CPFA have been identified as mildly carcinogenic and strongly cocarcinogenic towards rainbow trout (Salmo gairdneri). The mechanism by which CPFA enhance carcinogenesis is currently not understood. The research in this thesis has therefore been directed toward obtaining a better understanding as to how CPFA induce toxic responses in rainbow trout. Hepatic plasma membranes were isolated from both control trout and trout which had consumed dietary CPFA. The plasma membranes were then compared via the use of electron microscopy, chromatographic analysis of phospholipid and fatty acid content, two dimensional polyacrylamide gel electrophoresis of proteins, and Western blot analysis of concanavalin A sensitive glycoproteins. Electron micrographs revealed that control plasma membranes appeared more homogeneous than CPFA membranes and were characterized by more membrane sheets and less vesicularization. The analysis of enzyme activities revealed that CPFA caused a decrease in whole liver glucose-6-phosphatase activity and that control plasma membranes expressed slightly higher glucose-6-phosphatase and 5'-nucleotidase activities as compared to CPFA membranes. Although dietary CPFA appeared to have no effect on the phospholipid content of the plasma membranes, significant alterations in the fatty acid profiles of ethanolamine and choline phospholipids were observed. CPFA caused a decrease in palmitic, palmitoleic and oleic acids while the level of stearic and docosahexaenoic acids subsequently increased. Differences between the protein content of control and CPFA plasma membranes were made clear through the analysis of electrophoretic and Western blotting data. Membranes isolated from fish fed CPFA contained several proteins of high molecular weight (above 66,000 daltons) and other proteins of high isoelectric point that were not present in control plasma membranes. Additionally, two families of glycoproteins which had previously been identified as microsomal in origin were detected only in CPFA plasma membranes. A discussion concerning the possible causes and biological ramifications of the observed subcellular alterations caused by CPFA insult is also presented in this thesis.<br>Graduation date: 1989

Introduction: The relationship between pancreatitis and dyslipidaemia is unclear and has never been studied in a South African context. Patients and methods: A prospective evaluation of all admissions with acute pancreatitis to a regional hospital general surgical service was performed to ascertain its relationship to dyslipidaemia. Aetiology was determined by history and ultrasound assessment. Disease severity was assessed using a modified Imrie score and an organ failure score. Body mass index was calculated. A lipid profile was obtained. Abnormal profiles were repeated. Secondary causes of dyslipidaemia were noted. A comparison of the demographic profile, aetiology, disease severity scores, complications and deaths were made in relationship to the lipid profiles. Results: From June 2001 to May 2005, there were 230 admissions, of whom 31% were women and 69% men. The median age was 38 years(range 13- 73). The pancreatitis was associated with alcohol in 146(63%), gallstones in 42(19%) and idiopathic in 27(12%). The amylase was significantly higher with a gallstone aetiology (p<br>Thesis (MMedSc)-University of KwaZulu-Natal, 2006.

Sleep is a condition with reduction or absence of life supporting mechanism like alimentation, self-defence, or reproduction. It occupies about a third of the human life span and has been found in all animals including insects. Despite the intense research in recent years, several mysteries of sleep still need to be revealed. This manuscript aims to increase our knowledge of the effect of obstructive sleep apnea (OSA) on components of human homeostasis.

Curcumin is an active ingredient of traditional herbal remedy Curcuma longa L. and possesses a wide array of metabolic benefits including anti-obesity, glucose lowering, insulin sensitising, lipid lowering and renoprotective effects. Furthermore, curcumin also protects against variety of chemical-induced renal injury. Thus, in the present thesis, it is hypothesised that curcumin improves renal function by restoring the altered lipid metabolism through blocking or reversing the pathological progress of dyslipidaemia associated with Chronic kidney disease CKD. The proposed research project is based on the hypothesis that long-term curcumin (CUR) supplementation may improve lipid metabolism and reduce the risk of cardiovascular disease in CKD. The general objective of this thesis is to investigate the in vivo effects of curcumin on dyslipidaemia of adenine-induced chronic kidney disease in a rat model (CHAPTER 2). The specific objectives of this thesis are (i) elucidate the possible mechanism(s) of action of curcumin on cholesterol-rich lipoproteins metabolism in adenine-induced chronic kidney disease (CHAPTER 3), (ii) elucidate the possible mechanism(s) of action of curcumin on triglyceride-rich lipoproteins metabolism in adenine-induced chronic kidney disease (CHAPTER 4) and (iii) elucidate the possible mechanism(s) of action of curcumin on high density lipoprotein (HDL) abnormalities in adenine-induced chronic kidney disease (CHAPTER 5). Overall, this study demonstrated that (i) chronic feeding of adenine-rich diet (0.75 % w/w) to rats caused renal damage which mimics to human chronic kidney disease and precipitates the metabolic dyslipidaemia. (ii) chronic curcumin supplementation to CKD rats improved hepatic lipid profile and kidney function tests. (iii) curcumin supplementation to CKD rats improved cholesterol-rich lipoprotein (such as LDL) metabolism by regulating of hepatic HMG-CoA reductase, LDL receptor and ACAT2 proteins, triglyceride-rich lipoprotein (such as VLDL) metabolism by regulating of hepatic and lipoprotein lipases and alleviates HDL abnormalities by regulating of LCAT protein expression. Thus, the protective effects of curcumin may be therapeutically useful in the prevention or management of CKD-induced dyslipidaemia.

Obesity is the result of genetic, behavioral, environmental, physiological, social and cultural factors that result in energy imbalance and promote excessive fat deposition. In particular, in Western society, which is characterized by sedentary lifestyles combined with excess energy intake, many people often try to lose weight with caloric restriction. However it is well known that the weight regain after caloric restriction results in accelerated storage of adipose tissue (De Andrade et al. 2015). The high efficiency of the recovery of the energy depots of body fat probably evolved in ancient times, when the food availability was intermittent and it was necessary to face up to long periods of famine. Nowadays, this efficiency is the key factor causing higher body fat gain relative to lean tissue, and the preferential catch-up fat phenomenon has also been linked to the hyperinsulinemic state of catch-up growth and the associated risks for later development of metabolic syndrome (Crescenzo et al. 2003; Dulloo et al.2006). Several studies on refeeding after caloric restriction have been conducted on laboratory rats, a very good model for obesity studies, because energy intake, diet composition and the level of physical activity can be easily monitored, and also, considering the standard housing conditions, laboratory rats exhibit a sedentary behavior, similarly to what happens in humans (Aydin et al. 2014; Buettner et al. 2007; Spangenberg et al. 2005): it was observed that during the refeeding with low fat diet the rats showed a reduction in energy expenditure and an increase in metabolic efficiency, causing high body fat deposition, even in absence of hyperphagia (Dulloo et al. 2008). In addition it has been shown that this high metabolic efficiency that drives catch-up fat on a low fat diet is exacerbated by refeeding on HFD (Crescenzo et al. 2003; Dulloo & Gerardier, 1992), although in different ways depending on the type of fat included in the diet (Dulloo et al. 2005). In this thesis, I assessed whether changing the type of fat, in the context of a high fat dietary regimen, could differently affect whole body homeostasis. For this reason I investigated the effect of two kind of HFD, rich in lard or safflower/linseed oil. I used two different experimental designs. In the first one, rats were divided in two groups with the same mean body weight and were pair fed with 380 kJ metabolisable energy (ME)/day (corresponding to the spontaneous energy intake of the same rats, as assessed in the days before the experiment) with a lard-based (SFA-MUFA, mainly monounsaturated and saturated fatty acids) or safflower-linseed based (PUFA, polyunsaturated fatty acids of ω-6 and ω-3 series) diets for 2 weeks. The two HFDs contained 58.2% energy from fat, 21.1% energy from protein and 20.7% energy from carbohydrate. During the treatments, body weight, food, and water intake were monitored daily. Faces and urine were collected daily and the respective energy content assessed with a bomb calorimeter. The day before the sacrifice, 24-h VO2 and VCO2 of the rats were recorded with a four-chamber indirect open-circuit calorimeter (Panlab S.r.l., Cornella, Spain). Measurements were performed for the whole 24 h-period every 15 min for 3 min in each cage. Urine was collected for the whole (24-h) period and urinary nitrogen levels were measured by an enzymatic colorimetric method (FAR S.r.l., Settimo di Pescantina, Italy). Daily energy expenditure and substrate oxidation rates were calculated for the whole 24-h period from VO2, VCO2, and urinary nitrogen according to Even et al. The day before the euthanasia, rats were fasted for 6 h and small blood samples were taken from the tail vein, placed in EDTA coated tubes and transferred on ice. Plasma glucose concentration was measured by colorimetric enzymatic method (Pokler Italia, Italy), while plasma insulin concentration was measured using an ELISA kit (Mercodia AB, Sweden). Basal postabsorptive values of plasma glucose and insulin were used to calculate Homeostatic Model Assessment (HOMA) index as [Glucose (mg/dL) × Insulin (mU/L)]/405. After the treatment, body composition and energy balance were measured. Plasma concentrations of triglycerides, cholesterol, non esterified fatty acids (NEFA), alanine aminotransferase (ALT) and lipid peroxidation were measured. Liver composition and oxidation, as well as respiratory capacity, oxidative status of liver mitochondria were measured. Quantification of uncoupling protein (UCP-1) in interscapular brown adipose tissue (IBAT) was performed. In the second experimental design, rats were food restricted for 14 days at approximately 50% of spontaneous intake. At the end of the semistarvation period, all the rats were separated into 3 groups (n = 8): one group was immediately euthanized by decapitation to calculate the body composition of restricted rats, while the other two groups were refed isocaloric amounts of two different HFDs (58.2% by energy), rich in lard or safflower/linseed oil. The amount of dietary energy provided to the refed animals corresponds to the metabolisable energy intake of spontaneously growing (non-restricted) weight-matched control animals fed on chow diet, as previously reported (Crescenzo et al., 2003). Furthermore, the level of fat in the HFDs utilized here (i.e., 58% of energy intake) corresponds to dietary fat levels often utilized in rehabilitation (energy-dense) diets of malnourished infants and children in order to meet their high energy requirements for catch-up growth (Prentice and Paul, 2000). In this second design, I performed the same measurement of the first one, but I also investigated the regulation of the pathway of de novo lipogenesis in liver, white adipose tissue (WAT) and brown adipose tissue (BAT). I also evaluated liver and muscle composition, mitochondrial activity, as well as parameters of oxidative stress and inflammation, since both these tissues are major contributors to daily metabolic rate (Rolfe & Brown, 1997) and it has been proposed that skeletal muscle is involved in the suppression of thermogenesis that underlines the high metabolic efficiency for accelerated body fat recovery after caloric restriction (Dulloo, 2005; Crescenzo et al. 2006). The results of the first experimental design suggest that, at the end of the treatment, the percent of body lipid doubled both in SFA-MUFA and in PUFA rats, although the final value was significantly lower in PUFA than in SFA-MUFA rats; conversely the percentage of body protein was maintained constant in PUFA rats, while it significantly decreased in SFA-MUFA rats. The percent of body epididymal and visceral WAT increased, reaching a final value that was significantly lower in PUFA rats than in SFA-MUFA rats. The percent of body IBAT was significantly higher in PUFA than in SFA-MUFA rats and its content of UCP-1 was markedly increased in PUFA rats compared to SFA-MUFA rats. PUFA rats exhibit lower lipid gain but higher protein gain compared to SFA-MUFA rats. The analysis of fuel oxidation showed that PUFA rats had reduced protein oxidation but higher lipid oxidation compared to SFA-MUFA rats. Plasma metabolic characterisation evidenced higher alanine aminotransferase activity in PUFA rats compared to SFA-MUFA rats. Livers from PUFA rats showed higher degree of steatosis and had higher lipid content, triglycerides and cholesterol, as well as higher lipid peroxidation, compared to SFA-MUFA rats. Liver mitochondria from PUFA rats displayed a significant decrease in basal and fatty acid-induced proton leak compared to SFA-MUFA rats. Both NAD, FAD and lipid-linked maximal oxidative capacities were significantly higher in isolated liver mitochondria from rats PUFA compared to SFA-MUFA rats. Evaluation of oxidative status showed that lipid peroxidation was significantly higher in mitochondria from PUFA rats compared to SFA-MUFA rats. The results of the second experimental design suggest that rats refed the PUFA-enriched diet had lower body lipids and higher body proteins compared to rats refed the SFA-MUFA-enriched diet, as well as lower amount of visceral and epididymal WAT, but higher amount of IBAT. Rats refed PUFA diet gained significantly less lipids and more proteins. At the end of the 2 weeks refeeding period, rats refed PUFA diet exhibited higher NPRQ values and non-protein energy utilisation was fulfilled by using proportionally more carbohydrates and less fat compared to rats refed SFA-MUFA diet, lower HOMA index and plasma insulin levels in the fasting and the fed state, together with lower plasma triglycerides and cholesterol levels. Plasma lipid peroxidation was not significantly different between the two groups of rats, while a significant decrease in ALT was found in rat refed PUFA diet. FAS activity, the rate-limiting enzyme in the pathway of de novo lipogenesis, was found to be significantly higher in liver, e-WAT and IBAT in rat refed PUFA diet. In the liver, higher triglyceride content was found in rat refed PUFA diet, but hepatic lipid peroxidation was significantly lower. SOD activity was found to be significantly higher in liver mitochondria of rat refed PUFA diet while no difference was found in skeletal muscle mitochondria. Lower degree of hepatic inflammation and unchanged hepatic content of the proinflammatory mediator TNF-α was found in rat refed PUFA diet. Expression of the UCP-1 protein in BAT was found significantly increased in rat refed PUFA diet. In conclusion, in this thesis I provide evidence that not only the amount, but also the type of dietary fat, is a primary obesogenic factor. In particular, when considering the composition of high fat diets for nutritional rehabilitation, the inclusion of PUFA could be useful for improving protein deposition and maintaining glucose homeostasis, while limiting lipid storage in adipose tissue and oxidative stress and inflammation in the liver.