Dissertations / Theses on the topic 'L-carnitine'

This set of experiments evaluated the physiological responses of intensively managed ostriches to L-carnitine. In experiment 1, 32 female and 16 male Zimbabwean Blue Neck and South African Black Neck breeders (n=48 of each sub-species; eight years old), were investigated in 16 breeder units of two females and one male (Trio), in a completely randomised design within four treatments and four replicates over an 8-month period during the breeding season. The same basal diet was fed supplemented with 0 (T0, control), 125 (T125), 250 (T250) or 600 (T600) mg/kg L-carnitine. T600 improved the egg production percentage, egg fertility percentage and the hatchability of set eggs for Black-Necks and Blue-Necks, respectively, and the hatchability of fertile eggs in Black Necks. L-carnitine did not affect egg shape index, defective eggs, egg weight, embryonic and post-hatch mortality. In experiment 2, 12 Black Neck males (5.5 years old) were allocated to three treatments (T0, T250 and T500) and four replicates. Semen samples were collected once a month over three months. L-carnitine had a significant effect on semen volume, sperm motility, live sperm percentage and sperm count, but had no significant effect on abnormal sperm percentage. In experiment 3, 32 day-old Black Neck ostrich chicks were allocated to treatments T0, T125, T250 and T600 with four replicates of two chicks. Chicks were vaccinated against inactive Newcastle Disease (ND) vaccine at day 30 as primary, and at day 51 as booster immunisation. ND antibody responses in the sera were monitored over three phases at 51, 70 and 80 days. Anti-NDV antibodies were detected using a modified chicken anti-NDV enzyme-like immunosorbent assay (ELISA). The treatments and the time periods and their interactions influenced ND antibody responses. T125 and T250 had the highest level of ND antibody response compare to the other groups. There were no differences in ND antibody response between T0 and T600 as well as T125 and T250. The highest ND antibody responses were recorded at day 70. Experiment 4 was designed the same as 3, to determine chicks’ growth responses over the 60-day period. Live weight and live weight gain values of T125 and T250 did not differ from those of T0. T600 had the lowest feed conversion ratio (FCR) during the total period. Feed intake (FI) was reduced in the T125 and T600 treatments compared to T0 and T250 over the total period. T125 gave the lowest FI and FCR responses over the total period, whereas there was no difference between T0 and T250. These results suggest that dietary T600 can have a beneficial effect on egg production, fertility and hatchability in the Black and Blue Neck breeders and T250 might improve sperm quality in males. In ostrich chicks T125 and T250 had positive effects on immune responses and T125 can improve the performance by decreasing the FCR. In contrast, the suppressive effect of a high inclusion level (T600) might indicate that ostrich chicks are sensitive to high inclusion levels that could cause adverse effects.<br>gm2013<br>Animal and Wildlife Sciences<br>Thesis (PhD)--University of Pretoria, 2013.<br>Unrestricted

Dans ce memoire de these, nous avons etudie l'effet d'un pretraitement par la l-carnitine sur deux modeles d'ischemie experimentale: l'ischemie regionale permanente realisee in vivo par ligature de l'artere coronaire gauche et l'ischemie globale partielle de courte duree realisee in vitro sur cur isole perfuse. Dans la premiere partie de ce travail, nous avons utilise le modele d'ischemie in vivo et nous avons demontre qu'un pretraitement par la l-carnitine entraine une limitation veritable de la taille de l'infarctus, associee a une moindre deterioration fonctionnelle 48 heures apres occlusion coronaire, l'etat metabolique de la zone myocardique saine restant inchange. La seule modification metabolique associee a cet effet protecteur apparait etre l'augmentation tres marquee, chez les rats traites, de la carnitinemie, dont nous demontrons egalement qu'elle doit atteindre un certain seuil pour qu'un effet protecteur se manifeste. Dans la deuxieme partie, nous avons realise une etude morphometrique en microscopie electronique sur des curs pretraites par la l-carnitine, perfuses par voie aortique, puis soumis a une periode de 30 minutes d'ischemie globale partielle in vitro et nous avons retrouve l'effet protecteur de la l-carnitine au niveau de l'ultrastructure mitochondriale

L'hypertrophie cardiaque obtenue chez le rat par realisation d'une fistule aorto-cave entraine une reduction de l'oxydation des acides gras a longues chaines. Des mesures, realisees sur mitochondries provenant de coeurs hypertrophies, montrent une diminution de 30 % de l'utilisation du palmitate par rapport aux controles. Par contraste, les niveaux d'oxydation du palmitoylcarnitine et du palmitoyl-coa sont normaux. Ces resultats mettent en cause la navette carnitine. L'etude de la palmitoyl-coa synthetase montre une conservation des proprietes cinetiques de cette enzyme. Par contre, la carnitine palmitoyltransferase externe presente une plus grande sensibilite a l'inhibition exercee par le palmitoyl-coa. Ce phenomene n'apparaissant qu'a concentrations elevees en palmitoyl-coa, il est peu probable que cette defaillance soit le seul facteur responsables des troubles metaboliques observes chez les coeurs hypertrophies. Une depletion de 30 % de la l-carnitine, cofacteur indispensable a la translocation des acides gras a travers les membranes mitochondriales, est observee au niveau des coeurs hypertrophies. L'etude du mecanisme de transport de la l-carnitine par des vesicules de sarcolemme montre que ce processus est independant du sodium mais que le transporteur est capable de catalyser un echange entre la l-carnitine externe et la butyrobetaine endogene. Ce systeme se revele sensible a l'hypertrophie cardiaque. Une augmentation du km et une chute de la vmax de la capture de la l-carnitine sont observees sur des vesicules de sarcolemme isolees a partir de coeurs hypertrophies. Notre etude sur des organites subcellulaires a mis en evidence l'alteration des proprietes cinetiques des enzymes membranaires telles que la carnitine palmitoyltransferase externe et le transporteur de la l-carnitine. Ces deux facteurs vont provoquer la defaillance du metabolisme des acides gras a longues chaines observee au niveau du coeur hypertrophie.

L'hydrogenase cytoplasmique d'alcaligenes eutrophus h16 (ec 1. 12. 1. 2) est utilisee pour regenerer le cofacteur nadh. Une etude de la stabilite de cette enzyme met en evidence sa rapide denaturation en presence d'agents reducteurs, ou de nadh. Paradoxalement, l'oxygene agissant comme stabilisateur de l'enzyme, semble etre tres nefaste lors de la reaction en milieu reducteur. On constate une degradation du cofacteur lors de la mise en oeuvre d'un reacteur en absence d'oxygene, en vue de la production de l-lactate avec l'hydrogenase et la l-lactate deshydrogenase (ec 1. 1. 1. 27) coimmobilisees sur des rafles de mais. L'application a la synthese de l-carnitine, a l'aide de la carnitine deshydrogenase (ec 1. 1. 1. 108) est envisagee. Un taux de recyclage de 270 est atteint au bout de 23 h. La degradation du cofacteur dans le milieu reactionnel peut etre ralentie par l'utilisation d'un derive macromoleculaire hydrosoluble, le n**(6)-(6-aminohexyl)-carbamoyl methyl-nad**(+) greffe sur de l'acide poly-l-glutamique. Une decarboxylation spontanee du substrat en milieu basique, la 3-dehydrocarnitine, impose un rendement de bioconversion de 100 %, qui a ete atteint pendant 24 h avec des ajouts reguliers d'hydrogenase.

Introdução: A acidemia 3-hidroxi-3-metilglutárica é causada pela deficiência da 3-hidroxi-3-metil-glutaril-CoA-liase, uma enzima do metabolismo da leucina, levando ao acúmulo, especialmente, do ácido 3-hidroxi-3-metilglutárico nos tecidos. Estudos sugerem que o estresse oxidativo pode contribuir para os danos neurológicos observados em algumas acidúrias orgânicas. Objetivo: Avaliar parâmetros de estresse oxidativo em pacientes com acidúria 3-hidroxi-3-metilglutárica antes e após o tratamento. Materiais e Métodos: Amostras de sangue e urina foram coletadas de pacientes no momento do diagnóstico e após tratamento com dieta com restrição de proteínas e suplementação de L-carnitina (100mg/kg/dia) e de controles. O TBA, um subproduto final da peroxidação lipídica, foi medido no plasma. A determinação do teor de carbonilas e de grupos sulfidrila, marcadores de dano oxidativo a proteínas, foi realizada no plasma. Para avaliar na urina a oxidação de proteínas, os níveis de di-tirosina foram medidos por autofluorescência. O ensaio da capacidade antioxidante urinária foi realizado utilizando um kit comercial. Os níveis de carnitina livre e isovalerilcarnitina foram analisados em amostras de sangue por espectrometria de massas em tandem usando o método de monitorização de reação múltipla (MRM). A concentração de proteínas foi determinada pelo método de biureto em amostras de plasma usando um kit comercial. Resultados e Discussão: Os resultados demonstraram um aumento significativo nos níveis de isovalerilcarnitina em sangue total, das concentrações plasmáticas de malondialdeído e urinárias de di-tirosina, além de uma redução significativa da capacidade antioxidante urinária e dos níveis sanguíneos de carnitina livre nos pacientes no momento do diagnóstico em relação aos controles. Verificou-se uma diminuição nas concentrações do malondialdeído plasmático e da di-tirosina na urina dos pacientes tratados, o que sugere um efeito de proteção do tratamento sobre a peroxidação de lípidos e do dano oxidativo a proteínas, bem como uma normalização dos níveis de L-carnitina durante o tratamento. Conclusões: Esses resultados permitem sugerir que o estresse oxidativo ocorre em pacientes com acidemia 3-hidroxi-3-metilglutárica e que o tratamento com a dieta restrita de proteína e suplementada com L-carnitina pode oferecer proteção contra o dano oxidativo a biomoléculas.<br>Introduction: The 3-hydroxy-3-methylglutaric acidemia is caused by the deficiency of 3-hydroxy-3-methyl-glutaryl-CoA lyase, an enzyme of leucine metabolism, leading to accumulation of 3-hydroxy-3-methylglutaric acid in tissues. Studies have suggested that oxidative stress may contribute to the neurological damage observed in some organic acidurias. Objective: Evaluate oxidative stress parameters in patients with 3-hydroxy-3-methylglutaric aciduria patiets before and after treatment. Materials and Methods: Blood and urine samples were collected from patients at diagnosis and after treatment with restricted protein diet and supplemented with L-carnitine (100mg/kg/dia) and from controls. TBA , an end subproduct of lipid peroxidation, was measured in plasma. Determination of carbonyl and sulphydryl content, biomarkers of oxidative damage to proteins, was done in plasma. To assess urine protein oxidation, levels of di-tyrosine were measured by autofluorescence. The assay of antioxidant urinary capacity was performed using a commercial kit. The levels of free carnitine and isovalerylcarnitine were analyzed in blood samples by tandem mass spectrometry using the method of multiple reaction monitoring (MRM). Protein content was determined by the biuret method for plasma samples using a commercial kit. Results and Discussion: The results demonstrated a significant increase of total blood isovalerylcarnitine, malondialdehyde plasma concentrations and di-tyrosine urinary levels and a significant reduction of the urinary antioxidant capacity and free-carnitine blood levels in pacients at diagnosis compared to controls. It was verified a decrease in plasma malondialdehyde concentrations and urinary di-tyrosine levels in treated patients, suggesting a protective effect of the treatment on lipid peroxidation and protein oxidative damage, as well as a normalization of L-carnitine levels during treatment. Conclusions: These results allow to suggest that oxidative stress occurs in 3-hydroxy-3-methyl-glutaryl-CoA lyase deficient patients and treatment with restricted protein diet and L-carnitine may offer protection against oxidative damage.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2017-11-24T13:23:07Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Lidiane Pescke Pereira.pdf: 897636 bytes, checksum: 92549067a59b73ac61dbce09ee1c1ee0 (MD5)<br>Made available in DSpace on 2017-11-24T13:23:07Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Lidiane Pescke Pereira.pdf: 897636 bytes, checksum: 92549067a59b73ac61dbce09ee1c1ee0 (MD5) Previous issue date: 2017-09-20<br>Com o aumento da produtividade e da demanda nutricional pela fêmea suína, o uso de moduladores L-carnitina, L-arginina, cromo, somatotropina e ractopamina tem sido uma alternativa para melhorar os índices produtivos. Entretanto, a variabilidade nas informações e a complexidade dos estudos envolvendo o tema exige uma abordagem mais sistêmica. Objetivou-se por meio desta meta-análise determinar o efeito do uso de moduladores nutricionais no desempenho reprodutivo e das leitegadas de porcas em gestação e lactação. A base de dados utilizada incluiu 83 artigos publicados entre os anos de 1989 e 2017, totalizando 22.608 porcas em 534 tratamentos. Critérios foram estabelecidos para a seleção dos artigos: uso de moduladores nutricionais: L-carnitina, L-arginina, cromo, somatotropina e ractopamina; conter as variáveis corporais e reprodutivas de porcas gestantes e lactantes. A meta-análise envolveu as análises de heterogeneidade, gráfica, correlação, variância e de resíduos. Não houve correlação (P>0,05) entre o uso de moduladores nutricionais e as variáveis corporais das porcas. No estudo de correlações verificou-se que a suplementação com L-carnitina, L-arginina e cromo aumentam (>0,450; P<0,05) o peso do leitão ao nascer e número de leitões nascidos vivos e o peso dos leitões ao nascer. Já o uso de somatotropina aumenta o número de leitões desmamados (0,985; P<0,01). Não houve diferença significativa (P>0,05) entre as médias dos grupos dos tratamentos com L-carnitina, cromo, e somatotropina para o consumo de ração e condição corporal das porcas. O uso de ractopamina aumentou em 3,41 % (P<0,05) a espessura de toucinho ao parto. A suplementação com L-carnitina e cromo aumentaram em 2,30 % e 4,73 % (P<0,05) o número de leitões nascidos vivos, respectivamente. O uso da L-carnitina, arginina e somatotropina proporcionaram, em média, leitões mais pesados ao nascer em relação ao controle (1,48 vs. 1,43kg; P<0,05). A administração da somatotropina aumentou em 9,01 % (P<0,05) o número de leitões desmamados em relação ao controle. Os estudos sobre o uso de moduladores nutricionais encontrados na literatura são pouco explorados quanto a condição corporal e nutricional, o que impossibilita conclusões sobre o uso adequado destes aditivos para ajustes nutricionais em porcas gestantes e lactantes. Entretanto, os moduladores nutricionais L-carnitina, L-arginina, cromo e somatotropina podem melhorar o desempenho produtivo das porcas e de suas leitegadas.<br>The increase in productivity and nutritional demand by sows, the use of modulators L-carnitine, L-arginine, chromium, somatotropin and ractopamine has been an alternative to improve the productive indexes. However, the variability in information and the complexity of studies involving the subject requires a more systemic approach. The objective of this meta-analysis was to determine the effect of the use of nutritional modulators on the reproductive performance and litter of sows in gestation and lactation. The database used included 83 articles published between 1989 and 2017, totaling 22,608 sows in 534 treatments. Criteria were established for the selection of articles: use of nutritional modulators: L-carnitine, L-arginine, chromium, somatotropin and ractopamine; contain the body and reproductive variables of pregnant and lactating sows. The meta-analysis involved analyzes of heterogeneity, graph, correlation, variance and residuals. Don´t were significant correlations (P>0.05) between the body variables of the sows and nutritional modulators and their use. In correlation study, the L-carnitine, L-arginine and chromium supplementation increases (>0.450; P<0.05) the birth piglets weight and liveborn number. Somatotropin administration increased the weaner piglets number (0.985; P<0.01). There were no significant difference (P>0.05) between the means of groups with L-carnitine, chromium, and somatotropin for feed intake and body condition of the sows. Ractopamine use increased in 3.41% (P<0.05) the backfat thickness at farrowing. Supplementation with L-carnitine and chromium increased in 2.30 % e 4.73 % (P<0.05) the alive piglets number, respectively. The use of L-carnitine, L-arginine and somatotropin provided heavier piglets at birth in relation to control groups (1.48 vs. 1.43kg; P<0.05). Somatotropin administration increased in 9.01% (P<0.05) the of weaned piglets number in relation to control group. Studies on the use of nutritional modulators found in the literature are poorly explored in body and nutritional condition terms, which makes it impossible to reach conclusions about the proper use of these additives for nutritional adjustments in pregnant and lactating sows. However, the nutritional modulators L-carnitine, L-arginine, chromium and somatotropin can improve the productive performance of sows and their litters.

Sickness rate of obesity has been 57% increased in recent years, and it occurs in 80% of endocrine patients. Prevalence of chronic pancreatitis (CP) in patients with obesity constitutes from 45% to 80%.

La sindrome dell’ovaio policistico (PCOS) è un comune disordine endocrinologico e riproduttivo che si riscontra nel 6-10% della popolazione femminile. In generale, è considerata una malattia metabolica multifattoriale caratterizzata da varie manifestazioni cliniche quali iperandrogenismo, ovaie policistiche e disfunzioni ovulatorie, che la rendono la causa più comune di infertilità da anovulazione nelle donne, ma anche da problemi metabolici come obesità, insulino-resistenza, iperinsulinemia e diabete di tipo II, che la associano a complicanze cardiovascolari, neurologiche e psicologiche, come ansia e depressione. Come è emerso recentemente, un aumento di AGE ha un ruolo chiave nelle disfunzioni ovariche e nella riduzione della fertilità associata alla PCOS. I risultati presentati in questa tesi dimostrano, per la prima volta, che una condizione di stress glicativo MG-dipendente si instaura nell’ambiente ovarico di topi PCOS. Questa condizione risulta associata a cambiamenti della funzione di SIRT1 nella regolazione della fisiologia mitocondriale e della sopravvivenza cellulare. Nel nostro studio, ci siamo basati su un modello ben studiato di topo PCOS indotto da DHEA e abbiamo approfondito la caratterizzazione del micro-ambiente ovarico nei topi DHEA. Abbiamo studiato, inoltre, l’efficacia di diverse formulazioni di carnitine (L-Carnitina, Acetil-L-Carnitina e Propionil-L-Carnitina) su un modello in vitro murino, sottoposto a danno ossidativo, noto essere una delle possibili cause della ridotta qualità ovocitaria associata alla PCOS. Infine è stata valutata l’efficacia di queste formulazioni di carnitine sui topi DHEA. Le carnitine sono essenziali nel metabolismo degli acidi grassi e possono agire proteggendo dal danno mitocondriale e da condizioni di bilancio energetico alterato come quelle presenti nella sindrome dell'ovaio policistico (PCOS), evidenziate anche dai ridotti livelli di L-carnitina nel siero delle pazienti affette da questa patologia. Ripristinare l'equilibrio energetico e fornire adeguate riserve di energia all’ovocita durante la follicologenesi e la maturazione può rappresentare un’importante strategia per migliorare l’ambiente intraovarico e aumentare la probabilità di gravidanza. In questo contesto, composti metabolici, come le carnitine, con effetti positivi sull’attività mitocondriale e sullo scavenging dei radicali liberi, possono contribuire a mitigare gli effetti di questa sindrome.

Esta Tesis Doctoral recoge el trabajo de investigación que se ha realizado en dos líneas desarrolladas de forma paralela sobre Escherichia coli. Por un lado, la optimización de un proceso de biotransformación para mejorar la síntesis de L( )-carnitina mediante técnicas de ingeniería metabólica. Y por otro, la determinación de los principales efectos que provoca la exposición prolongada a altas concentraciones de sal y su respuesta de adaptación, principalmente cuando las fuentes de carbono pueden contener altas concentraciones de sal y tanto el sustrato como el producto son osmoprotectores. Para ello, se han aplicado técnicas utilizadas por la biología de sistemas y la ingeniería metabólica. La importancia de L( )-carnitina viene determinada por el papel que desempeña en el metabolismo energético, de hecho su deficiencia está asociada a diversas patologías. Varios trabajos centrados en la aplicación terapéutica de L( )-carnitina han demostrado que su administración puede ayudar a suplir dicha carencia. A partir de este punto, comienza una creciente actividad investigadora centrada en la producción de L( )-carnitina. Este trabajo presenta un método alternativo basado en la utilización de E. coli para llevar a cabo la biotransformación de compuestos de bajo valor añadido como puede ser D(+)-carnitina y/o crotonobetaína en L( )-carnitina. Por medio de técnicas de biología molecular se ha modificado genéticamente una cepa de E. coli, consiguiendo la sobreexpresión de caiC y mejorando el rendimiento de la cepa silvestre. Además, para optimizar la producción de L( )-carnitina se han estudiado aspectos relacionados con el metabolismo de carnitina como la disponibilidad de coenzima A o la inhibición de una ruta metabólica que favorezca la transformación del sustrato en L( )-carnitina. Posteriormente, se obtuvo una cepa modificada más estable y con una alta capacidad para la producción de L( )-carnitina, para ello se han implementado diversas estrategias de ingeniería metabólica. Las mutaciones implicadas en la mejora de esta cepa fueron: a) la deleción del gen aceK para incrementar el flujo hacia el ciclo de los ácidos tricarboxílicos, b) la deleción del gen caiA para impedir la síntesis de γ-butyrobetaína (productos del metabolismo de carnitina), y c) la sustitución del promotor natural altamente regulado del operón cai por un promotor artificial constitutivo. Con dichas mutaciones implementadas en una misma cepa, no sólo se consiguió aproximadamente un 100 % de conversión de crotonobetaína en L( )-carnitina en las condiciones de ensayo, sino que también la limitación impuesta por la presencia de oxígeno fue superada por este mutante, lo que indica la importancia de la ingeniería metabólica en la mejora de procesos biotecnológicos. L(-)-carnitina o compuestos similares son utilizados como osmoprotectores, acumulándolos en el interior celular, para evitar la deshidratación cuando la osmolaridad del medio se incrementa. Ante estas situaciones, los microorganismos pueden adaptarse y dar una respuesta pasajera, o realizar un proceso de adaptación para mantener su supervivencia mientras el estrés está presente. En este trabajo, se observó la evolución y la respuesta generada de una cepa de E. coli cultivada en un reactor continuo y sometida a tres concentraciones crecientes de sal (moderada, alta y muy alta). La medida de las actividades enzimáticas de las principales rutas metabólicas, así como la determinación de los metabolitos fermentativos producidos, resaltaron el importante papel ejercido por el metabolismo central en la adaptación y en la supervivencia celular tras una larga exposición a estrés salino, así como la necesidad de disponer de precursores biosintéticos y de energía en forma de ATP. Además, se profundizó en el estudio del comportamiento celular realizando una aproximación desde la biología de sistemas, integrando los niveles metabolómico, flujómico y transcriptómico. Se debe destacar que se observaron dos conjuntos de respuestas consecuencia de la concentración de sal presente en el medio. Uno dirigido a mantener unos niveles energéticos umbrales en la célula, basado tanto en el incremento de los flujos metabólicos hacia rutas que permitían la generación de energía, como en la reducción de procesos no esenciales para la supervivencia. Y otro, una respuesta característica en las células expuestas a alta o a muy alta concentración de sal, que estuvo caracterizada no sólo por cambios en los patrones de fermentación metabólica sino también por una alteración significativa del estado redox celular. Así, con el uso de técnicas apropiadas se han podido detectar un gran número de cambios en la fisiología y el metabolismo de E. coli. Además, la aproximación de la biología de sistemas ofrece una forma de obtener e integrar gran cantidad de información, que de otra forma se perdería por la cantidad de información que se obtiene. Finalmente, la ingeniería metabólica y la biología de sistemas han aportado una excelente manera de mejorar y conocer las características de los microorganismos involucrados en los procesos biotecnológicos relacionados con la producción de L( )-carnitina.<br>Two parallel research aims addressed on Escherichia coli are shown in this PhD thesis. On one hand, the optimization of a biotransformation process in order to improve L( )-carnitine synthesis by using metabolic engineering techniques is explained within the first chapters. On the other hand, in the following chapters, the main effects provoked by long-term high salt concentrations and the adaptative response to osmotic stress were determined using different techniques related to systems biology. L( )-carnitine is an important trimethylammonium compound because of its role in the energetic metabolism, in humans, several pathologies are related with deficiencies of carnitine level. Several works focused on the therapeutic application of L( )-carnitine, showed that administration of this compound could be a solution as opposed to its absence. Once different carnitine production ways were revised, this work shows an alternative method using Escherichia coli to carry out the biotransformation from D(+)-carnitine and/or crotonobetaine into L( )-carnitine. By using molecular biology techniques a strain of E. coli was engineered, obtaining caiC overexpression and enhancing the production yield respect to the wild type strain. Moreover, several aspects related with carnitine metabolism, such as coenzyme A availability and the inhibition of specific metabolic pathways were studied to optimize the carnitine production. Afterwards, various metabolic engineering strategies were implemented, obtaining a stable engineered strain with high capacity to produce L( )-carnitine. The modifications carried out were: a) deletion of the aceK gene (encoding a bifunctional protein phosphatase/kinase which performs post-translational control of isocitrate dehydrogenase) in order to increase the metabolic flux towards TCA cycle, b) deletion of the caiA gene (encoding the crotonobetainyl-CoA reductase) to avoid synthesis of γ-butyrobetaine (byproduct of the carnitine metabolism), and c) replacement of the highly regulated natural promoter of the cai operon by a constitutive promoter. These mutations implemented in the same strain led to obtaining almost 100% conversion from crotonobetaine to L( )-carnitine in the assay conditions. Moreover, the main restrictions impossed to the aerobic expression of the carnitine metabolism were eliminated producing L( ) carnitine in the presence of oxygen. Therefore, this work emphasizes the important role of metabolic engineering to improve any biotechnological process. On the other hand, L( )-carnitine and similar compounds are used as osmoprotectors, which are accumulated in high concentrations, either through the uptake from the medium or through de novo synthesis inside the cells, to avoid dehydratation when the osmolarity of the culture medium increases. Under these conditions, microorganisms have different response to an environmental stress, short-term or shock and long-term adaptation. In this work, evolution and response to long-term adaptation were analyzed in a E. coli strain growing in continuous reactors supplemented with a gradually increasing concentration of NaCl (moderate, high and very high). Enzyme activities from the main metabolic pathways and fermentative metabolites were analyzed, highlighting important role of central metabolism on adaptation and cellular survival after salt stress exposition. Furthermore, the need of biosynthetic precursors and energy as ATP were shown. In addition, a systems biology approach was conducted to study cellular behavior. In order to estimate the critical modifications undergone to overcome stress and to develop tolerance to salt, the metabolism was examined at several levels using different techniques (metabolomics, fluxomics and transcriptomics). Under salt stress conditions two set of responses were shown. One of them was focused to maintain the energetic threshold in cells, thus, either an increment of the metabolic pathways which could produce energy or a decrease of no-essential processes to survive were shown. On the other hand, cells under high or very high salt concentrations showed another similar response characterized by both changing on pattern of fermentative pathways and redox state. Therefore, using suitable techniques many changes in the physiology and metabolism of the E. coli strain in use were detected. Moreover, the systems biology approach offered a way to obtain and integrate a large amount of information, preventing some of the information being overlooked by the massive amount of data. Both the metabolic engineering and systems biology approaches have provided excellent ways to improve and know features of microorganisms involved in biotechnological processes related with the L( )-carnitine production.

Master of Science<br>Department of Animal Sciences and Industry<br>Barry J. Bradford<br>Dairy cows undergo many homeorhetic adaptations during the transition to lactation. Although many of the physiological processes - including increased lipolysis and postpartum inflammation - are adaptive, exaggerated responses can contribute to metabolic disease and reduced milk production. L-carnitine has been shown to increase hepatic oxidation of fatty acids and reduce hepatic lipid accumulation in early lactation cows; however, L-carnitine is degraded in the rumen. An experiment using 4 ruminally-cannulated Holstein heifers in a split plot design demonstrated that the relative bioavailability of L-carnitine was greater when delivered abomasally than ruminally. There was a dose × route interaction and a route effect for increases in plasma carnitine above baseline, with increases above baseline being greater across all dose levels (1, 3, and 6 g L-carnitine/d) when infused abomasally compared to ruminally. A second experiment used 56 lactating Holstein cows in a randomized complete block design to evaluate 2 rumen-protected products (40COAT and 60COAT) compared to crystalline L-carnitine at doses targeting 3 and 6 g/d carnitine. Although crystalline and 40COAT were effective in linearly increasing carnitine concentrations, only subtle responses were seen for the 60COAT, which were less than that for crystalline carnitine in plasma, milk, and urine. Ineffectiveness of rumen-protected products to increase carnitine concentrations beyond crystalline may have been due to over-encapsulation that hindered liberation of the carnitine and its absorption in the small intestine. Although L-carnitine has the potential to reduce postpartum hepatic lipidosis, effective rumen protection of L-carnitine while maintaining intestinal availability needs further investigation. Plant polyphenols have anti-inflammatory properties and when administered during the transition period, have been shown to increase milk production. An experiment used 122 multiparous Holstein cows in a randomized block design to determine the effect of short term (5-d; SBE5) and long term (60-d; SBE60) administration of Scutellaria baicalensis extract (SBE)on whole-lactation milk yield, 120-d milk component yield, and early lactation milk markers of inflammation. Whole-lactation milk yield was increased for SBE60 compared to control, but was not different for SBE5 compared to control. Greater total pellet intake, milk lactose yield, and reduced SCC during wk 1-9 for SBE60 compared to control, all could have contributed to the observed sustained increase in milk yield. Milk production parameters were not different for SBE5 compared to control. No treatment effects were observed for BCS or milk markers of inflammation (haptoglobin) and metabolic function (β-hydroxybutyrate). Overall, long term administration of S. baicalensis effectively increased milk production, however the mechanism by which this was achieved is unknown. Although routes of administration to effectively achieve their physiological responses were different between L-carnitine (abomasal delivery) and SBE (feeding), both bioactive nutrients can improve the metabolic function of early lactation dairy cows.

Two experiments were done to study the effects of L-carnitine supplementation (CNsup) during exercise. EXP 1, examined the effect of CNsup on lipid oxidation and muscle glycogen utilization during submaximal EX. Triglycerides were elevated by a fat feeding (90g fat), 3 h later subjects cycled for 60 min at 70% VO2max (CON). Muscle biopsies were obtained preEX, after 30 and 60 min of EX. Blood samples were taken preEX and every 15 min of EX. Subjects randomly completed two additional trials following 7 and 14 days of CNsup (6 g/day). During one of the trials, subjects received 2000 units of heparin 15 min prior to EX to elevate FFA (CNhep). There were no differences in V02, RER, HR, g of CHO and fat oxidized among the three trials. Serum total acid soluble (TASC) and free carnitine (FC) increased with CNsup (CON, 71.3 ± 2.9; CN, 92.8 ± 5.4; CNhep, 109.8 ± 3.5 mol·g'). Muscle carnitine concentration at rest was unaffected by CNsup. During EX, TASC did not change, FC decreased (p<0.05) and SCAC increased (p<0.05). With CNsup the decrease in FC was less (~50%) (p<0.05) and the increase in SCAC was greater (~200-300%) (p<0.05) compared to CON (free 65%; SCAC 150%). Pre and postEX muscle glycogens were not different. EXP 2, examined the effects of CNsup on blood lactate accumulation during maximal EX. Subjects cycled for 4 min at ~100% VO2max (CON). Exercise was repeated following 6 and 13 days of CNsup (6 g/day). Serum TASC and FC were elevated due to CNsup. Blood Lactate was measured prior to and 0, 3, 5, and 7 min postEX. CNsup resulted in less (p<0.05) lactate accumulation compared to CON. There were no differences between DAY-6 and DAY-13.<br>Human Performance Laboratory

Les cardiopaties congènites que cursen amb hiperaflux pulmonar resulten en una disfunció endotelial progressiva de la vasculatura pulmonar, que en part és dependent d’una disminució en la via de senyalització de l’òxid nítric (NO). En un model animal oví de cardiopatia congènita amb hiperafluxe pulmonar hem demostrat prèviament una disrupció en l’homeòstasis de la carnitina, que s’associa a disfunció mitocondrial i que contribueix tant al desacoblament de la sintassa d’òxid nítric endotelial (eNOS) com a una reducció del NO disponible. Aquest projecte de tesi doctoral té com a objectiu testar la hipòtesi que el tractament precoç amb L-carnitina mantindrà l’homeòstasis de la carnitina, la funció mitocondrial i la senyalització del NO en un model animal oví de cardiopatia congènita amb hiperaflux pulmonar. Mètodes: En l’última fase de la gestació, es va inserir intra-úter una comunicació aorto-pulmonar en 13 fetus de bens. Després d’un part espontani, els xais van rebre tractament diari amb L-carnitina (n = 7; 100 mg / kg / dia) o el seu vehicle (n = 6). Un grup addicional d’11 xais amb flux pulmonar normal va servir de control. Resultats: En comparació als xais amb hiperaflux pulmonar tractats amb vehicle, els xais tractats amb L-carnitina van presentar a les 4 setmanes d’edat postnatal una disminució en els nivells d’acilcarnitina i en la raó acilcarnitina/carnitina lliure. Aquests canvis es van correlacionar amb un augment en els nivells i en l’activitat de l’enzim carnitina acetiltransferasa (CRAT), alhora que amb una disminució dels nivells de CRAT nitrada i de la raó làctic/pirúvic. A més, els xais tractats amb carnitina van presentar una reducció significativa dels nivells de la proteïna Hsp70, en correlació amb un augment de les interaccions eNOS/Hsp90, de l’activitat NOS i dels nivells NOx, a la vegada que amb una disminució dels nivells de superòxid derivat d’eNOS. Finalment, l’administració d’acetilcolina va disminuir significativament la resistència pulmonar vascular esquerra només en els xais tractats amb L-carnitina. Conclusions: El tractament precoç amb L-carnitina pot millorar i/o atenuar el declivi de la funció endotelial que caracteritza els nens amb cardiopaties congènites associades a hiperaflux pulmonar, amb potencials implicacions clíniques rellevants que justifiquen continuar investigant.<br>Las cardiopatías congénitas que cursan con hiperaflujo pulmonar resultan en una disfunción endotelial progresiva de la vasculatura pulmonar, que en parte es dependiente de una disminución en la vía de señalización del óxido nítrico (NO). En un modelo animal ovino de cardiopatía congénita con hiperaflujo pulmonar, hemos demostrado previamente una disrupción en la homeostasis de la carnitina, que se asocia a una disfunción mitocondrial i que contribuye tanto al desacoplamiento de la sintasa del óxido nítrico endotelial (eNOS) como a una reducción del NO disponible. Este proyecto de tesis doctoral tiene como objectivo testar la hipótesis de que el tratamiento precoz con L-carnitina mantendrá la homeostasis de la carnitina, la función mitocondrial y la señalización del NO en un modelo animal ovino de cardiopatía congénita con hiperaflujo pulmonar. Métodos: En la última fase de la gestación, se insertó intra-útero una comunicación aorto-pulmonar en 13 fetos de corderos. Tras un parto espontáneo, los corderos recibieron tratamiento diario con L-carnitina (n=7; 100 mg/kg/día) o su vehículo (n=6). Un grupo adicional de 11 corderos con flujo pulmonar normal sirvió de control. Resultados: En comparación a los corderos con hiperaflujo pulmonar tratados con vehículo, los corderos tratados con L-carnitina presentaron a las 4 semanas de edad post-natal una disminución en los niveles de acilcarnitina y en la razón de acil-carnitine/carnitina libre. Estos cambios se correlacionaron con un aumento en los niveles y en la actividad de la enzima carnitina acetil-transferasa (CrAT), a la vez que con una disminución de los niveles de CrAT nitrada y de la razón láctico/pirúvico. Además, los corderos tratados con L-carnitina presentaron una reducción significativa de los niveles de la proteína Hsp70, en correlación con un aumento de las interacciones eNOS/Hsp90, de la actividad NOS y de los niveles NOx, a la vez que con una disminución de los niveles de superóxido derivado de eNOS. Por último, la administración de acetilcolina disminuyó significativamente la resistencia pulmonar vascular izquierda solo en los corderos tratados con L-carnitina. Conclusiones: El tratamiento precoz con L-carnitina puede mejorar y/o atenuar el declive de la función endotelial que caracteriza a los niños con cardiopatías congénitas asociadas a hiperaflujo pulmonar, con potenciales implicaciones clínicas relevantes que justifican continuar investigando.<br>Congenital heart disease (CHD) with increased pulmonary blood flow (PBF) results in a progressive pulmonary vascular endothelial dysfunction that is partly dependent on decreased nitric oxide (NO) signaling. In a lamb model of CHD with increased PBF, we have previously shown a disruption in carnitine homeostasis, associated with mitochondrial dysfunction that contributes to eNOS uncoupling and decreased bioavailable NO. This study aims to test the hypothesis that Lcarnitine therapy would maintain carnitine homeostasis, mitochondrial function and NO signaling in our lamb model of increased PBF. Methods: 13 fetal lambs underwent in-utero placement of an aorto-pulmonary graft (shunt). Immediately following spontaneous delivery, lambs received daily treatment with oral L-carnitine (n=7; 100 mg/kg/day) or its vehicle (n=6). An additional group of eleven lambs with normal PBF served as controls. Results: At 4-weeks of age, L-carnitine-treated shunt lambs had decreased levels of acylcarnitine and a reduced acylcarnitine/free carnitine ratio compared to vehicle-treated shunt lambs. These changes correlated with increased carnitine acetyl-transferase (CrAT) protein and enzyme activity, as well as decreased levels of nitrated CrAT. The lactate/pyruvate ratio was also decreased in L-carnitine-treated shunt lambs. Furthermore, Hsp70 protein levels were significantly decreased in L-carnitine-treated shunt lambs, which correlated with a significant increase in eNOS/Hsp90 interactions, NOS activity, and NOx levels, as well as with a significant decrease in eNOS derived superoxide. Further, acetylcholine significantly decreased left pulmonary vascular resistance (PVR) only in L-carnitine-treated shunt lambs. Conclusions: Early L-carnitine therapy may improve and/or attenuate the decline in endothelial function noted in children with CHD associated with pulmonary overflow, and thus has potentially important clinical implications that warrant further investigation.<br>Universitat Autònoma de Barcelona. Programa de Doctorat en Pediatria, Obstetrícia i Ginecologia

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-10-11T14:31:19Z No. of bitstreams: 2 Tese - Tiago Omar Diesel - 2018.pdf: 2037910 bytes, checksum: e5037a6e126e6597f8f92b2754602731 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)<br>Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-15T11:00:19Z (GMT) No. of bitstreams: 2 Tese - Tiago Omar Diesel - 2018.pdf: 2037910 bytes, checksum: e5037a6e126e6597f8f92b2754602731 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)<br>Made available in DSpace on 2018-10-15T11:00:19Z (GMT). No. of bitstreams: 2 Tese - Tiago Omar Diesel - 2018.pdf: 2037910 bytes, checksum: e5037a6e126e6597f8f92b2754602731 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-09-13<br>Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG<br>Chemical delipidation has been used as an alternative to improve the cryotolerance of in vitro produced embryos (IVP). The aim of this study was evaluate the effect of L-carnitine (LC) on the development and survival of vitrified IVP bovine embryos by the Cryotop method in the first assay, and in the second trial the effect of LC and Forskolin on Cryotop cryopreserved embryos Experiment 1), or by modified slow freezing (Experiment 2), so mitochondrial activity, intracytoplasmic lipid (LI) content, cellular apoptosis (NCA) and hatching after heating were evaluated. In the first essay LC was used at the concentration of 0,6 mg/mL in maturation culture medium (IVM), embryo culture (IVC) and / or post-thawing (REC), in four treatments: without LC (Control), LC added to CIV (LCiv), LC to CIV + LC to REC (LCivR), and LC to MIV / CIV + LC to REC (LMivCR). The addition of LC increased the production of blastocysts in D7 by 28.6% (LCiv) and the amount of embryos grade I by 36.9% (LCivR), the re-expansion rate in 22,7% and hatching in 20.1% (LCiv), and mitochondrial activity was 1.9 times higher (P <0.001) (LCivR) than Control. The LI quantity was 29% lower in LCiv and LCivR and 50.2% in LMivCR compared Control (P <0.001). In the second experiment the embryos were cultured without addition of delipidators (Control), in the presence of 10μM of Forskolin added to the IVC in D5 (FORSK) or L-carnitine (0.6 mg / mL) added to the IVC and in post-thawing (LC). LC supplementation increased the production of blastocysts in D7 by 22.0% and grade I embryos by 30.1% (P <0.05), in relation to Control and FORSK. In Experiment 1, the re-expansion rate in LC increased (P <0.05) 28.9% in relation to FORSK. In Experiment 2, two Control treatments were used for slow freezing (Classic and Modified). Hatching after 48 hours was greater (P <0.05) in LC compared to FORSK and Classical and Modified Controls (77.5%, 41.9%, 40.5%, 40.8% respectively). In the LC treatment, there was a decrease (P <0.05) of 64.7% in the degenerate embryo rate in relation to the Classical Control. Treatment with delipidators reduced LI content (P <0.001) by 2.2 fold in FORSK and four times in the LC compared to Control. The addition of 0.6 mg / mL of L-carnitine to the culture medium and the post-thawing increased the rate of in vitro production of bovine embryos acting positively on mitochondrial potential, reducing the amount of intracellular lipids and cellular apoptosis and increasing cryotolerance of embryos submitted to the modified slow freezing protocol.<br>A delipidação química tem sido utilizada como alternativa para a melhoria da criotolerância em embriões produzidos in vitro (PIV). Este estudo foi realizado objetivando avaliar o efeito da Lcarnitina (LC) sobre o desenvolvimento e a sobrevivência de embriões bovinos PIV vitrificados pelo método Cryotop no primeiro ensaio, e no segundo ensaio o efeito comparado da LC e Forskolin em embriões criopreservados por Cryotop (Experimento 1), ou por congelamento lento modificado (Experimento 2). Para isto foram avaliadas a atividade mitocondrial, o conteúdo de lipídeos intracitoplasmático (LI), a apoptose celular e a eclosão após o aquecimento. No primeiro ensaio a LC foi utilizada na concentração de 0,6 mg/mL no meio para maturação (MIV), cultivo (CIV) e/ou recultivo embrionário (REC), em quatro tratamentos: sem LC (Controle), LC adicionado ao CIV (LCiv), LC ao CIV+LC ao REC (LCivR), e LC ao MIV/CIV+ LC ao REC (LMivCR). A adição de LC aumentou (P <0,05) a produção de blastocistos em D7 em 28,6% (LCiv), a quantidade de embriões grau I em 36,9% (LCivR), a taxa de re-expansão em 22,7%, a eclosão em 20,1% (LCiv) e a atividade mitocondrial foi 1,9 vezes maior (P <0,001) (LCivR) em relação ao Controle. A quantidade LI foi 29% menor em LCiv e LCivR e 50,2% em LMivCR comparado Controle (P <0,001). No segundo ensaio os embriões foram cultivados sem adição de delipidadores (Controle), na presença de 10µM de Forskolin adicionado ao CIV no D5 (FORSK) ou L-carnitina (0,6 mg/mL) adicionada ao CIV e ao recultivo (LC). A suplementação com LC aumentou a produção de blastocistos em D7 em 22,0% e de embriões grau I em 30,1% (P <0,05), em relação ao Controle e ao FORSK. No Experimento 1 a taxa de re-expansão no LC aumentou (P <0,05) 28,9% em relação ao FORSK. No Experimento 2 foram utilizados dois tratamentos Controle para congelamento lento (Clássico e Modificado). A eclosão após 48 horas foi maior (P < 0,05) no LC em comparação ao FORSK e aos Controles Clássico e Modificado (77,5%, 41,9%, 40,5%, 40,8% respectivamente). No tratamento LC foi observada diminuição (P < 0,05) de 64,7% na taxa de embriões degenerados em relação ao Controle Clássico. O tratamento com delipidadores reduziu o conteúdo de LI (P < 0,001) em 2,2 vezes em FORSK e quatro vezes no LC comparados ao Controle. A adição de 0,6 mg/mL de L-carnitina aos meios de cultivo e recultivo aumentou a taxa de produção in vitro de embriões bovinos atuando positivamente sobre a atividade mitocondrial, reduzindo a quantidade de lipídeos intracelulares e a apoptose e aumentando a criotolerância dos embriões submetidos ao protocolo de congelamento lento modificado.

La capacité des cœurs hypertrophiés à la suite d'une surcharge volémique (rat porteur d'une fistule aorto-cave de 3 mois) à utiliser les acides gras exogènes a été approchée par les mesures de la production du 14CO2 à partir du U-14C-palmitate et du l-14C­octanoate. Les cœurs ont été isolés et perfusés sous les conditions de travail modéré et élevé. Leurs turnover énergétiques et leurs performances mécaniques ont été évalués à partir de la consommation d'oxygène et du travail de pression. Selon nos résultats, la production de 14CO2 à partir du palmitate exogène est abaissée de 30% chez les coeurs hypertrophiés. La consommation d'oxygène et les performances mécaniques sont diminuées dans les mêmes proportions. Quand l'octanoate constitue le principal substrat exogène, la consommation d'oxygène et la production du 14CO2 des cœurs hypertrophiés deviennent comparables à celles des coeurs normaux. De même, les performances mécaniques des cœurs hypertrophiés semblent être rétablies au cours des perfusions avec l'octanoate exogène. Ces résultats suggèrent qu'une partie de la défaillance mécanique observée chez les cœurs hypertrophiés perfusés avec les acides gras à longues chaînes est secondaire à la dysfonction de la carnitine-acyl carnitine translocase, qui serait elle-même secondaire à une déplétion de la L-carnitine tissulaire. Cette dernière serait la conséquence de la diminution de l'affinité du transporteur membranaire pour la L-carnitine circulante. Une diminution significative du Km a été en effet constatée dans notre étude du transport actif de la L-14C-carnitine dans les cœurs hypertrophiés.

Aging can be described as an extremely complex occurrence from which no organism can be excluded. Intrinsic and extrinsic aging make out the two components of skin aging and they differ on the macromolecular level while sharing specific molecular characteristics which include elevated levels of reactive oxygen species (ROS) and matrix metalloproteinase (MMP) while collagen synthesis decreases. The skin functions as a protective barrier against the harsh environment and is essential for regulating body temperature. The stratum corneum (SC) is responsible for the main resistance to the penetration of most compounds; nevertheless the skin represents as an appropriate target for delivery. The target site for anti-aging treatment includes the epidermal and dermal layers of the skin. Calendula oil and L-carnitine L-tartrate was utilised as the cosmeceutical actives as they can be classified as a mixed category of compounds/products that lie between cosmetics and drugs. Both show excellent properties which can prove valuable during anti-aging treatment, whether it is due to the scavenging of ROS (calendula oil), moisturising effects (calendula oil and L-carnitine L-tartrate) or the improvement of the skin turnover rate (L-carnitine L-tartrate). The Pheroid™ delivery system can enhance the absorption of a selection of active ingredients. The aim of this study was to determine whether the Pheroid™ delivery system will enhance the flux and/or delivery of the named actives to the target site by performing Franz cell diffusion studies over an 8 h period, followed by tape stripping experiments. The Pheroid™ results of the actives were compared to the results obtained when 1 00 % calendula oil was applied and the L-carnitine L-tartrate was dissolved in phosphate buffer solution (PBS), respectively. In the case of calendula oil only a qualitative gas chromatography mass spectrometry (GC/MS) method could be employed. No calendula oil was observed to permeate through the skin, but linoleic acid (marker compound) was present in the epidermis and dermis layers. Components in the Pheroid™ delivery system hampered the results as the marker compound identified is a fundamental component of the Pheroid™, making it difficult to determine whether or not the Pheroid™ delivery system enhanced calendula oil's penetration. The aqueous solubility and log D partition coefficient of L-carnitine L-tartrate was determined. Inspection of the log D value of -1.35 indicated that the compound is unfavourable to penetrate the skin, whereas the aqueous solubility of 16.63 mg/ml in PBS at a temperature of 32º C indicated favourable penetration. During the Franz cell diffusion and tape stripping studies it was determined by liquid chromatography mass spectrometry (LC/MS) that carnitine may be inherent to human skin. Pheroid™ enhanced the flux (average of 0.0361 µg/cm2.h, median of 0.0393 µg/cm2.h) of the L-carnitine L-tartrate when compared to PBS (average of 0.0180 µg/cm2.h, median of 0.0142 µg/cm2.h ) for the time interval of 2 -8 h. The PBS was more effective in delivering the active to the target site (0.270 µg/ml in the epidermis and 2.403 µg/ml in the dermis) than Pheroid™ (0.111 µg/ml and 1.641 µg/ml in the epidermis and dermis respectively). Confocal laser scanning microscopy (CLSM) confirmed the entrapment of L-carnitine L-tartrate in the Pheroid™ vesicle, while in the case of calendula oil it was impossible to differentiate between the oil and the Pheroid™ components.<br>Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Zusammenfassung: Effekte der L-Carnitinsupplementierung auf das metabolische Profil adipöser und insulinre- sistenter Ponys im Verlaufe einer mehrwöchigen Körpergewichtsreduktion Author: Uta Schmengler Institut für Tierernährung, Ernährungsschäden und Diätetik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im September 2012 76 S., 16 Abb., 23 Tab., 169 Lit., Anhang Einleitung: Das ”Equine Metabolische Syndrom” ist gekennzeichnet durch eine regionale oder generalisierte Adipositas, eine periphere Insulinresistenz sowie akute oder chronische Hufreheschübe. Die Ursache ist in einer bedarfsübersteigenden, hochkalorischen Fütterung und einem relativen Bewegungsmangel zu suchen, wobei auch der genetischen Prädisposition spezieller Rassen eine gewisse Bedeutung zukommt. Ziel dieser Studie war die Untersuchung der Effekte einer L-Carnitinsupplementierung in Kombination mit einer restriktiven Füt- terung und täglicher moderater Bewegung auf Körpermasseverlust, Insulinsensitivität und ausgewählte Parameter des Energiestoffwechsels adipöser und insulinresistenter Ponys. Material und Methoden: Für die placebokontrollierte Doppelblindstudie wurden 16 adipöse Ponys per Losverfahren in zwei Gruppen (N=8) eingeteilt. Zu Versuchsbeginn wiesen die Ponys einen mittleren Body Condition Score von 8,0±2,0 (Skala 1-9) und einen mittleren Cresty Neck Score von 4,0±1,0 (Skala 0-5) auf. Während des 14-wöchigen Körpermassere- duktionsprogramms wurden die Ponys restriktiv gefüttert mit 1 - 1,2 kg Heu/100 kg KM/d. Zusätzlich erhielten 8 Ponys eine L-Carnitin-Zulage (1,3 g/100 kg KM/2d) und 8 Tiere ein Placebo in Form einer Kieselsäureverbindung (1,3 g/100 kg KM/ 2d). Die Ergänzungen wur- den in einem Gemisch aus Grünmehl (50 g/2d) und Mineralfutter verabreicht. Über die 14-wöchige Versuchszeit wurde ein Bewegungsprogramm an sechs Tagen in der Woche durch- geführt, das 25 Minuten Schritt und 15 Minuten Trab beinhaltete. Zu Versuchsbeginn und nach Versuchsende wurde mit beiden Versuchsgruppen ein Frequently sampled intravenous glucose tolerance test (FSIGTT) zur Überprüfung der Insulinsensitivität durchgeführt. Über die gesamte Versuchszeit wurden wöchentlich Blutproben gewonnen zur Bestimmung der ba- salen Serum-Insulinaktivität und Plasma-Glucosekonzentration sowie der Konzentration der Freien Fettsäuren (FFS), Triacylglyceride (TAG), Harnstoff und Betahydroxybutyrat (BHB) im Serum. Die Körpermasseverluste wurden über wöchentliche Wägungen sowie Ermittlung von BCS und CNS kontrolliert. Die statistische Überprüfung wurde anhand parametrischer (ANOVA) und nicht-parametrischer Tests (Wilcoxon signed rank test) durchgeführt, die Kal- kulation der Insulinsensitivität erfolgte über das Minimalmodell anhand eines Computerpro- gramms (MINMOD). Ergebnisse: Im Mittel verloren die Ponys über den Versuchszeitraum von 14 Wochen 1- 3% ihrer Körpermasse pro Woche (Zeit: p &lt; 0, 01, Behandlung: p=0,79), was einem totalen Körpermasseverlust von 14,3±% entsprach. Der BCS reduzierte sich in beiden Versuchs- gruppen um eine Differenz von 3 Einheiten, der CNS verringerte sich in der Carnitingrup- pe (GC ) um eine Differenz von 1,4 und in der Placebogruppe (GP ) um eine Differenz von 1,9 Einheiten. Der Körpermasseverlust war von einer signifikanten Verbesserung der Insu- linsensitivität (Zeit p &lt; 0, 01, Behandlung: p=0,39) begleitet. Die Kalkulation der Insulin- sensitivität im Minimalmodell zeigte eine signifikante Erhöhung der SI-Werte am Versuch- sende in beiden Versuchsgruppen (Beginn Studie GC : 0,76±0,88 l/min/μU*10−4 und GP : 1,61±1,31 l/min/μU*10−4 ; Ende Studie GC : 5,45±0,81 l/min/μU*10−4 und GP : 6,08±2,98 l/min/μU*10−4 ). Signifikante, zeitabhängige Veränderungen wurden auch für die metabo- lischen Parameter beobachtet: Plasma-Glucose und Serum-Insulin reagierten mit einem si- gnifikanten Abfall (Glucose GC : 4,5±0,32 mmol/l vs. 4,21±0,61 mmol/l und Glucose GP : 4,34±0,62 mmol/l vs. 3,86±0,34 mmol/l; Insulin GC : 23,71±32,77 μU/ml vs. 3,67±3,94 μU/ml und GP : 13,55±12,67 μU/ml vs. 1,01±1,09 μU/ml). Dabei kam es zu einem signi- fikanten Anstieg des Serum-Harnstoffs (GC : 3,47±0,73 mmol/l vs. 4,31±1,06 mmol/l und GP : 3,71±0,79 mmol/l vs. 4,9±1,23 mmol/l) sowie der Serum-FFS (GC : 157±95 μmol/l vs. 731±138 μmol/l und GP : 113±63 μmol/l vs. 686±142 μmol/l) und Serum-TAG (GC : 0,53±0,28 mmol/l vs. 0,94±0,61 mmol/l und GP : 0,45±0,23 mmol/l vs. 0,64±0,25 mmol/l). Bezüglich der L-Carnitinsupplementierung wurden keine weiteren Effekte verzeichnet. Schlussfolgerungen: Die restriktive Energiezufuhr von 7 MJ DE/100 kg KM entspre- chend einer Heuzulage von 1 kg/100 kg KM führte zu KM-Verlusten von 1-3 %. Eine Kör- permassereduktion zeigte deutliche Auswirkungen auf den Glucose- und Lipidmetabolismus und führte zu einer signifikanten Verbesserung der Insulinsensitivität, wohingegen die L- Carnitinsupplementierung keine weiteren Effekte auf den Glucosestoffwechsel herbeiführte. Eine bedarfsdeckende Eigensynthese von L-Carnitin ist beim Pony offensichtlich auch im Zu- stand der Insulinresistenz gewährleistet und reicht aus um die obligatorischen Funktionen L-Carnitins im Energiestoffwechsel zu erfüllen<br>Summary: The effects of L-carnitine supplementation on body weight losses and metabolic profile in obese and insulin resistant ponies during a several weeks lasting bodyweight reduction pro- gramme Author: Uta Schmengler Institute of Animal Nutrition, Nutrition Diseases and Dietetics, Faculty of Veterinary Medi- cine, University of Leipzig Submitted in September 2012 76 p., 16 fig., 23 tab., 169 ref., appendix Introduction: Insulin resistance, local or general adiposity and the predisposition towards acute or chronical laminitis are components of the equine metabolic syndrome. Contributing factors for this syndrome are the intake and the quality of a high caloric feed by a lack of physical exersice. Howewer, the genetically predisposition of so called ”easy keepers” seems to play a role in pathogenesis. The objective of this study was to investigate the effects of L- carnitine supplementation in combination with a body weight reduction programme (BWRP) on body weight (BW) losses, insulin sensitivity and selected metabolic parameters in obese and insulin resistant ponies. Material und methods: 16 obese ponies (mean BCS = 8.0±2.0, mean CNS = 4.0±1.0) were assigned to a randomized double blind, placebo-controlled study. The ponies werde di- vided into two equal groups (N=8). During a 14 weeks lasting BWRP the ponies were fed 1.0-1.2 kg hay/100 kg BW daily. Additionally, 8 ponies were supplemented with L-carnitine (1.3g/100 kg BW) and 8 ponies were supplemented with a placebo (1.3g/100 kg BW). The supplements were offered in a mixture of 50 g grass meal and 50 g of a commercial mineral mixture, twice a day. During BWRP ponies were exercised a low-intensity protocol 6 days a week (daily 25 min walk and 15 min trot across the countryside). A frequently sampled intravenous glucose tolerance test (FSIGTT) was undertaken in order to assess insulin sen- sitivity at the beginning and the end of the study. Routine blood samples were collected for analysis of plasma glucose, serum insulin, free fatty acids (FFA), triglycerides (TG), urea and beta-hydroxybutyrate (BHB). Ponies were weighed weekly after 12 h of feed restriction by using an electronic scale for large animals. BCS and CNS were recorded weekly by the same 2 observers throughout the study. The statistical analysis was performed by parametric and non-parametric tests (ANOVA and Wilcoxon ranked test). The minimal modell calcu- lation of insulin sensitivity (SI) from FSIGTT was calculated by the computer programme (MINMOD). Results: Ponies lost 1-3% BW per week over the BWRP (time P&lt;0.01, L-carnitine supple- mentation P=0.79), meaning a total body weight loss of 14.3%. BCS decreased in both groups with a difference of three points and CNS was reduced with a difference of 1.4-1.9 points. BW losses were accompanied by a significant improvement in insulin sensitivity (Time: P&lt;0.01, L-carnitine supplementation: P=0.39). The calculation for SI-values by the minimalmodell showed a significant increase in L-carnitine group (GC ) and placebo group (GP ) in the end of the study. (GC : 0.76±0.88 L/min/μU*10−4 to 5.45±0.81 L/min/μU*10−4 , GP : 1.61±1.31 L/min/μU*10−4 to 6.08±2.98 L/min/μU*10−4 ). Significant time related decreases were observed for plasma glucose (GC : 4.5±0.32 mmol/L to 4.21±0.61 mmol/L, GP : 4.34±0.62 mmol/L to 3.86±0.34 mmol/L) and serum insulin (GC : 23.71±32.77 μU/mL to 3.67±3.94 μU/mL, GP : 13.55±12.67 μU/mL to 1.01±1.09 μU/mL). A significant increase was observed for serum urea (GC : 3.47±0.73 mmol/L to 4.31±1.06 mmol/L, GP : 3.71±0.79 mmol/L to 4.9±1.23 mmol/L), FFA (GC : 157±95 μmol/L to 731±138 μmol/L und GP : 113±63 μmol/L to 686±142 μmol/L) and TG (GC : 0.53±0.28 mmol/L to 0.94±0.61 mmol/L, GP : 0.45±0.23 mmol/L to 0.64±0.25 mmol/L) during BWRP. There was no further improvement in metabolic responses by L-carnitine supplementation. Conclusions: Energy intake of 7 MJ DE/100 kg BW leads to bodyweight losses of 1- 3%, herby improving insulin sensitivity and glucose metabolism. L-carnitine supplementation does not further improve glucose or fat metabolism, suggesting that endogenous L-carnitine synthesis was sufficient to facilitate energy metabolism in obese and insulin resistant ponies

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and become one of the most common causes of chronic liver disease over the last decade in developed countries. The general prevalence of NAFLD is reported ranging between 20–30 % and 87 % in obese people. It is commonly associated with visceral obesity, type 2 diabetes mellitus, dyslipidemia and hypertension, all components of the metabolic syndrome, so that NAFLD might be considered an additional component of metabolic syndrome itself. As the rate of obesity, diabetes, and metabolic syndrome continue to increase, NAFLD will bring a tremendous impact on health care in the upcoming years. The underlying causes of the disease progression in NAFLD are unclear. Recent evidences suggest the development of lipid droplets (steatosis), subsequent generation of reactive oxygen species (ROS) and fibrosis deposition in the progression to non-alcoholic steatohepatitis (NASH). Moreover, continued elucidation has needed to understand fibrosis progression and regression. The paradigm of hepatic stellate cell (HSCs) activation remains the foundation for defining key translational challenges in order to accelerate the development of new therapies for patients with chronic liver disease. L-Carnitine (LCARN) is an essential nutrient that converts fat into energy in mitochondria. LCARN plays an important role in lipid metabolism; it acts as an essential cofactor for the β-oxidation of fatty acids. Very recently, LCARN has been proposed for the treatment of various diseases, including liver injury and several studies have shown that LCARN administration can ameliorate or prevent liver damage of various etiologies. We investigated the potential antioxidant and antifibrotic role of LCARN supplementation on methionine choline deficient (MCD)-diet-induced NAFLD in C57BL/6 mice. Mice were divided into three groups of CONTR (normal diet without any treatment), MCDD (MCD diet only), MCDD+LCARN 200 mg/kg/die group. Liver and heart weight, histological changes and fibrosis progression were assessed after 6 weeks of experiments. The MCD-diet induced severe hepatic fatty accumulation, but the fatty change was reduced in the MCDD+LCARN group. LCARN supplementation showed a role in controlling liver ROS generation and consequently coordinating the HSCs activation. Additionally, the same antioxidant and antifibrotic effect was observed in the myocardium. In conclusion, our findings indicate that LCARN has a potential role in control NAFLD progression to NASH. Therefore, our data suggest that LCARN may acts as a novel and potent supplementation agent against NAFLD cardiac complications.

У хворих із неалкогольною жировою хворобою печінки, яким додатково до стандартної лікувальної програми призначили L-карнітин, упродовж двох тижнів лікування ефективніше коригувалися показники, які відображають процеси цитолізу, холестазу та інтоксикаційного синдрому. Застосування L-карнітину сприяло достовірному зменшенню вмісту фактора некрозу пухлин-альфа, що свідчить про зниження інтенсивності процесів запалення, а також зниженню вмісту лептину на тлі зростання концентрації адипонектину у крові, що вказує на нормалізацію адипокінового дисбалансу.<br>Кафедра пропедевтики внутрішніх хвороб

This study examined the effects of 14 days of L-carnitine supplementation on blood and muscle lactate concentrations, and carnitine fractions, during high intensity sprint cycling exercise. Eight subjects performed three experimental trials - control I (CON I, 0 days), control II (CON II, 14 days), and L-carnitine (LCN, 28 days). Each trial consisted of a 4 min ride at 90% VO2max, followed by a rest period of 20 min, and then 5 x 1 min rides at 115% VO2max (2 min restbetween each). Following CON II, all subjects began dietary supplementation of L-carnitine for a period of 14 days (4 g/day). L-carnitine supplementation had no significant effect on either muscle carnitine or lactate concentrations following the 4 min 90% ride. Plasma total acid soluable and free carnitine concentrations were significantly higher at all time points following supplementation. Differences observed in blood hydrogen ion and lactate concentrations between CON I and CON II appear to be the result of an order effect. The data from the present investigation indicate that L-carnitine supplementation has no significant effect on blood or muscle lactate accumulation following high intensity sprint cycling exercise.<br>School of Physical Education

Submitted by Amauri Alves (amauri.alves@ufv.br) on 2015-10-16T11:25:49Z No. of bitstreams: 1 texto completo.pdf: 566274 bytes, checksum: 435b70c31d30f285a894df8e6609cd67 (MD5)<br>Made available in DSpace on 2015-10-16T11:25:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 566274 bytes, checksum: 435b70c31d30f285a894df8e6609cd67 (MD5) Previous issue date: 2015-06-26<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>O objetivo deste trabalho foi avaliar os efeitos da adição de L-carnitina, um acelerador do metabolismo lipídico, e ácido linoléico conjugado (trans-10, cis-12) em diferentes fases da produção in vitro de embriões sobre o desenvolvimento e criotolerância embrionária. Em todos os experimentos, embriões foram produzidos in vitro utilizando-se complexos cumulos-oócitos (CCO) provenientes de ovários coletados em abatedouro. Foram calculadas as taxas de clivagem (dia 3 do cultivo), taxa de formação de blastocistos expandidos e blastocistos em estágio avançado de desenvolvimento (dia 7 do cultivo) em função do total de CCO inseminados. Embriões em estágio de blastocisto expandido foram coletados de cada tratamento no dia 7 do cultivo in vitro e submetidos ao congelamento lento. Avaliou-se a taxa de reexpansão e eclosão embrionária 24, 48 e 72 h após o descongelamento em função do total de embriões descongelados. No experimento 1, oócitos fertilizados foram incubados em meio de cultivo contendo diferentes concentrações de L-carnitina (0,00; 0,75; 1,50 ou 3,03 mM) suplementado ou não com 5 % de soro fetal bovino (SFB). A adição de L-carnitina ao meio de cultivo na concentração de 1,5 mM melhorou a taxa de formação de blastocistos em estágio avançado de desenvolvimento quando comparado ao grupo controle (15,2±2,0 vs. 11,2±1,5; P<0,05). A L-carnitina também apresentou efeito positivo sobre a reexpansão embrionária 24 e 48 h pós-descongelamento quando adicionada na concentração de 0,75 e 3,03 mM (77,4±4,5; 80,1±4,0 e 75,0±5,5; 78,0±4,2 vs. 64,0±4,7; 67,4±4,7) ao meio de cultivo (P<0,05). Não houve interação entre os efeitos da adição de L-carnitina e SFB ao meio de cultivo embrionário. Apesar da suplementação do meio de cultivo com SFB ter melhorado o desenvolvimento embrionário (27,2±1,1 vs. 19,4±0,9; P<0,01), houve uma redução das taxas de reexpansão 24, 48 e 72 h pós-descongelamento (65,8±2,9; 67,8±2,8; 66,0±3,0 vs. 78,1±3,8; 81,4±3,0; 79,1±3,5; P<0,01). No experimento 2, os embriões foram cultivados em meio contendo L-carnitina (0,75 mM) ou ácido linoleíco conjugado (CLA – 100 mM) durante as primeiras 96 h, últimas 72 h ou durante todo o cultivo in vitro. A suplementação do meio de cultivo com L-carnitina ou CLA durante diferentes vifases do cultivo in vitro não afetou o desenvolvimento e a criotolerância embrionária. No experimento 3, avaliou-se o desenvolvimento e a crioresistência embrionária quando oócitos foram maturados em meio suplementado com L-carnitina (3.03 mM) e/ou CLA (100 mM). L-carnitina e CLA não afetaram o desenvolvimento embrionário quando adicionados ao meio de maturação. Apesar da L-carnitina não ter afetado a a taxa de reexpansão embrionária pós-descongelamento, o CLA apresentou efeito negativo sobre a eclosão embrionária 72h pós-descongelamento (53,3±3,6 vs. 65,1±4,3; P<0,05) quando adicionado ao meio de maturação oócitaria. Como conclusão, a L- carnitina melhora a criotolerância de embriões produzidos in vitro quando adicionada à concentração de 0,75 mM ao meio de cultivo embrionário. Já o CLA apresenta efeito negativo sobre a sobrevivência embrionária após o descongelamento quando adicionado ao meio de maturação oócitaria.<br>High lipid content in embryo is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine and trans-10, cis-12 (t10, c12) conjugated linoleic acid (CLA) on in-vitro development and cryotolerance of bovine embryos when added during different stages of in vitro production of embryos. For all experiments, embryos were produced in vitro using slaughterhouse cows oocytes. Cleavage rates on Day 3, blastocyst and advanced blastocyst (hatching/hatched blastocyst) formation rates on Day 7 were calculated from the total number of oocytes subjected to in vitro fertilization (IVF). Expanded blastocysts-stage embryos from each treatment were harvested on Day 7 and subjected to slow freezing. Embryo viability was assessed 24, 48 and 72 h after thawing. In experiment 1, fertilized oocytes were incubated with different L-carnitine concentrations (0.0, 0.75, 1.50 or 3.03 mM) in the presence or absence of fetal bovine serum (FBS). There was an improvement (P<0.05) on embryo development when 1.5 mM of L-carnitine was added to in vitro culture (IVC) medium. L-carnitine had also a positive effect (P<0.05) on post thaw embryo competence when supplemented at 0.75 and 3.03 mM during IVC. There was no interaction (P>0.05) between the effects of L-carnitine and FBS supplementation on IVC on embryo development and cryosurvival. Although FBS supplementation had increased blastocyst development (P<0.05), it reduced the reexpansion rates at 24, 48 and 72 h post thawing. In experiment 2, L-carnitine (0.75 mM) or CLA (100 mM) were supplemented during the first 96 h, last 72 h or throughout the entire IVC period. There was no effect of L-carnitine or CLA supplementation during different periods of IVC on embryo development and cryotolerance. In experiment 3, embryo development and cryosurvival were evaluated when oocytes were maturated in medium supplemented with L-carnitine (3.03 mM) or/and CLA (100 mM). No effect of L-carnitine and CLA supplementation during in vitro maturation (IVM) on IVP embryo development was detected. Although there was no effect (P>0.05) of L-carnitine supplementation on embryo cryotolerance, CLA showed a negative effect (P<0.05) on embryo cryosurvival when added during IVM. In conclusion, L-carnitine improved embryo cryosurvival viiiwhen added at 0.75 mM during IVC and CLA supplementation during IVM has a negative effect on post thaw embryo survival.

Neurofibromatosis type 1 (NF1), previously termed von Recklinghausen’s disease, is a genetic disorder characterised by the development of tumours on the nerves. NF1 can also affect muscle, with hypotonia, muscle weakness and fatigue able to have a profound impact on paediatric quality of life. This thesis investigates an underlying metabolic myopathy, and the potential of dietary intervention to treat the muscular symptomology in NF1. In Chapter 2, we utilised the Nf1Prx1-/- mouse model to test a range of dietary interventions, including 1) a medium-chain fatty acid (MCFA)/L-carnitine combination, 2) MCFA/L-carnitine with reduced dietary compliance (5/7 days/week), 3) MCFA alone, 4) L-carnitine alone, 5) a “mitochondrial cocktail” (L-carnitine, riboflavin, CoQ10 and creatine), and 6) a low fat diet. Oil Red O (ORO) analysis revealed a significant reduction of intramyocellular lipid (IMCL) accumulation following every dietary intervention, excluding the low fat diet. The MCFA/L-carnitine combination resulted in a significant reduction of IMCL within four weeks, but this was not sustained upon reversion to a standard diet. Lipidome analysis revealed consistent and separable changes in muscle lipidome profiles, by genotype and dietary treatment. Nf1-deficient muscle demonstrated significantly elevated acylcarnitine levels (particular C16 and C18:1) compared to WT. Taken together, these data support the theory of an underling metabolic problem that can be treated by dietary intervention. In Chapter 3, we used a qualitative approach to explore the decision-making process of families who chose to self-supplement their child with L-carnitine. Thematic analysis revealed that primary muscular symptoms and psychosocial repercussions, the ineffectiveness of current treatment strategies, and research availability and accessibility were driving factors in their decision. During L-carnitine supplementation, every parent perceived a significant improvement in their child’s physical abilities. Further guidance around the optimum dose for their child, research accessibility and sharing health findings, and the development of a clinical trial were queried. This study hints at the potential of L-carnitine supplementation in the context of NF1. In Chapter 4, we conducted the first Phase 2a clinical trial of L-carnitine supplementation for NF1-associated muscle weakness and fatigue. Six children aged between 8-12 years with a confirmed diagnosis of NF1, history of muscle weakness and fatigue, and naïve to L-carnitine supplementation were enrolled in the study. There were no adverse events or side effects reported throughout the study with kidney and liver function tests confirming the safety profile, and compliance rate was high. There was evidence of improved foot strength (plantarflexion and dorsiflexion movements), as well as long jump and six minute walk distances. Plasma acylcarnitines were low, but not within a range clinically linked to carnitine deficiency. Upon three-month follow up, four out of six families elected to continue L-carnitine supplementation. These data support a Phase 3 clinical trial of L-carnitine supplementation in NF1.

Very long-chain fatty acids, especially n-3 polyunsaturated fatty acids (PUFAs), play an integral role in several physiological processes and are essential components of phospholipids in membranes. Fatty acid elongation in the cytoplasm (endoplasmic reticulum) and in the peroxisomes utilizes malonyl-CoA as a carbon source, whereas mitochondria1 elongation uses acetylcarnitine or acetyl-CoA. Fatty acids are involved in the biosynthesis of docosahexaenoic acid (DHA, 22:6n-3) and the formation of acetylcarnitine. DHA's last biosynthesis step consists of one peroxisomal P-oxidation cycle of its precursor, C24:6n-3. After a few cycles of peroxisomal P-oxidation, the fatty acid is transported into the mitochondrion for further P-oxidation. Previous research showed that MPTP causes a decrease of acetyl-CoA production due to the inhibition of the acyl-CoA dehydrogenase enzymes, ETF, or ETF-QO. Experimental animals treated with MPTP developed neurological damage which leads to clinical symptoms similar to Parkinson's disease and a metabolic profile similar to GA II, suggesting that it may be possible to induce a deficiency similar to GA II. GA II may in several ways be linked to extrapyramidal symptoms, such as a decrease of acetyl-CoA production which is characteristic of GA II. We argued that if the neurological symptoms are the result of deceased acetyl-CoA production, which could prevent fatty acid elongation, the problem could possibly be rectified by supplementation with acetylcarnitine. Acetylcarnitine supplementation had already been shown to prevent the development of the clinical symptoms associated with MPTP treatment, but it has not been known if acetylcarnitine will increase the fatty acid elongation. Our first objective of this study was to determine whether ALCAR plays a role in very long-chain fatty acid metabolism by influencing the fatty acid elongation process. To test this hypothesis, rats were treated with ALCAR and their serum analysed for certain metabolites. The fatty acid concentrations were expressed in consequential ratios (C24:C22 and C26:C22), which provide a more sensitive criterion than concentrations per se to indicate the possible defective enzymes involved in the fatty acid elongation pathway. The results showed that ALCAR had no prominent role in long-chain fatty acid metabolism and could therefore not be responsible for the preservation of peroxisomal P-oxidation or fatty acid elongation. However, concentrations of the C22:0 and C24:0 fatty acids were increased on certain days. A statistical significant higher concentration in the experimental group was observed in the C22:0concentrations on day 1, 7 and 13 and for the C24:0 concentrations on day 7, 13 and 14. Our second objective was to determine acetyl- and acylcamitine concentrations in ALCAR treated rats before and after a single treatment with the GA II inducing chemical, MPTP. The acetylcarnitine concentration of the experimental group was statistically significant elevated on day 1, but thereafter the concentrations of both groups stabilized and were similar. The glutarylcarnitine concentrations of the experimental group significantly increased after MPTP treatment. Our data suggest that the influence of ALCAR on VLCFA metabolism is not significant enough to substantiate the hypothesis that ALCAR plays an appreciable role in preserving peroxisomal P-oxidation and fatty acid elongation. ALCAR does not influence the VLCFA biosynthesis under normal circumstances, but it does activate VLCFA biosynthesis when their concentrations are decreased. ALCAR treatment can however be considered as safe in the treatment of VLCFA and neurodegenerative defects, since it has little/no effect on the VLCFA biosynthesis. ALCAR promoted the formation of some acylcamitines (especially glutarylcamitine), conjugates of the GA II metabolites, after MPTP treatment. Thus, it can be concluded that ALCAR possibly play an important role in the detoxification of GA II metabolites.<br>Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2006.

Introdução. A criopreservação de oócitos é importante, tanto para a tentativa de preservação da fertilidade feminina, como nos tratamentos de reprodução assistida. Hipotetizamos que novas formulações de meios crioprotetores, contendo biomoléculas que participam da estrutura e funcionalidade celular, em particular da função mitocondrial e da dinâmica do sistema de membranas, poderiam melhorar a criotolerância e segurança da vitrificação. Objetivos. Este estudo teve como objetivo investigar os efeitos do meio padrão de vitrificação (T4) e T4 suplementado com L-carnitina (LC), LC-ácidos graxos (T4-AG) e LC-AGfosfatidilcolina (T4-PC) sobre a sobrevida e qualidade de oócitos criopreservados, mensurada por parâmetros do desenvolvimento in vitro de embriões, como número de núcleos totais (NT), de células na massa celular interna (MCI) e trofectoderma (TE) dos blastocistos oriundos de oócitos vitrificados nestes meios, assim como sobre os padrões de atividade mitocondrial oocitária. Materiais e métodos. Estudo experimental usando o camundongo da cepa C57BL/6 como modelo. Oócitos maturados in vivo, foram distribuídos em 5 grupos: controle a fresco (CT) e 4 grupos vitrificados: T4, T4-LC, T4-AG e T4-PC. Após a desvitrificação, foi analisada a sobrevida oocitária e os oócitos viáveis foram submetidos a fertilização in vitro ou utilizados para análise da atividade mitocondrial, por meio da análise de espécies reativas de oxigênio (EROs) intracelular por Intensidade de Fluorescência emitida pelo diacetato de 2,7- diclorodihidrofluoresceína, metabolismo oxidativo pela autofluorescência de dois fluoróforos endógenos [dinucleótideo de flavina adenina oxidado e o dinucleotideo reduzido de nicotinamida adenina (fosfato)] e potencial de membrana mitocondrial por Intensidade de Fluorescência (IF) emitida pelo JC1. Os oócitos dos 5 grupos submetidos a FIV foram cultivados por 96 horas, sendo comparados entre os grupos: taxa de fertilização e formação de blastocisto, assim como o número de NT, células MCI e TE e tamanho dos blastocistos. Resultados. A taxa de sobrevivência dos oócitos vitrificados em T4 foi superior a dos demais grupos T4-LC, T4-AG e T4-PC (respectivamente 100%, 97,07%, 96,75% e 97,95%). A taxa de fertilização do grupo CT (77,5%) foi superior a dos grupos T4, T4-LC e T4-PC, não diferindo do grupo T4-AG. Não foram evidenciadas diferenças estatisticamente significativas nas taxas de formação de blastocistos entre os grupos CT, T4, T4-LC, T4-AG e T4-PC (58,07%, 48,05%, 47,44%, 57,89% e 51,06%,respectivamente). Comparando-se o número NT e de células do TE dos blastocistos, observou-se que o os valores do grupo controle foram maiores do que o dos outros 4 grupos, que não apresentaram diferenças entre si. O número de células da MCI dos blastocistos do grupo controle foi superior ao dos grupos T4 e T4-LC e similar ao dos grupos T4-AG e T4- PC. Para o perfil de atividade mitocondrial, foram analisados 15 oócitos/grupo. Os níveis de EROs foram maiores no grupo CT comparado ao grupo T4 e menor quando comparado aos grupos T4-LC e T4-AG, mas sem diferença significativa em relação ao grupo T4-PC. Para Resumo analise do estado redox, o grupo CT teve maiores valores do que a dos grupos T4, T4-LC e T4- AG e o grupo T4-PC foi maior do que a dos outros quatro grupos. Para o potencial de membrana mitocondrial, o grupo CT teve maiores valores do que a do grupo T4-LC, menor do que a do grupo T4 e sem diferença estatisticamente significativa com a dos grupos T4-AG e T4-PC. Conclusão. O número de NT, células do TE e tamanho do blastocistos foram inferiores nos grupos vitrificados/desvitrificados em meio padrão e suplementados comparados ao controle. Porém, o número de células da MCI não foi diferente entre o grupo controle e os vitrificados T4- AG e T4-PC, sugerindo que a suplementação dos meios padrão com LC, AG e PC possa melhorar a competência do oócito e a subsequente qualidade embrionária, o que precisa ser melhor investigado antes da aplicação clínica dos novos meios. Apesar dos oócitos vitrificados no meio T4 terem apresentado taxa de sobrevivência estatisticamente superior a dos demais grupos, sugerimos que estas diferenças não apresentariam potencial relevância clínica. Os resultados de atividade mitocondrial revelam que há uma diferença importante entre oócitos controle e vitrificados com ou sem suplementos na eficiência da função mitocondrial, regulação do estado redox e controle intracelular das EROs, demonstrando que mais trabalhos são necessários para entender esta via metabólica e os diferentes efeitos dos meios de vitrificação.<br>Introduction. Cryopreservation of oocytes is important, both for the attempt to preserve female fertility and for assisted reproduction treatments. We hypothesize that novel cryoprotective formulations containing biomolecules that participate in cellular structure and functionality, particularly mitochondrial function and membrane system dynamics, could improve cryotolerance and safety of vitrification. Objeticve. The objective of this study was to investigate the effects of the standard vitrification medium (T4) and T4 supplemented with L-carnitine (LC), LC-fatty acids (T4-AG) and LC-AGphosphatidylcholine (T4-PC) on survival and quality of cryopreserved oocytes, measured by in vitro embryo development parameters, such as number of total nuclei (NT), cells in the internal cell mass (ICM) and trophoectoderma (TE) of the blastocysts derived from vitrified oocytes in these media, as well as on the patterns of oocyte mitochondrial activity. Materials and methods. Experimental study using the C57BL/6 mouse as a model. Matured in vivo oocytes were distributed into 5 groups: fresh (CT) control and four vitrified groups: T4, T4-LC, T4-AG and T4-PC. After devitrification, oocyte survival was analyzed and viable oocytes were submitted to in vitro fertilization or used for analysis of mitochondrial activity, through the analysis of intracellular reactive oxygen species (ROS) by Fluorescence Intensity emitted by diacetate of 2, 7-dichlorodihydrofluorescein, oxidative metabolism by the autofluorescence of two endogenous fluorophores [oxidized flavin adenine dinucleotide and the reduced dinucleotide of nicotinamide adenine (phosphate)] and mitochondrial membrane potential by Fluorescence Intensity (FI) emitted by JC1. The oocytes of the 5 groups submitted to IVF were cultured for 96 hours, being compared between the groups: fertilization rate and blastocyst formation, as well as number of NT, MCI and TE cells and size of blastocysts. Results. The survival rate of vitrified oocytes in T4 was higher than in the other groups T4-LC, T4-AG and T4-PC (respectively 100%, 97.07%, 96.75% and 97.95%). The fertilization rate of the CT group (77.5%) was higher than that of the T4, T4-LC and T4-PC groups, not differing from the T4-AG group. There were no statistically significant differences in the rates of blastocyst formation between the groups CT, T4, T4-LC, T4-AG and T4-PC (58.07%, 48.05%, 47.44%, 57.89% and 51.06%, respectively). Comparing the NT and TE cells of the blastocysts, it was observed that the values of the control group were higher than that of the other 4 groups, which did not present differences between them. The number of MCI cells from the blastocysts of the control group was higher than that of the T4 and T4-LC groups and similar to that of the T4-AG and T4-PC groups. For the mitochondrial activity profile, 15 oocytes/group were analysed. The levels of EROs were higher in the CT group compared to the T4 group and lower when compared to the T4-LC and T4-AG groups, but without significant difference in relation to the T4-PC group. For the analysis of the redox state, the CT group had higher values than the T4, T4-LC and T4-AG groups and the T4-PC group was higher than the other four groups. For the mitochondrial membrane potential, the CT group had higher values than the T4-LC group, lower than the T4 group and without a statistically significant difference with the T4-AG and T4-PC groups. Conclusion. The number of NT, TE cells and blastocyst size were lower in the vitrified/devitrified groups in standard medium and supplemented compared to control. However, the MCI cell number was not different between the control group and the vitrified T4-AG and T4-PC, suggesting that the supplementation of the standard media with LC, GA and CP could improve oocyte competence and subsequent embryo quality, which needs to be better investigated before the clinical application of the new media. Although vitrified oocytes in the T4 medium had a statistically higher survival rate than the other groups, we suggested that these differences would not present potential clinical relevance. The results of mitochondrial activity reveal that there is an important difference between control and vitrified oocytes with or without supplements in the efficiency of mitochondrial function, regulation of the redox state and intracellular control of ROS, demonstrating that more work is needed to understand this metabolic pathway and the different effects of the vitrification media.

Master of Science<br>Department of Animal Sciences and Industry<br>Joel DeRouchey<br>Mike Tokach<br>Six experiments using 3,862 pigs were conducted to evaluate effects of ractopamine HCl (RAC) feeding programs, dietary L-Carnitine and dried distillers grains with solubles (DDGS) on growth, carcass traits, loin and jowl fat quality of pigs, and energy and protein sources in nursery diets. In Exp. 1 and 2, RAC-fed pigs had greater (P<0.05) ADG, G:F and HCW compared with the control. Within RAC treatments, there were no differences in growth. Pigs fed step-up RAC had increased (P<0.01) percentage lean, fat-free lean index and loin depth but decreased (P<0.01) backfat than the control or constant treatment. In Exp. 2, pigs fed step-up RAC program had greater (P<0.05) ADG and G:F than the constant treatment. Pigs fed constant RAC had greater (P=0.002) carcass yield than controls. There were no overall differences in other carcass traits among treatments. In Exp. 3, dietary L-Carnitine improved (P<0.02) ADG and final BW. A DDGS × L-Carnitine interaction (quadratic, P<0.01) was observed for G:F. Pigs not fed DDGS had similar G:F, but in DDGS diets pigs fed 50 ppm L-Carnitine had worse G:F than those fed 100 ppm. Pigs fed L-Carnitine had greater (P<0.02) HCW compared with those not fed L-Carnitine. Increasing L-Carnitine up to 100 ppm increased HCW (quadratic, P<0.03) and backfat (quadratic, P<0.04), with the maximum response at 50 ppm dietary L-Carnitine. Increasing L-Carnitine increased (linear, P<0.04) purge loss of loin. Feeding DDGS increased (P<0.001) linoleic acid and iodine value of jowl fat compared with feeding no DDGS. However, feeding L-Carnitine did not change jowl fatty acid composition. In Exp. 4, 5 and 6, nursery pigs fed choice white grease (CWG) had improved (P<0.02) G:F than pigs fed a control diet or an alcohol based energy source. Also, pigs fed CWG had greater (P<0.04) ADG in Exp. 4 and 6 and had reduced (P<0.01) ADFI in Exp. 5. The alcohol based energy source improved (P<0.04) ADG and ADFI with no change in G:F in Exp. 4; but did not affect growth in Exp. 5 and 6. In Exp. 6, pigs fed AV-E Digest had equal performance as nursery pigs fed other specialty proteins.

The objectives were to: 1) determine if im treatments of Lutalyse expedited the training of sexually inexperienced boars for semen collection and increased spermatozoal output, and 2) determine the effects of dietary L-carnitine supplementation on boar libido, semen quality, sperm production, and maintenance of sperm motility during liquid storage. Experiment 1 utilized lean-type, terminal-line boars (National Pig Development, Roanoke Rapids, NC) (n = 40; 177.4 ± 2.4 d of age and 112.8 ± 2.0 kg body weight) that had not previously experienced natural mating. Boars were individually moved twice weekly for 6 weeks (total of 12 training sessions) to a semen collection room equipped with an artificial sow. Upon entering the semen collection room, boars received in treatments of either deionized water (4 mL, n = 10) or Lutalyse at doses of 5 mg (n = 10), 10 mg (n = 10), or 20 mg (n = 10), and subsequently received a libido score of 1 to 5 (1 = no interest in the artificial sow; 5 = mounting the artificial sow and allowing semen collection). The percentages of boars successfully trained for semen collection during the experimental period were similar (P > 0.05) for controls (20%) and boars receiving 5 mg (30%), 10 mg (20%), or 20 mg (10%) of Lutalyse. Average libido score for boars receiving 10 mg Lutalyse (2.35 ± 0.08) was greater (P < 0.05) than for controls (2.14 ± 0.06). Libido score for the 20 mg treatment group were (1.78 ± 0.06) lower (P < 0.05) compared to the other treatment groups. Characteristics of ejaculates (volume, gel weight, sperm concentration, total spermatozoa) from control boars and boars treated with Lutalyse at doses of 5, 10, or 20 mg were similar (P > 0.05). For Exp. 2, the same group of boars was utilized in two similar trials (Trial 1, 1a, 1b: n = 9 for control and L-carnitine-treated boars; Trial 2, 2a, 2b: n = 10 for control and L-carnitine-treated boars). Boars were fed a fortified, corn and soybean meal-based diet at a rate of 2 kg/d. Boars that were randomly selected for L-carnitine treatment received the same diet mixed with L-carnitine to achieve supplementation of 500 mg/d. For 16 wk, semen was collected weekly via the gloved hand method and was analyzed for gel-free volume, gel weight, sperm concentration, sperm per ejaculate, and characteristics of sperm motility. Time to ejaculation (reaction time), duration of ejaculation, and number of false mounts were also recorded for each collection. Trials 1a and 2a were conducted during weeks 16 and 17 for each respective trial. Boars were collected once on 4 consecutive days, allowed 4 d of rest, and then collected again, to estimate daily spermatozoal production. At the end of 16 wk, a semen sample was also processed and extended in Beltsville Thawing Solution (BTS) to achieve a dilution of 3 x 109 spermatozoa/100 mL-dose for Trials 1b and 2b. The extended semen was stored in plastic bottles at 18°C and motility was evaluated daily for 7 d post collection. L-carnitine supplementation for 16 wk had no effects on semen volume, gel weight, total number of sperm cells per ejaculate, reaction time, or sperm motility (P > 0.1). Boars receiving the L-carnitine-supplemented diet displayed an increase in the number of false mounts before ejaculating and an increase in sperm concentration (P < 0.05) in Trial 2. A treatment by week interaction was detected for sperm concentration in Trial 2 (P < 0.005). Increased sperm concentrations in L-carnitine-treated boars were demonstrated after only one week of feeding the respective diets. Given that the production of a mature sperm cell requires 7 to 8 wk in boars, it is therefore difficult to conclude that differences in sperm concentration were due solely to treatment. Daily spermatozoal production was similar between control boars and boars supplemented with L-carnitine (P > 0.1) for both Trials 1a and 2a. L-carnitine supplementation did not affect percent motility in Trials 1b and 2b or sperm progressive motility in Trial 2b during 7 d storage (P > 0.1). A treatment by day interaction was determined for sperm velocity (P < 0.05) in Trial 2b. L-carnitine supplementation decreased mean sperm velocity significantly after 2 d of storage. Overall, L-carnitine had no beneficial effects on boar libido, semen quality, sperm production, or maintenance of sperm motility during liquid storage. However, Lutalyse increased libido scores, but did not affect the number of boars trained for semen collection or number of spermatozoa ejaculated.<br>Master of Science

A doença da urina do xarope do bordo (MSUD) é causada pela deficiência na atividade do complexo da desidrogenase dos U-cetoácidos de cadeia ramificada (BCKAD), promovendo o acúmulo dos aminoácidos de cadeia ramificada (BCAA) leucina (Leu), isoleucina (Ile) e valina (Val) e seus U-cetoácidos correspondentes (BCKA). A MSUD caracteriza-se por cetoacidose, ataxia, coma, retardo mental e psicomotor. Estudos em animais demonstraram que BCAA e BCKA estimulam a lipoperoxidação e reduzem capacidade antioxidante cerebral em ratos. Também há evidências de que o estresse oxidativo ocorra em pacientes com MSUD no diagnóstico e durante o tratamento e que devido à terapia com dieta restrita e hipoproteica eles possuam deficiência de L-carnitina (L-car), um importante composto para o metabolismo energético. Recentemente, estudos demonstraram o papel antioxidante e anti-inflamatório da L-car, através de sua ação antiperoxidativa, sequestradora de espécies reativas e efeito estabilizador de danos às membranas celulares. Considerando que a fisiopatologia da MSUD ainda é pouco compreendida e que existe um crescente número de estudos enfatizando o envolvimento do estresse oxidativo na doença, neste trabalho foi investigado o efeito in vitro e in vivo da L-car sobre o estresse oxidativo e o dano inflamatório na MSUD tendo como objetivos: A) estudar a indução ao dano oxidativo pelos metabólitos acumulados na MSUD, verificando o possível papel antioxidante da L-car sobre o dano ao DNA in vitro; B) avaliar o efeito in vivo da suplementação de 50 mg/kg/dia de L-car sobre: b.1) a indução do dano ao DNA em leucócitos de pacientes com a MSUD tratados com dieta de restrição proteica, correlacionando as concentrações dos principais metabólitos acumulados nesta doença e verificando o possível papel antioxidante da suplementação da Lcar; b.2) a concentração de citocinas pró-inflamatórias em plasma de pacientes com MSUD tratados com dieta de restrição proteica e a correlação com o estresse oxidativo; b.3) os parâmetros de dano oxidativo à biomoléculas em urina de pacientes com MSUD sob dieta de restrição proteica; C) avaliar o efeito da L-car sobre o estresse oxidativo causado pelos metabólitos acumulados na MSUD em córtex cerebral e cerebelo de ratos Wistar, através de um modelo crônico de indução química da doença. Verificou-se que a Leu e o seu - cetoácido correspondente, o ácido -cetoisocapróico (KIC), causaram danos ao DNA in vitro e L-car foi capaz de diminuir significativamente essas alterações, principalmente as causadas pelo KIC. Quando testado o efeito da suplementação de L-car sobre o dano ao DNA em pacientes MSUD, observou-se um aumento significativo de lesões ao DNA em pacientes com dieta de restrição proteica quando comparados aos controles e a terapia com L-car foi capaz de diminuir significativamente os níveis desses danos. Também foram verificadas correlações do tipo negativa entre as concentrações de L-car e os índices de dano ao DNA e do tipo positiva entre as lesões ao DNA e níveis de MDA, marcador de lipoperoxidação, explicitando uma relação entre o dano ao DNA observado nos pacientes com MSUD, estresse oxidativo e o benefício da suplementação de L-car. Também averiguou-se o efeito da terapia de L-car sobre as citocinas pró-inflamatórias interleucina 1Y (IL-1Y), interleucina 6 (IL-6) e interferon gama (INF- Z). Constatou-se aumentos significativos de IL-1Y, IL-6 e INF- Z no plasma de pacientes com MSUD antes da suplementação de L-car e uma reversão completa desses valores aos níveis dos controles para IL-1Y e INF- Z após a administração de L-car. Ainda, verificou-se que a L-car pode auxiliar na defesa celular contra a inflamação e o estresse oxidativo, observando-se uma correlação negativa entre todas citocinas testadas e as concentrações de L-car, e uma correlação positiva entre o conteúdo de MDA e níveis de IL-1Y e IL-6. Constatou-se também que as medidas de di-tirosina (dano oxidativo a proteínas) e isoprostanos (dano de lipoperoxidação) estavam aumentadas e a capacidade antioxidante total diminuída na urina de pacientes com MSUD sem terapia com L-car e a suplementação deste composto induziu efeitos benéficos sobre estes parâmetros, reduzindo os níveis de di-tirosina e isoprostanos e aumentando a capacidade antioxidante medida em urina. Foi também observado um aumento de KIC urinário após dois meses de tratamento com L-car, quando comparado com o grupo controle, demonstrando um incremento da excreção deste metabolito tóxico. Desta forma, esses resultados sugerem um efeito de reversão de dano oxidativo pela L-car e que a urina pode ser utilizada para monitorar este tipo de lesão em pacientes afetados pela MSUD. Por fim, foram analisados em córtex cerebral e cerebelo de ratos Wistar submetidos ao modelo crônico de MSUD: espécies reativas ao ácido tiobarbitúrico (TBARS), para avaliar lipoperoxidação, conteúdo de carbonilas (dano oxidativo proteico), oxidação de diclorofluoresceína (DCF), para quantificar produção de espécies reativas teciduais, conteúdo de glutationa reduzida (GSH) que é um importante antioxidante não enzimático e a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx) e glicose-6-fosfato-desidrogenase (G6PD). Os resultados mostraram que a administração crônica de BCAA estimulou a lipoperoxidação, o dano oxidativo proteico, aumento de espécies reativas e diminuição das defesas antioxidantes enzimáticas e não enzimáticas, especialmente em córtex cerebral e o tratamento com L-car foi capaz de prevenir estes efeitos, exceto o dano oxidativo a proteínas. Em conjunto, estes resultados demonstram que os metabólitos acumulados na MSUD induzem dano oxidativo a biomoléculas (lipídios, proteínas e DNA), diminuem o status antioxidante e promovem aumento de processos inflamatórios. Ainda, estes dados podem contribuir para a compreensão dos mecanismos de ação dos efeitos citotóxicos dos metabólitos acumulados na MSUD e evidenciar o papel do estresse oxidativo e da inflamação na neuropatofiosiologia desta doença, além do efeito protetor da L-car sobre este processo. O estudo de antioxidantes, como a L-car, pode propor uma abordagem terapêutica adicional ao que é empregado atualmente para pacientes com MSUD, que é essencialmente dietética e, portanto, de difícil manejo.<br>Maple syrup urine disease (MSUD) is caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain U-ketoacid dehydrogenase (BCKAD). The metabolic defect leads to accumulation of the branched chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val) and the corresponding branched-chain U-keto acids. The clinical features of MSUD include ketoacidosis, seizures, coma, psychomotor delay and mental retardation. Treatment consists in Leu, Val and Ile restricted diet. Studies in animals have demonstrated that lipid peroxidation is stimulated by BCAA and BCKA in brain of rats and these metabolites reduce in vitro and in vivo the cerebral capacity to modulate the damage associated to increased free radical production. Also, there is evidence that oxidative stress occurs in MSUD patients at diagnosis and during treatment and that due to terapy with protein restricted diet they present L-carnitine (L-car) deficiency, an important compound for energy metabolism. Recent studies have demonstrated the antioxidant and anti-inflammatory role of L-carnitine (L-car), through its action against peroxidation in different tissues by various mechanisms, a scavenger of reactive oxygen species and the stabilizing effect of damage to cell membranes. Considering that the pathophysiology of MSUD is still poorly understood, and that there is an increasing number of studies emphasizing the oxidative stress involvement in the disease, this study investigated the in vitro and in vivo effect of L-car on oxidative stress and inflammatory damage in MSUD with the following purposes: A) to study the induction of damage by accumulated metabolites in MSUD, analyzing the possible antioxidant role of L-car on DNA damage in vitro; B) to evaluate the in vivo effect of 50 mg/kg/day of L-car supplementation about: b.1) the induction of DNA damage in leukocytes of MSUD patients treated with protein-restricted diet, correlating this damage with the concentrations of the major metabolites accumulated in this disorder and checking the possible antioxidant role of L-car supplementation; b.2) plasma inflammatory cytokines in treated MSUD patients with protein-restricted diet and the correlation with oxidative stress; b.3) oxidative damage parameters in urine of MSUD patients with protein-restricted diet supplemented with L-car; C) to investigate the BCAA effect on some oxidative stress parameters and evaluate the L-car efficacy against these possible pro-oxidant effects in cerebral cortex and cerebellum of rats submitted to a chronic chemically-induced model of MSUD. DNA damage index (DI) showed that Leu and -ketoisocaproic acid (KIC) groups was significantly higher than that of the control group, and that L-car was able to significantly prevent this damage, especially that due to KIC. Accordingly, DNA DI in MSUD patients under BCAA-restricted diet was significantly increased as compared to controls and L-car supplementation was able to significantly decrease this parameter. It was also verified a significant positive correlation between DNA DI and MDA content, a marker of lipid peroxidation. Furthermore, we found an inverse significant correlation between DI and L-car levels. These results strengthen a relationship between DNA damage observed in MSUD patients, oxidative stress and the L-car supplementation benefit. The role of L-car on plasma inflammatory cytokines interleukin-1Y (IL-1Y), interleukin-6 (IL-6) and interferon-gamma (INF- Z) was also evaluated in these patients. Significant increases of IL-1Y, IL-6, and INF- Z were observed before the treatment with L-car. Moreover, there is a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1Y and IL-6 values after L-car supplementation. It was also demonstrated that the oxidative stress parameters di-tyrosine (oxidative protein damage) and isoprostanes (lipid peroxidation assay) were increased and the antioxidant capacity was reduced in urine of MSUD patients without L-car therapy and that the supplementation of this compound induced beneficial effects on these parameters, so reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary KIC after 2 months of L-car treatment compared to control group, demonstrating an increased excretion of this toxic metabolite. In conclusion, these results suggest a reversion effect of the oxidative damage by L-car and that urine can be used to monitorize oxidative damage in patients affected by this disease. The following parameters were analysed in cerebral cortex and cerebellum of Wistar rats submitted to MSUD chemically-induced chronic model: thiobarbituric acid reactive species (TBA-RS), to evaluate lipid peroxidation, carbonyl content to evaluate protein oxidative damage, DCF oxidation to quantify reactive species production, reduced glutathione (GSH), an important non-enzymatic antioxidant and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD). The results showed that the chronic administration of BCAA was able to promote both lipid and protein oxidation, increase of reactive species production and decreased brain antioxidant defenses, especially in cerebral cortex and that L-car was able to prevent these effects, except for oxidative damage to proteins. Taken together, these results demonstrate that the metabolites accumulated in MSUD cause oxidative damage to biomolecules (lipids, proteins and DNA), decrease antioxidant status and promote increased inflammatory processes. These results may contribute to the understanding of the mechanism of action of the cytotoxic effect of the metabolites accumulated in MSUD and the role of oxidative stress and inflammation in the MSUD neuropathophysiology besides the protective effect of L-car on this process. The study of antioxidants like L-car can opens an additional therapeutic approach to that currently employed for MSUD patients, which is primarily dietary and therefore difficult to handle.

Thesis (MPhil)--Stellenbosch University, 2004.<br>ENGLISH ABSTRACT: 60-day growth experiment was conducted to investigate the effect of dietary Lcarnitine supplementation on the production performance parameters of Mozambique tilapia, Oreochromis mosambicus. A number of approximately 140 tilapia fry with average weight of l.4g ± 0.71g were stocked in each of 40 fine-meshed hapas (I mx 1mx 1.5m) submerged within a complete recirculation pond system. During the first 30 days of the experiment water temperatures ranged from 19 to 23°C where after it decreased to 16-20°C for the consecutive 30-day period. Dietary treatments consisted of 8 replicates of 5 levels of L-carnitine supplementation labelled as Co, C250,C500, C750 and C 1000represented Omg, 250mg, 500mg, 750mg and 1000mg L-carnitine supplementation per kg feed respectively. Results were analyzed for significant differences using one-way analysis of variance (ANOVA) and Tukey's pairwise comparison test for growth rate, feed intake (FI) and feed conversion efficiency. After completion of the trial 8 fish from each hapa were sacrificed and analyzed for cephalosomatic index (CSI), dress out percentage (viscera, gills and head excluded), viscerosomatic index (VSI) and hepatosomatic index (HSl). Poor production performance results were generally observed as water temperatures were sub-optimal, especially during the second 30-days period. Results from the trial indicate no significant differences (P>0.05) between treatments for weight gain, FCR, FI and VS!. A negative trend was observed for FCR with increasing level of L-carnitine supplementation for both the first 30-day period (1.50±0.07, 1.53±0.08, 1.58±0.09 and 1.61±0.17 for C250,C50Q,C750and C 1000)as well as for the consecutive lower temperature 30-day period (2.22±0.10, 2.25±0.ll, 2.27±0.28 and 2.29±0.2l for C250, C500, C750 and C 10(0)'Although statistically not significant, fish fed the C250showed better performance in dress out percentage weight either than the control or the higher levels. The increasing trend for head weight with increasing level of L-carnitine supplementation were significant (P<0.05) from Co and C250with and above C500.The decreasing trend for liver weight with increasing level of L-carnitine supplementation became significant (P<0.05) with and above C750.The results of the current study showed a trend in the improvement of L-carnitine on the production performance parameters. However, the natural content of L-carnitine in the basal diet impaired with the inclusion levels, thus further research at lower inclusion levels is recommended.<br>AFRIKAANSE OPSOMMING: 'n Proef oor 'n tydperk van 60-dae is onderneem om die effek van L-karniten aanvulling op produksie prestasie parameters van Mosambiek tilapia (0. mosambicusi te ondersoek. 140 tilapia vingerlinge met 'n gemiddelde massa van lAg ± 0.7lg is ewekansig uitgeplaas in 40 eksperimentele hapa-hokkies (lmxlmx1.5m) in "n hersirkulasie sementdam-stelsel. Gedurende die eerste 30 dae van die proef het water temperatuur gewissel tussen 19 to 23°C waarna dit gedaal het na tussen l6-20°C vir die opeenvolgende 30-dag periode. Proef-rantsoen behandelings het bestaan uit 8 herhalings van 5 vlakke van L-karnitien aanvulling, naamlik Co,C250, C500, C750 en CIOOOvir Omg, 250mg, 500mg, 750mg en 1000mg L-karnitien aanvulling per kg voer afsonderlik. Resultate was ontleed vir betekenisvolle verskille deur gebruik te maak van analise van variansie (ANOVA) ontleding en die Tukey se vergelykende toets vir groeitempo, voerinname en voeromsettingsverhouding. Aan die einde van die proefperiode is 8 visse van elke hapa ontleed vir liggaamskomponent-samestelling (kop-, ingewande- en hepatosomatiese indekse. Ondergemiddelde produksie resultate is waargeneem wat toegeskryf kan word aan onder-optimale water temperature, veral gedurende die tweede 30-dag periode van die proef. Proef resultate het geen betekenisvolle verskille (P>0.05) in massatoename, voeromsettingsverhouding (VOV) of visserosomatiese indeks tussen behandelings getoon nie. 'n Negatiewe neiging is waargeneem vir VOV met toenemende vlakke van L-kamitien insluiting vir beide die eerste 30 dag periode (1.50±0.07, 1.53±0.08, 1.58±0.09 and 1.61±0.17 for C250, C500, C750 and CIOOO) sowel as vir die opvolgende 30-day periode nie (2.22±0.10, 2.25±0.11, 2.27±0.28 and 2.29±0.21 for C250, C50o, C750 and CIOOO). 'n Toenemende neiging vir kop-massa met toenemende L-kamitien insluiting was betekenisvol (P<0.05) vanaf Co en C250 met en hoër as C500. 'n Dalende neiging vir lewermassa met toenemde L-kinsluiting was betekenisvol (P<0.05) met en hoër as C750. Resultate van die proef dui oor die algemeen op 'n neiging tot verbeterde produksie prestasie parameters van tilapia vingerlinge met toenemde insluiting van Lkamitien. Verdere navorsing word aanbeveel om die invloed van natuurlike Lkamitien in die proteïen-bronne van die basaalrantsoen te op die gebrek aan betekenisvolheid van hierdie neiging te verklaar.

La caquexia afecta negativamente a los pacientes con enfermedades crónicas y sobre todo en el cáncer. Las estrategias terapéuticas son aún limitadas. Los beta2-agonistas (formoterol) y el soporte nutricional (L-carnitina) pueden atenuar los efectos deletéreos en el músculo. En la presente tesis, el tratamiento con formoterol y L-carnitina indujo efectos beneficiosos (peso corporal y muscular, estructura, apoptosis, proteólisis y vías de señalización) en el diafragma y músculos de las extremidades en un modelo experimental de caquexia cancerosa (hepatoma ascitico Yoshida AH-130, en ratas). En ratones con caquexia cancerosa (células de adenocarcinoma del pulmón LP07), el tratamiento del tumor con anticuerpos monoclonales (anti-PD1, anti-CTLA4, anti-CD137, y anti-CD19) indujo efectos beneficiosos de la misma índole como consecuencia de la disminución del tamaño y la carga tumoral. En esta tesis se ha demostrado que diversas vías de señalización y mecanismos implicados en la degradación proteica y muscular se ven atenuadas, mejorando las características fenotípicas y funcionales de los músculos diafragma y periféricos en respuesta a diversas estrategias terapéuticas. (165 palabras)<br>Cachexia negatively affects patients with chronic diseases and especially in cancer. Therapeutic strategies are still limited. The beta2-agonists (formoterol) and the nutritional support (L-carnitine) can attenuate the deleterious effects in the muscle. In this thesis, treatment with formoterol and L-carnitine induced beneficial effects (total body and muscle weights, structure, apoptosis, proteolysis and signaling pathways) in the diaphragm and limb muscles in an experimental model of cancer cachexia (AH-130 Yoshida hepatoma ascites cells, in rats). In mice with cancer cachexia (LP07 lung adenocarcinoma cells), treatment of the tumor with monoclonal antibodies (anti-PD1, anti-CTLA4, anti-CD137, and anti-CD19) induced beneficial effects of the same kind as a consequence of the decrease in size and tumor burden. This thesis has shown that various signaling pathways and mechanisms involved in protein and muscle degradation are attenuated, improving the phenotypic and functional characteristics of the diaphragm and peripheral muscles in response to various therapeutic strategies. (149 words)

As acidemias D-2-hidroxiglutárica e L-2-hidroxiglutárica são duas distintas desordens neurometabólicas bioquimicamente caracterizadas por níveis aumentados dos ácidos D-2-hidroxiglutárico e L-2-hidroxiglutárico em tecidos e fluidos biológicos, respectivamente. Pacientes acometidos pela acidemia D-2-hidroxiglutárica são classificados em duas variantes, a D-2-hidroxiglutárica do tipo I ou a D-2-hidroxiglutárica do tipo II. A acidemia D-2-hidroxiglutárica do tipo I é causada por uma mutação no gene da D-2-hidroxiglutarato desidrogenase enquanto que a acidemia D-2-hidroxiglutárica do tipo II é causada por uma mutação de ganho de função no gene da isocitrato desidrogenase II. A acidemia L-2-hidroxiglutárica é causada por uma mutação no gene da L-2-hidroxiglutarato desidrogenase. Considerando que a fisiopatologia destas doenças não está totalmente elucidada e que muitos estudos têm demonstrado o envolvimento do estresse oxidativo em erros inatos do metabolismo, este trabalho tem por objetivo principal investigar parâmetros de estresse oxidativo e nitrativo na urina de pacientes com acidemia L-2-hidroxiglutárica e o dano ao DNA in vitro causado pelos ácidos acumulados em ambas as patologias, as acidemias D-2-hidroxiglutárica e L-2-hidroxiglutárica, bem como o efeito protetor da L-carnitina sobre o dano. Dessa forma, verificou-se que as concentrações de 50 μM do ácido D-2-hidroxiglutárico e 30 μM do ácido L-2-hidroxiglutárico induzem dano ao DNA e que concentrações de 30 μM e 150 μM de L-carnitina reduzem significativamente in vitro o dano ao DNA, comparado aos controles. Além disso, foram analisadas amostras de urina dos pacientes com acidemia L-2-hidroxiglutárica. Observou-se aumento significativo de espécies de guanina oxidadas, um marcador bioquímico de dano oxidativo ao DNA, bem como um aumento significativo da excreção de di-tirosina, indicando que os pacientes tem dano a proteínas. Entretanto, não houve diferença significativa nos níveis de isoprostanos urinários e nos níveis de espécies reativas do nitrogênio. Esses resultados sugerem, pelo menos em parte, dano oxidativo a proteínas e ao DNA e ressaltam o potencial antioxidante da L-carnitina como um promissor adjuvante no tratamento de pacientes afetados pelas acidemias L-2-hidroxiglutárica ou D-2-hidroxiglutárica.<br>D-2-hydroxyglutaric and L-2-hydroxyglutaric acidurias are two distinct neurometabolic disorders biochemically characterized by increased levels of D-2-hydroxyglutaric and L-2-hydroxyglutaric acids in biological fluids and tissues, respectively. Patients affected by D-2-hydroxyglutaric aciduria are classified into two variants, D-2-hydroxyglutaric aciduria type I or D-2-hydroxyglutaric aciduria type II. D-2-hydroxyglutaric aciduria type I is caused by mutation of D-2-hydroxyglutarate dehydrogenase gene while D-2-hydroxyglutaric aciduria type II is caused by a gain of function mutation in isocitrate dehydrogenase 2 gene. L-2-hydroxyglutaric aciduria is caused by mutation in the L-2-hydroxyglutarate dehydrogenase gene. Considering that the pathophysiology of these diseases is not fully understood and that many studies have been shown the involvement of oxidative stress in inborn errors of metabolism, the main objective of this work was investigate oxidative and nitrative stress parameters in the urine of L-2-hydroxyglutaric aciduria patients and to investigate the in vitro DNA damage caused by the accumulated acids of D-2-hydroxyglutaric and L-2-hydroxyglutaric acidurias as well as the protective effect of L-carnitine on this damage. It has been found that concentrations of 50 μM of D-2-hydroxyglutaric acid and 30 μM of L-2-hydroxyglutaric acid induce DNA damage and concentrations of 30 μM and 150 μM of L-carnitine significantly reduced the in vitro DNA damage compared to controls. In addition, urine samples from L-2-hydroxyglutaric aciduria patients were analyzed. It was observed a significant increase of oxidized guanine species, an oxidative DNA damage biomarker as well as a significant increase of urinary di-tyrosine level, indicating protein oxidative damage in the patients. However, there was no significant difference in the levels of urinary isoprostanes and reactive nitrogen species. These results suggest, at least in part, proteins and DNA oxidative damage and highlight the L-carnitine antioxidant potential as a promising adjuvant in the treatment of patients affected by L-2-hydroxyglutaric or D-2-hydroxyglutaric aciduria.

Orientadores: Anibal Eugenio Vercesi, Mariana Pinheiro Fernandes<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-08-15T13:38:04Z (GMT). No. of bitstreams: 1 Costa_RuteAlvesPereirae_M.pdf: 1931770 bytes, checksum: c11c3126a7042737485a72a01e8020a7 (MD5) Previous issue date: 2010<br>Resumo: Estatinas são fármacos amplamente utilizadas no tratamento das hipercolesterolemias. Elas são inibidores competitivos da 3-hidroxi 3-metilglutaril coenzima A (HMG-CoA) redutase impedindo, dessa forma, a síntese do colesterol. L-carnitina é sintetizada a partir dos aminoácidos essenciais lisina e metionina no fígado e no rim. Desempenha função importante na célula onde está envolvida na oxidação dos ácidos graxos agindo como um cofator no transporte de grupos acil através da membrana mitocondrial interna. Piracetam é uma droga nootropica cuja função é melhorar o desempenho cognitivo, e as funções envolvidas nos processos de aprendizagem, memória, atenção e consciência. O presente trabalho teve como objetivo avaliar a ação protetora da Lcarnitina ou piracetam contra a morte por necrose de células PC-3 induzida por sinvastatina 60 µM ou tert-butyl-hidroperóxido (t-BOOH) 500 µM. Tanto a sinvastatina quanto o t-BOOH causam transição de permeabilidade mitocondrial (TPM), seguida de morte celular por necrose. L-carnitina e piracetam protegeram contra a morte celular induzida por sinvastatina ou t-BOOH por meio de um mecanismo dose-dependente (1- 12 µM). Na avaliação da disfunção mitocondrial causada por sinvastatina ou t-BOOH, tanto a L-carnitina quanto o piracetam protegeram contra a perda de potencial de membrana mitocondrial de forma semelhante à ciclosporina A. As quedas nas velocidades de respiração de estado III e estado IV, (fosforilação e repouso), também foram prevenidas por L-carnitina, piracetam e ciclosporina A. Quando linhagens de células não tumorais (GN16-P6 e HaCaT) foram analisadas, observou-se que tanto Lcarnitina quanto o piracetam também protegeram contra a morte celular induzida por sinvastatina. Podemos concluir que nas células PC-3, estes compostos protegem contra necrose celular através da inibição da TPM e que em linhagens não tumorais estes compostos apresentam efeitos semelhantes.<br>Abstract: Statins are drugs widely used in the treatment of hypercholesterolemia. They are competitive inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase preventing in this way, the synthesis of cholesterol. L-carnitine is synthesized from the essential aminoacids lysine and methionine in liver and kidney and plays na important role in the cell, where it is involved in fatty acid oxidation by acting as a cofactor in the transport of acyl groups across the inner mitochondrial membrane. Piracetam is a nootropic drug which function is to improve cognitive performance, and the functions involved in the processes of learning, memory, attention and consciousness. This study aimed to evaluate the protective action of L-carnitine or piracetam against necrosis in PC-3 cells induced by 60 µM simvastatin or 500 µM tertbutyl- hydroperoxide (t-BOOH). Both simvastatin and t-BOOH causes mitochondrial permeability transition (MPT) followed by necrosis. L-carnitine and piracetam protected against cell death induced by simvastatin or t-BOOH by a dose-dependent mechanism (1-12 µM). In the assessment of mitochondrial dysfunction caused by simvastatin or t- BOOH, L-carnitine or piracetam similarly to cyclosporin A protected against the loss of mitochondrial membrane potential. The decrease in state III or state IV respiration rates, were also prevented. When non-tumor cell lines (GN16-P6 and HaCaT) were analyzed, it was observed that both L-carnitine and piracetam also protected against cell death induced by simvastatin. We can conclude that these compounds protected against cell necrosis by inhibiting MPT in both tumor or non tumor cell lines.<br>Mestrado<br>Biologia Estrutural, Celular, Molecular e do Desenvolvimento<br>Mestre em Fisiopatologia Médica

Traumatic injuries to the spinal cord and brachial plexus induce a significant inflammatory response in the nervous tissue with progressive degeneration of neurons and glial cells, and cause considerable physical and mental suffering in affected patients. This thesis investigates the effects of the antioxidants N-acetyl-cysteine (NAC) and acetyl-L- carnitine (ALC) on the survival of motoneurons in the brainstem and spinal cord, the expression of pro-apoptotic and pro-inflammatory cell markers, axonal sprouting and glial cell reactions after spinal hemisection in adult rats. In addition, a novel MRI protocol has been developed to analyse the extent of neuronal degeneration in the spinal cord. Rubrospinal neurons and tibial motoneurons were pre-labelled with the fluorescent tracer Fast Blue one week before cervical C3 or lumbar L5 spinal cord hemisection. The intrathecal treatment with the antioxidants NAC (2.4mg/day) or ALC (0.9 mg/day) was initiated immediately after injury using Alzet2002 osmotic mini pumps. Spinal cord injury increased the expression of apoptotic cell markers BAX and caspase 3, induced significant degeneration of rubrospinal neurons and spinal motoneurons with associated decrease in immunoreactivity for microtubule-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker glial fibrillary acidic protein and microglial markers OX42 and ED1 was markedly increased. Treatment with NAC and ALC attenuated levels of BAX, caspase 3, OX42 and ED1 expression after 2 weeks postoperatively. After 4-8 weeks of continuous intratheca ltreatment, NAC and ALC rescued approximately half of the rubrospinal neurons and spinal motoneurons destined to die, promoted axonal sprouting, restored the density of MAP2 and synaptophysin immunoreactivity and reduced the microglial reaction. However, antioxidant therapy did not affect the reactive astrocytes in the trauma zone. The inflammation modulating properties of ALC were also studied using cultures of human microglial cells. ALC increased the microglial production of interleukin IL-6 and BDNF, thereby possibly mediating the anti-inflammatory and pro-regenerative effects shown in vivo. To study degeneration in the spinal cord following pre-ganglionic and post-ganglionic brachial plexus injuries, adult rat models of ventral root avulsion and peripheral nerve injury were used. A novel MRI protocol was employed and the images were compared to morphological changes found in histological preparations. Ventral root avulsion caused degeneration of dendritic branches and axonal terminals in the spinal cord, followed by significant shrinkage of the ventral horn. Extensive astroglial and microglial reactions were detected in the histological preparations. Peripheral nerve injury reduced the density of dendritic branches but did not cause shrinkage of the ventral horn. Quantitative analysis of MRI images demonstrated changes in the ventral horn following ventral root avulsion only, thus validating the developed MRI technique as a possible tool for the differentiation of pre-ganglionic and post-ganglionic nerve injuries.

En esta tesis, se estudiaron los efectos del estrés por calor (EC) sobre la producción de ovejas lecheras Lacaune (Exp.1), así como la respuesta de cabras lecheras Murciano-Granadina bajo condiciones de EC a la L-carnitina (Exp. 2) y la metionina (Exp. 3). En los 3 Exp., los animales fueron alimentados con una ración única mezclada y se ordeñaron x2 al día. Las condiciones ambientales fueron: termo-neutralidad (TN; THI = 59-65) y EC (día, THI = 83; noche, THI = 75). El fotoperíodo (día-noche) fue constante (12-12 h). La temperatura rectal (TR), el ritmo respiratorio (RR), la IMS, el consumo de agua (CA) y la producción de leche (PL) se registraron diariamente, mientras que la leche para la composición se muestreó semanalmente y se registró el peso vivo (PV) al inicio y al final de cada período. En el Exp.1, las ovejas (n = 8) fueron expuestas a TN o EC en un diseño cruzado de 2 períodos (21 días cada uno). Además, a las ovejas se les administró glucosa, insulina y epinefrina para evaluar sus respuestas metabólicas. EC aumentó la TR, RR, CA y la pérdida de PV, pero redujo la IMS y el contenido de grasa y proteína de la leche sin afectar a PL. A pesar de la reducción de IMS por EC, los AGNE en sangre no cambiaron, y sin embargo los valores de creatinina aumentaron. La respuesta a los desafíos metabólicos indicó que las ovejas EC presentaban una rápida absorción de glucosa y una mayor resistencia a las señales lipolíticas en comparación con las ovejas TN. En los Exp.2 y 3 con cabras lecheras, el diseño fue un cuadrado latino 4 × 4, ya que se agregaron 2 factores dietéticos a las 2 condiciones ambientales. Las 2 condiciones dietéticas fueron control (CON) sin suplementación, versus L-carnitina protegida del rumen (CAR, Exp. 2) o metionina protegida del rumen (Met, Exp. 3). En Exp. 2, las cabras EC experimentaron un aumento de TR y RR. Además, las cabras EC sufrieron una pérdida del 26% en IMS, pero tendieron a comer partículas de tamaño más largo. La CAR aumentó drásticamente las concentraciones de carnitina libre, acetilo y total en sangre. A pesar de esta absorción eficiente, CAR no tuvo efecto sobre IMS, producción de leche o metabolitos en sangre en condiciones TN o EC. En el Exp.3, la IMS de las cabras TN se limitó a 2.0 kg/d, mientras que las cabras de EC se alimentaron ad libitum. Así pues, las cabras EC presentaron sólo un 9.8% menos IMS que TN, aunque significativo. En consecuencia, no se detectaron cambios en PL. Se observaron incrementos esperables en TR y RR debido al EC, pero Met redujo el RR por la mañana y RT en la tarde. Además, Met evitó la pérdida típica de PV en condiciones de EC. El perfil de aminoácidos en sangre (AA) reveló una menor concentración basal de Met, a pesar de los niveles comparables de IMS. Además, las cabras EC tenían poco glutamato, lo que podría estar relacionado con una inflamación y respuesta inmune a nivel gastrointestinal. La suplementación con Met ahorró glutamato, independientemente de la temperatura ambiente. En general, el EC afectó negativamente la producción de las ovejas lecheras. La adaptación metabólica de las ovejas lecheras al EC incluyó una reducción de la movilización de grasa corporal y el aumento de la degradación de las proteínas musculares. La metionina, pero no la L-carnitina, tuvo algunos efectos beneficiosos sobre el rendimiento de las cabras lecheras estresadas por el calor. Probablemente un poco más AA además de la metionina deberían ser suplementados en condiciones de EC.<br>In the current thesis the effects of heat stress (HS) on performance of Lacaune dairy ewes (Exp.1) as well as the response of HS Murciano-Granadina dairy goats to dietary L-carnitine (Exp. 2) and methionine (Exp. 3) were evaluated. In the 3 Exp., animals were fed a total mixed ration and milked x2 daily. The environmental conditions were: thermal neutral (TN; THI = 59-65) and HS (day, THI = 83; night, THI = 75). Photoperiod (light- dark) was constant (12-12 h). Rectal temperature (RT), respiratory rate (RR), DMI, water intake (WI) and milk yield (MY) were recorded daily, whereas milk for composition was sampled weekly and BW was registered at the start and the end of each period. In Exp.1, ewes (n = 8) were exposed to TN or HS in a crossover design with 2 periods (21 d each). Further, ewes were administered with glucose, insulin and epinephrine to evaluate the metabolic responses. HS increased RT, RR, WI and BW loss, but reduced DMI, and milk fat and protein contents without affecting MY. Despite the reduced DMI by HS, blood NEFA did not change, but creatinine values increased. Response to the metabolic challenges indicated that HS ewes had faster uptake of glucose and greater resistance to lipolytic signals compared to TN ewes. In Exp.2 & 3 with dairy goats, the design was 4 × 4 Latin square as 2 dietary factors were added to the 2 environmental conditions. The 2 dietary conditions were control (CON) without supplementation vs. rumen protected L-carnitine (CAR, Exp. 2) or rumen protected methionine (Met, Exp. 3). In Exp. 2, HS goats experienced increased RT and RR. Additionally, HS goats suffered 26% loss in DMI, but they tended to eat longer particle sizes. CAR dramatically increased blood free-, acetyl, and total-carnitine concentrations. Despite this efficient absorption, CAR had no effect on DMI, milk production or blood metabolites in TN or HS conditions. In Exp.3, DMI for TN goats was limited to 2.0 kg/d, whereas HS goats were kept feeding ad libitum. Consequently, HS goats had only 9.8% (although significant) less DMI than TN. Consequently, no changes in MY were detected. Expected increments in RT and RR due to HS were detected but Met resulted in less RR in the morning and lower RT in the afternoon. In addition, Met avoided the typical BW loss under HS conditions. The profile of blood amino acids (AA) revealed less basal Met concentration, despite the comparable DMI levels. Additionally, HS goats were in shortage of glutamate, which could be related to the inflammation and immune response at the gastrointestinal level. Met supplementation spared glutamate regardless the ambient temperature. Overall, HS negatively affected the performance of dairy ewes. Metabolic adaptations of dairy ewes to HS included reduced body fat mobilization and increased muscle protein breakdown. Methionine, but not L-carnitine, had some beneficial effects on the performance of heat-stressed dairy goats. Probably some more AA in addition to methionine should be supplemented under HS conditions.<br>Cette thèse, étude les effets du stress thermique (ST) sur les performances des brebis laitières Lacaune (Exp.1) ainsi que la réponse des chèvres laitières Murciano-Granadina à la L-carnitine (Exp.2) et à la méthionine (Exp. 3) sous conditions de ST. Dans les 3 Exp, les animaux ont reçu une ration totale mélangée et traitent x2 par jours. Les conditions environnementales étaient : thermoneutralité (TN; THI = 59-65) et ST (jour, THI = 83; nuit, THI = 75). La photopériode (jour-nuit) était constante (12-12 h). La température rectale (TR), le rythme respiratoire (RR), la MSI, la prise d’eau (PE) et la production de lait (PL) ont été enregistrés quotidiennement, tandis que le lait pour la composition a été échantillonné chaque semaine et PV a été enregistré au début et à la fin de chaque période. Dans Exp.1, les brebis (n = 8) ont été exposées au TN ou au ST avec permutation de 2 périodes (21 j chacune). En plus, les brebis ont été administrées avec du glucose, de l’insuline et de l’épinéphrine pour évaluer la réponse métabolique. Le ST a augmenté le TR, RR, PE et a réduit le PV, mais a réduit l’IMS et le contenu en matières grasses et en protéines du lait sans affecter la PL. Malgré la réduction de l’IMS par le ST, le AGNE sanguin n’a pas changé, mais les valeurs de créatinine ont augmenté. La réponse aux défis métaboliques a indiqué que les brebis ST avaient une absorption plus rapide du glucose et une plus grande résistance aux signaux lipolytiques que les brebis TN. Dans Exp.2 & 3 avec des chèvres laitières, le design expérimental était un carré latin 4 × 4 car 2 facteurs alimentaires ont été ajoutés aux 2 conditions environnementales. Les 2 conditions alimentaires étaient control (CON) sans supplémentation et une supplémentation avec la L-carnitine protégée du rumen (CAR, Exp. 2) ou avec la méthionine protégée du rumen (Met, Exp. 3). Dans Exp. 2, les chèvres ST ont demontré une augmentation du TR et RR accrues. De plus, les chèvres ST ont réduit de 26% l’IMS, mais elles avaient tendance à manger des particules plus longues. La CAR a considérablement augmenté les concentrations libres, d’acétyle et carnitine totale de sang. Malgré cette absorption efficace, la CAR n’a eu aucun effet sur l’IMS, la PL ou les métabolites sanguins dans les conditions TN ou ST. Dans Exp.3, l’IMS pour les chèvres TN était limité à 2,0 kg/j, tandis que les chèvres ST étaient nourries ad libitum. Par conséquent, les chèvres ST avaient seulement 9,8% (bien que significatif) de moins d’IMS que TN. Par conséquent, aucun changement dans PL n’a été détecté. Des augmentations attendues de la TR et du RR dues au ST ont été détectées, mais la Met a entraîné une diminution du RR le matin et une TR plus basse l’après-midi. De plus, Met a évité la perte de PV typique dans les conditions ST. Le profil des acides aminés du sang (AA) a révélé une concentration en Met basale inférieure, malgré des niveaux de DMI comparables. De plus, les chèvres ST manquaient de glutamate, ce qui pourrait être lié à l’inflammation et à la réponse immunitaire au niveau gastro-intestinal. La supplémentation rencontrée a épargné le glutamate quelle que soit la température ambiante. Globalement, le ST a affecté négativement la performance des brebis laitières. Les adaptations métaboliques des brebis laitières au ST comprenaient une mobilisation réduite des graisses corporelles et une dégradation accrue des protéines musculaires. La méthionine, mais pas la L-carnitine, a eu certains effets bénéfiques sur les performances des chèvres laitières soumises à un ST. Probablement un peu plus d’AA en plus de la méthionine devrait être supplémenté dans les conditions ST.

In the first phase of the current study, metabolic combinatorial solutions were in ovo administered in broiler eggs on Day 18 of incubation to investigate their effects in broiler chick tissue nutrient profiles until Day 10 of posthatch growout. In the second phase, the effects of in ovo injection of L-carnitine on Day 18 of incubation in broiler eggs were examined. The treatment solutions used in these studies were considered to play significant roles in various biochemical pathways, and were hence tested to determine whether they could potentiate the physiological growth and development of the broiler embryos and posthatch chicks. The in ovo injection of treatment solutions in both trials did not produce any significant increase in performance or slaughter yield in broiler chicks. However, positive trends were determined for rate of hatch and tissue nutrient profiles, which implied that the in ovo administration of nutrient supplements may be supportive of embryonic development and posthatch growout performance.