Dissertations / Theses on the topic 'Protein metabolism'

The weightless environment results in atrophy of the anti-gravity muscles. Hindlimb suspension is a model for weightlessness induced atrophy. This study evaluated the effects of hindlimb suspension, microgravity and exercise training followed by suspension on skeletal muscle. Soleus mass, myofibrillar and sarcoplasmic protein content were measured in one to four day hindlimb suspended animals. Protein synthesis was measured by intramuscular injection of ³H phenylalanine with correction for the difference between tRNA and intracellular specific activities. Myofibrillar protein loss was minimal after two days of unweighting but significant after three days. Although sarcoplasmic protein content showed no change, synthesis of both protein pools declined in parallel. Myofibrillar degradation increased during the first three days of unweighting, partially accounting for protein loss. The decline in degradation during day four explained the slower rate of protein loss at this time. Sarcoplasmic protein degradation increased slightly during the first two days of unweighting then declined sharply, thus explaining the sparing of sarcoplasmic proteins. Animals exposed to weightlessness showed soleus atrophy similar to suspended animals. The plantaris and gastrocnemius had reduced growth while the extensor digitorum longus and tibialis anterior grew normally in flight and suspended animals. Insulin stimulated glucose uptake was enhanced in soleus, but not extensor digitorum longus of flight and suspended animals. In situ insulin and IGF-1 stimulated 2-deoxyglucose uptake was greater after six days of suspension. Voluntary wheel training increased soleus mass, protein content and in vivo protein synthesis which plateaued by three weeks. Suspended or trained-suspended animals showed reductions in soleus mass, protein content and synthesis compared to trained animals. However, trained-suspended animals showed higher values for protein content and synthesis compared to suspended animals. In conclusion, these studies show that unweighting atrophy is characterized by decreased synthesis and increased degradation of myofibrillar proteins, and a sparing of sarcoplasmic proteins due to slower degradation. Tail-cast hindlimb suspension may be used as a ground based model to mimic the effects of weightlessness on muscle proteins. Wheel training causes muscle hypertrophy; and although training prior to suspension provides some protection against protein loss, it does not prevent atrophy.

Protein:protein interactions are central to the regulation of the intrinsic programmed cell death (apoptosis) pathway. Opposing members of the Bcl-2 family of proteins, which have distinct sequence features, interact with each other on the outer mitochondrial membrane to regulate apoptosis. Pro-survival proteins such as Bcl-2, Bcl-x[L], Bcl-w, Mcl-1 and A1 protect cells from apoptosis and contain up to four regions of homology to Bcl-2 (Bcl-2 homology domains 1 - 4, BH1-4). Pro-apoptotic BH3-only proteins such as Bim, Puma, Noxa, Bad, Bmf, and Bid promote apoptosis by interacting with and inactivating pro-survival proteins, and contain just the BH3-domain. The pro-apoptotic proteins Bax and Bak are essential for apoptosis and contain three regions of homology to Bcl-2 (the BH1-, BH2- and BH3-domains). In this study, two different sets of interactions involving pro-survival proteins were investigated. Initially, the pro-apoptotic protein Bnip3 was examined to determine if it was a mitochondrial anchor for the pro-survival protein Bcl-w. Secondly, to characterise the interactions between a pro-survival protein and different BH3-domains, structures were solved of the pro-survival protein A1 in complex with four different BH3-domains. In the structure of Bcl-w, the hydrophobic C-terminus is bound to its own BH3-domain binding groove. This location of the C-terminus is consistent with the observation that Bcl-w is only loosely associated with the outer mitochondrial membrane in healthy cells. Upon interaction of Bcl-w with a BH3-domain, Bcl-w becomes tightly associated with the mitochondrial membrane, presumably due to displacement of the C-terminal residues by the BH3-only protein. In healthy cells it has been suggested that Bcl-w is associated with the membrane due to an interaction with an unidentified membrane protein, which preliminary experiments suggested may be Bnip3. Protein interaction experiments performed in vitro and in vivo did not reveal an interaction between Bnip3 and Bcl-w. It was originally thought that each pro-apoptotic BH3-only protein could interact with all pro-survival proteins. However, it has recently become clear that there is selectivity within the pathway suggesting functional groupings. Bim and Puma behave as originally predicted and can interact with all pro-survival proteins and are potent killers. In contrast, Noxa and Bad interact with distinct subsets of pro-survival proteins. Noxa only binds Mcl-1 and A1, while Bad binds Bcl-2, Bcl-x[L] and Bcl-w. As a result, either Noxa or Bad acting alone is a weak killer, but together they are potent. Other BH3-only proteins bind tightly to some pro-survival proteins and weakly to others. The diversity that exists between BH3-domain sequences precludes sequence-based identification of the determinants of specificity. In this study, crystal structures of A1:Puma BH3-domain, A1:Bmf BH3-domain, A1:Bak BH3-domain and A1:Bid BH3-domain complexes have been solved. Differences identified between these structures explain some of the variation in affinities observed in pro-survival protein:BH3-domain complexes. These observations, in combination with published data, suggest that BH3-domains bind weakly when the optimal interactions with conserved residues cannot be formed. Additionally, differences were observed in the A1:Bak BH3-domain structure that may be functionally important for the regulation of Bak.

Les lipins són una família conservada evolutivament de fosfatases de fosfatidat (PAP1) dependents de Mg2+, que generen diacilglicerol per a la síntesi de fosfolípids i triacilglicerol. En mamífers, la família consta de lipina-1, lipina-2 i lipina-3. Mentre en ratolins la mutació del gen Lpin1 causa lipodistròfia, les mutacions deletèries en el gen LPIN1 en humans no afecten la distribució del greix. No obstant, persones amb diabetis tipus 2 mostren nivells reduïts de l'expressió de LPIN1 i de l'activitat PAP1. Aquesta tesi estudia el paper de les lipins en el teixit adipós humà, la adipogènesi i la lipòlisi. Descobrim que la expressió de gens i proteïnes lipin és alterada en el teixit adipós de les persones amb diabetis tipus 2. Silenciant cada membre de la família lipin en la línia cel•lular humana de preadipòcits del síndrome Simpson-Golabi-Behmel (SGBS), mostrem que mentre que els tres membres tenen un paper en el primers estadis de l’adipogènesi, els preadipòcits silenciats de lipin es diferencien i acumulen lípids neutres, la qual cosa condueix a la hipòtesi de l'existència de vies alternatives per a la síntesi de triacilglicerol en adipòcits humans quan es reprimeix l'expressió de les lipin. Les lipin participen també en el reciclatge d'àcids grassos alliberats mitjançant la via lipolítica. Després de la inducció de la lipòlisi, les lipines són defosforilades i es desplacen a la membrana del reticle endoplasmàtic, on exerceixen la seva funció enzimàtica. Aquesta activació és induïda pels àcids grassos alliberats i s'inverteix amb la presència d’albúmina o triacsin C. La inducció d’adipòcits silenciats de cada lipina demostra el seu paper en el metabolisme dels lípids neutres. En resum, les lipin semblen no tenir un paper imprescindible en la adipogènesi humana però sí poden comprometre el reciclatge d'àcids grassos, important per a la homeòstasis lipídica.
Las lipinas son una familia de fosfatasas de fosfatidato (PAP1) dependientes de Mg2+ evolutivamente conservadas, que generan diacilglicerol para la síntesis de fosfolípidos y triacilglicerol. En mamíferos, la familia consiste en lipina-1, lipina-2, y lipina-3. Mientras en ratones la mutación del gen Lpin1 causa lipodistrofia, las mutaciones deletéreas en el gen LPIN1 en humanos no afectan a la distribución de grasa. Sin embargo, los individuos con diabetes tipo 2 manifiestan niveles reducidos de expresión de LPIN1 y de actividad PAP1. En esta tesis doctoral se estudia la función de las lipinas en el tejido adiposo humano, la adipogénesis y la lipólisis. Descubrimos que la expresión génica y proteica de las lipinas está alterada en el tejido adiposo de individuos con diabetes tipo 2. La depleción de cada miembro de las lipinas en la línea celular humana de preadipocitos del síndrome Simpson–Golabi–Behmel (SGBS), mostró que, a pesar de que los tres miembros tienen un papel en la adipogénesis temprana, los adipocitos deplecionados de lipinas se diferencian y acumulan lípidos neutros, llevándonos a la hipótesis de la existencia de vías alternativas para la síntesis de triacilglicerol en adipocitos humanos cuando la expresión de las lipinas es reprimida. Las lipinas también intervienen en el reciclaje de los ácidos grasos liberados por la vía lipolítica. Tras la inducción de la lipólisis, las lipinas son defosforiladas y se desplazan a la membrana del retículo endoplásmico, donde ejercen su función. Esta activación es inducida por los ácidos grasos liberados, y revertida con albúmina o triacsin C. La depleción de cada lipina en adipocitos SGBS y posterior inducción de la lipólisis, demuestra su papel en el metabolismo de lípidos neutros. En resumen, las lipinas parecen no tener un papel indispensable en la adipogénesis humana pero sí comprometer el reciclaje de ácidos grasos, importante para la homeostasis lipídica.
Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatases (PAP1) that generate diacylglycerol for phospholipid and triacylglycerol synthesis. In mammals the Lipin family consists of lipin-1, lipin-2 and lipin-3. Whereas mutations in the Lpin1 gene cause lipodystrophy in mouse models, LPIN1 deleterious mutations in humans do not affect fat distribution. However, reduced LPIN1 expression and PAP1 activity have been described in participants with type 2 diabetes. In this doctoral thesis we investigate the roles of all lipin family members in human adipose tissue, adipogenesis and lipolysis. We found that adipose tissue gene and protein expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson–Golabi–Behmel syndrome (SGBS) pre-adipocyte cell line showed that even though all members alter early stages of adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids, pointing to the hypothesis of alternative pathways for triacylglycerol synthesis under repression of lipin expression. Lipins also have a role in the recycling of the fatty acids released by the lipolytic pathway. They become dephosphorylated upon lipolytic induction, and translocate to their active site, the endoplasmic reticulum membrane. This activation is induced by fatty acids and reversed with albumin or triacsin C. Depletion of every lipin member and subsequently stimulation of lipolysis in SGBS adipocytes revealed a role for lipins in neutral lipid metabolism. Overall, our data support that lipins may not have an indispensable role in adipogenesis, but their depletion compromise fatty acid recycling and lipid homeostasis.

The effect of cancer cachexia on protein metabolism has been studied in mice transplanted with the MAC16 adenocarcinoma. The progressive cachexia induced by the MAC16 tumour was characterised by a reduction in carcass nitrogen between 16-30% weight loss and a reciprocal increase in tumour nitrogen content. Carcass nitrogen loss was accompanied by a concomitant decrease in gastrocnemius muscle weight and nitrogen content and also by a decrease in liver nitrogen content. The loss of gastrocnemius muscle throughout the progression of cachexia was attributable to a 60% decrease in the rate of protein synthesis and a 240% increase in the rate of protein degradation. The loss of skeletal muscle protein that may be mediated by an increased rate of protein degradation has been correlated with a circulatory catabolic factor present only in cachectic tumour-bearing animals, that degrades host muscle in vitro. The proteolysis-inducing factor was found to be heat stable, not a serine protease and was inhibited by indomethacin and eicosapentaenoic acid (EPA) in a dose-related manner. The proteolytic factor induced prostaglandin E2 formation in the gastrocnemius muscle of non tumour-bearing animals and this effect was inhibited by indomethacin and EPA. In vivo studies show EPA (2.0g/kg-1 by gavage) to effectively reverse the decrease in body weight in animals bearing the MAC16 tumour with a concomitant reduction in tumour growth. Muscle from animals treated with EPA showed a decrease (60%) in protein degradation without an effect on protein synthesis. The action of the factor was largely mimicked by triarachidonin and trilinoleia. The increased serum levels of arachidonic acid in cachectic tumour-bearing animals may thus be responsible for increased protein degradation through prostanoid metabolism.

Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells. The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.

The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation.
In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein.
Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.

The organization of biological processes via protein-protein interactions and the subcellular localization of enzymes is believed to be fundamental to many aspects of metabolism. Although this organization has been demonstrated in several systems, the mechanisms by which it is established and regulated are still not well understood. The flavonoid biosynthetic pathway offers a unique system in which to study several important aspects of metabolism. Here we describe a novel toolset of mutant alleles within the flavonoid biosynthetic pathway. In addition, we discuss the use of several of these alleles together with a number of emerging technologies to probe the role of subcellular localization of chalcone synthase, the first committed flavonoid biosynthetic enzyme, on metabolic flux, and to characterize a novel chalcone synthase-interacting protein. The over-expression of this interacting protein induces novel phenotypes that are likely associated with the production or distribution of auxin. Further, interaction analyses between recombinant flavonoid biosynthetic enzymes point to the possibility that post-translational modifications play an important role in promoting interactions.
Ph. D.

Vitamin B12, or cobalamin, is a water soluble vitamin required as cofactor for two mammalian enzymatic processes: homocysteine remethylation to methionine in the cytoplasm using methionine synthase (MS), and fatty acid/amino acid metabolism in the mitochondrion using methylmalonyl-CoA mutase (MCM). Whereas the molecular nature of intracellular cobalamin metabolism in mammals remains poorly understood, the proteins MMACHC, MMADHC, LMBD1 and ABCD4 are implicated in its early uptake and processing. Due to the inherent challenges associated with the cellular utilization of cobalamin, we propose that these proteins mediate its early intracellular channeling; the objective of this thesis was to characterize the protein-protein interactions that coordinate this process.To gain insight into the function of MMADHC, recombinant isoforms were purified and low-resolution structural features were determined. MMADHC is monomeric and, in solution, adopts an extended conformation, with regions of disorder identified at the N-terminal domain. Panning combinatorial phage libraries against recombinant MMADHC allowed the mapping of putative sites of interaction on MMACHC. Kinetic analyses using surface plasmon resonance (SPR) confirmed a sub-micromolar affinity for the MMACHC–MMADHC interaction. Based on these studies, we propose that the function of MMADHC is exerted through its structured C-terminal domain via interactions with MMACHC in the cytoplasm.Clinical phenotypes and subcellular location of MS and MCM dictate that MMACHC functions in the cytoplasm while MMADHC functions at a branch point in the pathway in both the cytoplasm and the mitochondrion. To demonstrate that the MMACHC–MMADHC interaction is physiologically plausible, we used immunofluorescence and subcellular fractionation to confirm that MMACHC is cytoplasmic while MMADHC is dual-localized to the cytoplasm and mitochondria. Protein interaction analyses were extended by describing the recombinant production of the lysosomal membrane proteins LMBD1 and ABCD4. Detergent-solubilized LMBD1 and ABCD4 each formed homodimers in solution. SPR provided direct in vitro binding data for an LMBD1–ABCD4 interaction with low nanomolar affinity. Consistent with our phage display predictions, MMACHC interacted with LMBD1 and ABCD4 with high affinity.Our results support a model whereby membrane-bound LMBD1 and ABCD4 regulate the vectorial delivery of lysosomal cobalamin to cytoplasmic MMACHC, preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions. Subsequent formation of a cytoplasmic MMACHC–MMADHC complex then processes and partitions this cofactor to the downstream enzymes MCM and MS. These studies identify and characterize multiprotein complexes, advancing our basic understanding of early intracellular cobalamin metabolism.
La vitamine B12, ou bien la cobalamine, est une vitamine soluble requise pour deux processus enzymatiques distincts chez les mammifères; la production de l'acide aminée méthionine par la méthionine synthase (MS), et le métabolisme d'acides gras et d'acides aminées par la méthylmalonyl-CoA mutase (MCM). Malgré le fait que les procédées métaboliques intracellulaires de la cobalamine restes peu bien caractérisés, les protéines dont MMACHC, MMADHC, LMBD1, et ABCD4 jouent un rôle dans l'acquisition et le traitement de ce cofacteur. Vu les difficultés intrinsèques de l'utilisation cellulaire de la cobalamine, nous proposons que ces protéines assurent l'efficacité de son canalisation. Cette thèse avait pour objectif de caractériser les interactions protéine-protéine impliquées dans ce processus.Pour pouvoir caractériser la fonction de MMADHC, des isoformes protéiques ont été purifiées et leurs traits structurales ont étés déterminés à basse résolution. MMADHC se trouve à être monomérique et adopte une conformation étendue en solution, avec des régions non structurées dans la terminaison aminée de la protéine. Ensuite, des librairies combinatoires de phages ont été utilisées comme substrats pour tracer des sites d'interactions potentiels avec MMADHC. Les analyses kinésiques des interactions MMACHC–MMADHC ont été faites à l'aide de la résonance plasmonique de surface (SPR) et ont confirmées une intéraction d'affinité sub-micromolaire. Avec ces résultats, nous proposons que la fonction de MMADHC se fasse par sa terminaison acidique en interagissant avec MMACHC dans le cytoplasme.Les phénotypes cliniques et la localisation subcellulaire de MS et de MCM envisagent que MMACHC joue un rôle dans le cytoplasme et que MMADHC se trouve à être impliquée dans le processus au niveau de la mitochondrie et du milieu cytoplasmique. Pour démontrer que l'interaction MMACHC–MMADHC est physiologique, nous avons utilisé l'immunofluorescence et la fractionnement subcellulaire pour confirmer que MMACHC est cytoplasmique et que MMADHC se retrouvent au cytoplasme et au mitochondrie.Des analyses protéiques ont également engendré LMBD1 et ABCD4. Solubilisés à l'aide de détergent, ces deux protéines prennent la conformation d'homodimères en solution. Une interaction d'affinité nanomolaire entre LMBD1 et ABCD4 a été confirmée en SPR. En lien avec nos analyses de phages, MMACHC interagit avec haute affinité avec LMBD1 et ABCD4.Nos résultats supportent un modèle dans lequel LMBD1 et ABCD4, tous deux liés dans la membrane, régularisent l'octroi de la cobalamine lysosomale à MMACHC en prévenant la dilution de ce cofacteur dans le milieu cytoplasmique et en protégeant contre des réactions inactivant. La dissociation et le recrutement de la MMADHC cytoplasmique à MMACHC facilitent le transfert de la cobalamine vers les réactions enzymatiques catalysées par MCM et MS. L'identification et la caractérisation de ces complexes multiprotéiques font en sorte d'avancer nos connaissances générales sur le métabolisme de la cobalamine.

A series of in vivo experiments was conducted in sheep, to evaluate the effects of limiting amino acids, growth hormone and the β-adrenergic agonists, cimaterol and clenbuterol, on protein metabolism. As a complement to substrate studies, investigations into bicarbonate kinetics were performed. A multi-tracer amino acid model was developed, based on the large-dose procedure to measure protein synthesis. The response in nitrogen retention to a parenteral infusion of methionine, lysine and histidine was evaluated. This experiment was an attempt to define an experimental model to evaluate the responsiveness of peripheral tissue to supplies of limiting amino acids under conditions which minimise alterations in systemic substrate and hormonal conditions. The animals were probably not dietary-nitrogen limited, as initially calculated, and a poor responsive model was created. In a series of three experiments, the extent of ^13CO_2 sequestration in both the whole body and across the hind-leg preparation was studied in order to establish correction factors for planned substrate oxidation studies. Mean whole body sequestration was 30% with a fractional sequestration across the hind-limb of 3%. The results from these experiments suggested that the fraction of the bicarbonate sequestered did not appear to vary with either the level of intake or the duration of the infusion. The involvement of rumen microorganisms in the carbon dioxide sequestration was also excluded. The effects of two levels of protein nutrition on the response to exogenous bovine growth hormone was evaluated. Growth hormone infusion significantly increased nitrogen retention at both levels of intake, over an 8d period, suggesting that, under the established experimental conditions, the level of protein supply in the diet was not a factor limiting the hormone-induced increases in protein accretion. Rates of muscle protein synthesis (k_s %d) were measured by flooding with L-[1-^13C]phenylalanine, L-[1-^13C]leucine and L-[1-^13C]valine. The fractional synthesis rates obtained with the three amino acids were not significantly different. The results of these study were of particular relevance since it was important to establish a model which allowed serial measurements in the same animal.

Chronic renal failure (CRF) is associated with loss of lean body mass, a high incidence of malnutrition, and with insulin resistance. CRF is often complicated by metabolic acidosis. Metabolic acidosis is known to alter both protein and carbohydrate metabolism. A series of studies have been undertaken to investigate the effect of metabolic acidosis on protein metabolism in both normal and CRF human subjects, and to study whether metabolic acidosis in CRF affects insulin sensitivity. Protein turnover was studied using the technique of primed constant infusions of L-[1-¹³C]leucine. Normal subjects were studied before and after ammonium chloride induced metabolic acidosis. Acidosis was associated with increased protein turnover and amino acid oxidation. In CRF subjects, correction of acidosis with sodium bicarbonate decreased protein turnover and amino acid oxidation. The effect of acidosis in CRF on insulin mediated carbohydrate metabolism was studied using the technique of the hyperinsulinaemic euglycaemic clamp. Insulin sensitivity increased with correction of acidosis. By combining L-[1-¹³C]leucine infusions with hyperinsulinaemic euglycaemic clamps, the response of protein metabolism to hyperinsulinaemia was measured before and after correction of acidosis. The presence of acidosis did not impair the ability of insulin to modulate protein metabolism. There is therefore, dissociation between the effects of acidosis in CRF on insulin mediated carbohydrate metabolism and insulin mediated protein metabolism. In summary, metabolic acidosis increases protein catabolism in both normal and CRF man and may contribute to the loss of lean body mass characteristic of CRF. Insulin resistance in CRF improves with correction of acidosis. However the effects of acidosis on protein metabolism are not mediated via alterations in insulin sensitivity.

AMP-activated protein kinase (AMPK) is a major regulator of skeletal muscle metabolism with relevance to agriculture and human health. During the conversion of muscle to meat, the rate and extent of postmortem metabolism and pH decline largely determine pork quality development. Pigs with the AMPKγ3 R200Q mutation generate pork with low ultimate pH (pHu); this is attributed to high glycogen content, and greater â potentialâ to produce lactate and H+. We hypothesized that decreasing muscle phosphocreatine and creatine would decrease ATP buffering capacity, resulting in earlier termination of glycolysis and pH decline. Dietary supplementation with the creatine analogue, β-GPA, decreased muscle total creatine but negatively affected performance. Another experiment was conducted using control or β-GPA diet and wild type and AMPKγ3R200Q pigs in a 2à 2 factorial design. The loss of muscle total creatine was important in maintenance of ATP levels in AMPKγ3R200Q muscle early postmortem. Moreover, elevated glycogen did not affect pHu, supporting that energetic modifications induced by feed restriction and β-GPA supplementation influence extent of pH decline. Next, we utilized a line of pigs selected for differences in pHu. Another AMPKγ3 mutation (V199I), which is associated with higher pHu and lower glycolytic potential, was prevalent. The 199II genotype increased pHu in castrated males only. The wild type VV genotype increased glycolytic potential, but neither glycolytic potential nor lactate predicted pHu. In humans, AMPK activation is at least partly responsible for the beneficial effects of exercise on glucose transport and increased oxidative capacity in skeletal muscle. An inverse relationship exists between skeletal muscle fiber cross-sectional area and oxidative capacity, which suggests muscle fibers hypertrophy at the expense of oxidative capacity. Therefore, we utilized pigs possessing mutations associated with increased oxidative capacity (AMP-activated protein kinase, AMPKγ3R200Q) or fiber hypertrophy (ryanodine receptor 1, RyR1R615C) to determine if these events occur in parallel. RyR1R615C increased muscle fiber size; AMPKγ3R200Q increased oxidative capacity, evidenced by enhanced enzyme activity, mitochondrial function, and expression of mitochondrial proteins. Thus, pigs with both AMPKγ3R200Q and RyR1R615C possess increased fiber size and oxidative capacity, suggesting hypertrophy and oxidative capacity can occur simultaneously in skeletal muscle.
Ph. D.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

One of the major unresolved questions in cancer biology is why the majority of tumour cells express mutant p53 proteins. p53 is considered the prototype tumour suppressor protein, whose inactivation is the most frequent single genetic event in human cancer (Bourdon et al., 2005). Genetically-engineered p53-null knockout mice acquire multiple tumours very early on in life and human Li-Fraumeni families who carry germline mutations in p53 are highly cancer-prone (reviewed in Vousden and Lane, 2007). p53 mutant proteins have been found to acquire novel functions that promote cancer cell proliferation and survival, yet exactly why mutant p53s acquire oncogenic activity is still poorly understood. Mutant p53 has also been found to complex with wildtype p53, thus acting in a dominant negative way. However, this inhibition is incomplete since many cancers with mutant p53 alleles also have a loss of the second wild-type p53 allele and thus only express the mutant p53 (Baker et al., 1989). An N-terminal truncated p53 isoform, p47, arising from alternative splicing of the p53 gene (Ghosh et al., 2004) or by alternative initiation sites for translation (Yin et al. , 2002), has been described. Alternative splicing was found to be universal in all human multi-exon genes (Wang et al., 2008) and therefore determining the role of the p47 isoform with respect to the p53 gene is essential. Evidence in this study suggests that mutant p53 (p53RI75H) has a similar structure and function as p47, including the ability to complex with and impair both p53 and p73. Therefore, in addition to expressing a tumour suppressor protein, the p53 gene can also express an onco-protein (p47). This study therefore argues that tumours select for mutant p53 because it has gained the ability to function like p47, a wild-type p53 isoform.

The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.

A dextran sulphate sodium (DSS)- piglet model of colitis was used to investigate the effect of moderate (MD) and severe (SD) protein deficiency on disease severity in distal and spiral colon, growth, body composition, nitrogen balance and fractional synthesis rate (FSR) of proteins in plasma, liver and tissues. Moderate protein deficiency did not impair linear growth and chest circumference of DSS-colitis piglets although weight gain was restricted in MD piglets compared to well-nourished (WN) piglets. However, SD dramatically compromised body weight gain, linear growth and body composition of DSS-colitis piglets. Protein status did not affect distal colon histopathology whereas SD drastically compromised spiral colon integrity. MD piglets could maintain the synthesis of liver-derived and most tissue proteins. In contrast, SD remarkably restricted protein synthesis in visceral tissues, measured by a constant infusion of the isotopically labeled tracer L-[ring-2H5]phenylalanine, by approximately 50-70% compared to WN piglets. SD, but not MD, severely impaired growth and aggravated spiral colon inflammation severity in this piglet model of colitis.
Un modèle de colite expérimentale provoquée par le dextran sulphate de sodium (DSS) chez le porcelet a été employé pour étudier sur les effets d'une déficience modérée (DM) ou sévère (DS) en protéines sur la sévérité de la maladie dans le colon distal et spiral, la croissance, la composition corporelle, l'équilibre azoté et les taux de synthèse fractionnelle des protéines plasmatiques, du foie et tissus. DM n'a pas affecté la croissance linéaire ni la circonférence de la poitrine. En revanche, DS a sévèrement affecté le gain de masse corporelle, la croissance et la composition corporelle des porcelets. L'histopathologie du colon distal n'a révélé aucun effet du statut protéinique, alors que DS a sévèrement compromis l'intégrité du colon spiral. Les porcelets MD ont pu maintenir les taux de synthèse de protéines hépatiques et de la majorité des tissus. Par contre, la synthèse de protéines des tissus viscéraux, mesurée par infusion constante du tracer isotopique L-[ring-2H5]phenylalanine, a été remarquablement diminuée par DS de 50 a 70% chez les porcelets DSS comparés aux porcelets WN. DS, et non DM, a sévèrement retardé la croissance et a aggravé la sévérité de l'inflammation du colon spiral dans ce modèle animal de colite.

Modification of proteins by ADP-ribose has been shown to be a versatile modification with respect to the amino acid side chain. The results described here will allow the study of the biological importance of ADP-ribose glycation and also allow differentiation on crude extracts between enzymatic modifications from protein ADP-ribose glycation that can occur due to the presence of NAD glycohydrolases.

At present, there is no clear consensus as to whether protein feeding harnesses any ergogenic benefit for endurance athletes. In this thesis demonstrate no effect of protein on endurance performance. Furthermore, data presented herein indicates that protein co-ingestion does not enhance recovery 24 hours after exercise. Consequently, there is currently no basis on which to recommend protein feeding for endurance performance and recovery. Nutrient strategies implemented after exercise can markedly alter the acute response of muscle protein synthesis and, potentially, long-term phenotypic adaptation. Protein nutrition has traditionally been considered in the context of resistance exercise. Endurance exercise followed by protein ingestion increases the synthesis of mixed muscle protein via increased mRNA translation. Herein, we demonstrate that protein feeding after endurance exercise elevates the synthetic rate of contractile proteins and specific mRNA signalling intermediates. Insulin resistance that precedes Type II diabetes is characterized by blunted sensitivity of the pancreas to glucose and impaired glucose uptake in skeletal muscle. Lifestyle interventions including nutrient and exercise have the potential to improve glycemic control. The final experimental chapter in this thesis provides mechanistic evidence to support the benefits of resistance exercise for lowering post-prandial glucose concentrations. Interestingly, protein ingestion did not augment the glucose-lowering effects of prior resistance exercise.

Elevated postprandial triacylglycerol (TAG) concentrations and increased C-reactive protein (CRP) concentrations are risk markers for cardiovascular disease. The effects of resistance exercise on postprandial TAG metabolism and CRP are uncertain. The studies reported here examined the influence of resistance exercise on postprandial lipaemia and CRP. All studies were approved by the University's Ethical Advisory Committee. Fifty-five male participants were recruited to five studies. The age, height and body mass of the participants were: 24.2 ± 3.8 yrs, 1.78 ± 0.06 m, 78.0 ± 10.8 kg (mean ± SD). The aim of the first study was to evaluate the hypothesis that a single session of resistance exercise would lower postprandial lipaemia.

Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

Postprandial urea production in subjects with insulin dependent diabetes mellitus (IDDM) on conventional insulin therapy is normal when the previous diet is high in protein, but there is an incomplete adaptive reduction in urea production following protein restriction. To evaluate the nutritional implications of restricted protein intake in human diabetes mellitus, it is first necessary to establish a reliable method to measure changes in urea production and amino acid catabolism in response to changes in dietary protein intake. We therefore tested (1) the accuracy of the urea production rate (Ra) to depict changes in urea production, (2) whether sulfate production can be accurately depicted using tracer or nontracer approaches, after establishing the use of electrospray tandem mass spectrometry to measure sulfate concentrations and 34SO4 enrichments following administration of the stable isotope tracer sodium [34S]sulfate, (3) the reproducibility of urea and sulfate measurements following a test meal low in protein (0.25 g/kg) in subjects previously adapted to high (1.5 g/kg.d) and low (0.3 g/kg.d) protein intakes, and compared the metabolic fate of [ 15N]alanine added to the test meal with that of [15N] Spirulina platensis, a 15N-labeled intact protein, and (4) whether we could identify the differences in postprandial urea and sulfate productions between normal subjects and persons with IDDM receiving conventional insulin therapy previously adapted to high protein intake, when the test meal was limiting in protein. Under basal conditions, steady state urea Ra is an accurate measure of urea production. Following changes in urea production, both the tracer and nontracer methods seriously underestimated total urea Ra. The tracer method overestimated sulfate production by 20%, but the nontracer method provided an accurate measure of sulfate production and, hence, sulfur amino acid catabolism. Postprandial changes in urea and sulfate productions following normal ada

El present treball es centra en l’estudi de la producció de proteïnes recombinants en línies cel·∙lulars de mamífer. Concretament, s’ha realitzat l’estudi de tres estratègies de bioprocés, totes elles basades en el cultiu de cèl·∙lules HEK293. Com a proteïna model per a l’expressió de proteïnes heteròlogues s’ha triat la proteïna CapPCV2, la qual conforma la càpsida viral del Circovirus porcí serotip 2 (PCV2). Aquest virus és l’agent causal de PCVDS (porcine circovirus diseases o malaties derivades de circovirus porcí). Aquest terme engloba un conjunt de malalties i síndromes que tenen un elevat impacte econòmic en la indústria porcina. El projecte s’ha enfocat des de la perspectiva de desenvolupament i optimització del bioprocés i, en conseqüència, l’increment de la producció volumètrica ha estat la força impulsora de tot el treball. En primer lloc es presenten els estudis per a la selecció del medi de cultiu i suplements nutricionals. El creixement cel·∙lular depèn en gran mesura de les característiques nutricionals i fisicoquímiques del medi en que se les cultiva. Per tant, trobar el medi adequat és un dels factors clau per a l’expansió del cultiu cel·∙lular. L’estudi inicial de medis de cultiu va permetre augmentar sis vegades la densitat de cèl·∙lules viables en comparació al medi original en que es cultivaven. D’altra banda, s’han explorat diferents estratègies de cultiu, i com a resultat s’ha implementat una estratègia de fed-­‐batch que ha permès arribar a densitats cel·∙lulars de 26.8x106 cell/mL. En el segon i tercer capítol de resultats, s’avaluen tres estratègies diferents per a la producció de la proteïna recombinant CapPCV2 (r-­‐CapPCV2). La primera estratègia ha estat la infecció de cèl·∙lules HEK293 amb un vector adenoviral que codifica el gen de la CapPCV2 (vector generat dins del treball d’aquesta tesis doctoral). Els paràmetres d’infecció s’han estudiat en profunditat per tal de trobar els paràmetres d’infecció (medi de cultiu, MOI (multiplicitat d’infecció), TOI (temps d’infecció) i TOH (temps de recollida)) per a la millora de la producció de la proteïna i el vector adenoviral. La segona i tercera estratègia estan basades en la generació de línies cel·∙lulars estables. Concretament, s’ha generat una línia cel·∙lular productora de r-­‐CapPCV2 a partir de la integració a l’atzar del vector plasmídic en el genoma de la cèl·∙lula. D’altra banda, s’han generat línies cel·∙lulars amb la integració dirigida del gen en llocs prèviament caracteritzats com d’altra transcripció genètica. La integració dirigida s’ha efectuat mitjançant la tecnologia RMCE (recombinant mediated cassette exchange, o bescanvi de casset mitjançada per recombinació). Després de la comparació de les productivitats específiques i volumètriques aconseguides amb cada estratègia, el millor productor va ser seleccionat. Nogensmenys, r-­‐CapPCV2 es produeix en quantitats molt baixes i per tant no ha sigut possible dissenyar un procés de producció rentable i altres alternatives de producció s’haurien d’estudiar en un futur. Finalment, l’estudi d’un comportament metabòlic particular observat en les cèl.lules en cultiu s’ha adreçat des d’una perspectiva fisiològica i metabòlica. A certes condicions extracel·∙lulars, s’ha observat que les cèl·∙lules HEK293 poden consumir de manera simultània glucosa i lactat durant el seu creixement exponencial. Després d’un ampli estudi d’aquestes condicions, s’ha determinat que el canvi de la producció d’àcid làctic (que és el principal problema dels cultius d’alta densitat de cèl·∙lules de mamífer) cap al consum d’aquest metabòlit pot ser generat des de el començament del cultiu quan el pH és de 6.6 i la concentració de lactat és de 4-­‐8mM. En aquestes condicions, ni el creixement cel·∙lular ni la producció de proteïna es veuen afectades negativament. A la llum d’aquests resultats, es genera la hipòtesi de que les cèl·∙lules HEK293 poden co-­‐transportar el lactat extracel.lular i els protons com un mecanisme de detoxificació del pH. D’altra banda, l’aplicació de l’anàlisi de balanç de fluxos (FBA) ha revelat que quan la glucosa i el lactat es consumeixen simultàniament s’aconsegueix un metabolisme “equilibrat”, és a dir els fluxos de la glicòlisi i el cicle TCA esdevenen similars, evitant l’acumulació de piruvat en el citosol, la seva transformació a làctic i finalment la secreció d’aquest metabòlit. Aquest comportament és totalment oposat al que s’observa de forma general en els cultius de cèl.lules de mamífer en creixement exponencial, on els elevats fluxos de la glicòlisi troben una limitació en els fluxos d’entrada a la mitocòndria (és a dir, del cicle TCA) i conseqüentment el lactat és produït i secretat al medi. La construcció d’un model metabòlic i l’aplicació de FBA permetrà fer prediccions in silico de comportaments metabòlics causats per la sobreexpressió o el silenciament de gens diana. Aquesta estratègia obre la possibilitat de generar línies cel·∙lulars que presentin un metabolisme optimitzat per tal d’estudiar estratègies de cultiu més eficients per a l’increment de la densitat cel·∙lular i productivitat de proteïna recombinant.
The thesis is focused on the study of recombinant protein production in mammalian cell lines. In particular, the study of three different approaches of different bioprocesses based on HEK293 cells has been addressed. As a model protein for recombinant expression, CapPCV2 has been selected. This protein makes up the viral capsid of Porcine circovirus serotype 2 (PCV2), which is the causative agent of PCVDs (porcine circovirus diseases), a group of diseases with major impact in pig’s industry worldwide. This project has been addressed from the perspective of bioprocess development and optimization and therefore, the increment of volumetric production of cells, virus and proteins have been the driving force of the research. Firstly, cell culture media and nutritional supplementation studies are presented. Cell growth relies in high extent to the nutritional and physicochemical characteristics of the media in which cells are cultured and therefore, finding the proper cell media is one of the key factors for cell culture expansion. The initial media study resulted in a 6-­‐fold increment of the maximal viable cell achieved in the original media. Besides, different cell culture strategies have been explored, which resulted in a fed-­‐batch strategy that allowed reaching maximal viable cell densities of 26.8x106 cell/mL, which represents 13-­‐fold increment on maximal viable cell density originally reached. In the second and third chapter of results, three different approaches for the expression of recombinant CapPCV2 (r-­‐CapPCV2) are evaluated and discussed. As a first approach, a viral recombinant adenovirus encoding for the gene CapPCV2 has been generated and used as viral vector for the production of the recombinant protein in HEK293 cells. Besides, a deep study of the main parameters that affect the infection performance has been carried out and discussed in order to find the best media, MOI (multiplicity of infection), TOI (time of infection) and TOH (time of harvest) for adenovirus and recombinant protein production. This study was performed with an adenovirus expressing the reporter gene GFP and thereafter, the best infection parameters encountered were applied for the production of r-­‐CapPCV2 (media: SFMTransFx-­‐293 supplemented with 4mM glutaMAX, 5% FBS and 10%CB5; MOI:1; TOI:1x106 cell/mL) and TOH:48hpi). The second and third strategies are both based on the generation of stable producer cell lines, but one strategy relies on illegitimate (or random) integration of the gene in the HEK293 genome ,whereas the other strategy is a site-­‐directed integration of the gene in previously characterized hot-­‐spots (i.e. high-­‐active transcribed regions from genome). The site-­‐directed integration was performed using RMCE technology (Recombinant mediated cassette exchange). After the comparison of the specific and volumetric productivities achieved with each approach, the best producer has been selected. Nevertheless, r-­‐CapPCV2 was poorly produced so it was unfeasible to develop/design a cost-­‐effective industrial bioprocess and other alternatives must be studied in the future. Finally, the study of an unexpected metabolic behaviour observed in HEK293 cells cultured in our lab has been addressed from a physiologic and metabolic perspective. HEK293 cells could concomitantly consume glucose and lactate in exponentially growing cultures at particular environmental conditions. After a deep study of these conditions, it was found out that the switch from lactate secretion (which is the main drawback of mammalian high cell density cultures) to lactate consumption can be triggered from the beginning of cell culture at pH0=6.6 together with the addition of 4-­‐12mM of lactate to media. Remarkably, under these conditions nor cell growth neither protein production were negatively affected. Form these results, we hypothesize that HEK293 can co-­‐transport lactate and H+ to the cytosol as a pH-­‐detoxification mechanism. Moreover, the application of flux balance analysis permitted to find out that when lactate and glucose are consumed together a “more balanced” metabolism is achieved, meaning that glycolytic and TCA fluxes became similar, avoiding pyruvate accumulation at the cytosol and consequently, lactate formation. This is totally opposed to the extensively observed metabolism of exponentially growing mammalian cell lines, where the high flux through the glycolytic pathway encounters a limitation on the fluxes entering the mitochondria (hence, the TCA cycle) and consequently lactate is produced and secreted to media. The construction of a HEK293 metabolic model and the application of FBA will allow making in silico predictions of metabolic beahaviours after the upregulation or downregulation of target genes. This strategy may open the possibility of generate engineered HEK293 cell lines with an optimised metabolism in order to study more efficient cell culture strategies towards the achievement of higher cell densities and product titres.

The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

Transforming growth factor beta (TGFbeta) and Angiotensin II (AngII) signaling occurs through two distinct receptor superfamilies, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Through diametric actions, TGFbeta and AngII regulate various biological responses, including cell proliferation and migration. Previously, we identified the G protein-coupled receptor kinase 2 (GRK2), which acts through a negative feedback loop mechanism to terminate Smad signaling. To investigate the impact of TGFbeta-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). We show that activation of the TGFbeta signaling cascade results in increased GRK2 expression levels, consequently inhibiting AngII-induced ERK phosphorylation and antagonizing AngII-induced VSMC proliferation and migration. The inhibitory effect of TGFbeta on AngII signaling occurs at the MEK-ERK interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFbeta signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation. GRK2 is a key factor in mediating this crosstalk.

Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways.
QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA.
Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes.
Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.

Introduction: Idiopathic Pulmonary arteriole hypertension (IPAH) is a rare but severely debilitating disease that strikes women to men at a ratio of 3:1. Endothelial cell (EC) dysfunction is a hallmark of the disease. This includes rapid growth of the ECs until the occlusion of the vasculature as well as decreased blood levels of vasodilators. Markedly increased levels of endothelin-1, a potent vasoconstrictor and smooth muscle mitogen, have been noted in IPAH patients.
Recently mutations in the bone morphogenic protein receptor type II (BMPRII) have been linked to the disease. Interestingly mutations in activin-like kinase-1 (ALK-1) and endoglin have been linked to hereditary haemorrhagic telangiectasia (HHT), a disease that results in PAH clinically indistinguishable from IPAH. All of these proteins are either receptors or co-receptors to members of the TGFbeta superfamily. The connection of these mutations to the disease still remains largely a mystery to researchers and the effects of either bone morphogenic proteins 2, 4, 7 or TGFbeta levels on endothelin-1(ET-1) production in human microvascular endothelial cells cultured from normal lungs (HMVEC-LBI) are unknown.
Methods: HMVEC-LBI cells were cultured in the presence of various concentrations of BMP 2,4,7 and TGFbeta, in complete media or serum starved conditions. After allotted time points the media was collected and assayed by ELISA, meanwhile the cells were lysed and protein content assayed for normalization purposes. Small Mothers against Decapentaplegic (SMAD) 1/5 phosphorylation was also measured.
Results and Conclusions: Despite evidence that all BMPs used were biologically active, namely through SMAD phosphorylation studies, only BMP7 at very high dosages increased ET-1 production levels. TGFbeta had a more pronounced effect at earlier time points with lower concentrations. The results provide insights on the effects of an important group of proteins, the BMPs and TGFbeta, on lung microvascular ECs and which are likely the key cellular player In IPAH development. These findings may have clinical relevance in terms of control of the disease and understanding the normal response of these cells BMPs and TGFbeta.

Activation of the Unfolded Protein Response (UPR) following stress in the Endoplasmic Reticulum (ER) is an important mechanism by which obesity results in insulin resistance and type II diabetes. We uncovered a role for the adaptor protein Nck in modulating the UPR. In this study, we report that obese Nck1-/- mice, which show lower levels of UPR in liver and adipose tissue, present improved insulin signalling in these tissues. We established that the effect of Nck1 is cell autonomous by showing that HepG2 cells treated with Nck1 siRNA have reduced ER stress-induced UPR and Insulin Receptor Substrate-1 (IRS-1) serine phosphorylation. In these cells, we observed that the IRS-1 levels and activation of signalling components downstream of the insulin receptor were increased. This correlates with enhanced cell survival to stress and insulin stimulated glycogen synthesis. Overall, we demonstrated that Nck1 participates in ER-stress-induced insulin resistance and regulation of IRS-1-dependent signalling.

Copy of author's previously published work inserted. Includes bibliographical references (leaves 160-182). Concentrates mainly on the characterisation of the molecular mechanism of action of the tec protein tyrosine kinase using biochemical and genetic approaches.