Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Divisió IV)
URI permanent per a aquesta col·leccióhttps://hdl.handle.net/2445/36270
Examinar
Enviaments recents
Mostrant 1 - 4 de 4
TesiPaper de la UGT1A6 en la resistència al metotrexat : estudi dels harpins de polipurines per disminuir l'expressió gènica(Universitat de Barcelona, 2010-12-13) Almagro García, Ma. Cristina de; Ciudad i Gómez, Carlos Julián; Noé Mata, Verónica; Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Divisió IV)[cat] El treball presentat en aquesta memòria es centra en l'estudi dels mecanismes pels quals el quimioteràpic metotrexat (MTX) desenvolupa resistència en cèl·lules canceroses. Dins d'aquest objectiu es creen dues línies d'estudi que són, trobar nous mecanismes d'inhibició de la dihidrofolat reductasa (DHFR), principal causant de la resistència al MTX, i la troballa de nous gens que puguin estar implicats en aquest procés de resistència. En el present estudi, hem analitzat la resistència al MTX en cèl·lules de càncer de mama. S'han realitzat microarrays d'expressió amb dues línies cel·lulars sensibles i resistents al MTX, els quals han originat com a únic gen en comú entre les cèl·lules resistents d'ambdues línies la família de les UDP-glucuronosil transferases (UGT1A). Hem estudiat aquesta família de gens, i s'ha identificat la UGT1A6 com la principal responsable de la sobrexpressió de les UGTs a les cèl·lules resistents. En aquest treball s'ha analitzat la implicació de la UGT1A6 en la resistència al MTX. També hem analitzat la inducció transcripcional del MTX sobre la UGT1A6, així com els factors transcripcionals involucrats en aquest procés. A més, s'han estudiat les repercussions terapèutiques que pot tenir la sobrexpressió de la UGT1A6 quan es dóna MTX en combinació amb altres fàrmacs glucuronidables. Dins de la recerca de nous mètodes per inhibir la DHFR diferents als quimioteràpics clàssics com el MTX, trobem els basats en teràpia gènica. En treballs anteriors del nostre grup s'havien desenvolupat diversos tipus de molècules per disminuir l'expressió de la dhfr, tal com oligonucleòtids antisentit (aODNs), siRNAs i Triplex forming oligonucleotides (TFOs). En aquest treball hem desenvolupat Template-PPRHs i Coding-PPRHs, hairpins destinats a unir-se a la cadena motlle o codificant del DNA i mRNA, respectivament. Hem estudiat la capacitat citotòxica que tenen els PPRHs en cèl·lules de càncer de mama utilitzant com a model el gen dhfr humà, així com el mecanisme d'acció pel qual aquestes molècules inhibeixen la DHFR. També hem analitzat la capacitat in vitro dels hairpins d'unir-se a la seva seqüència diana i la influència que té la presència d'interrupcions de purines en la cadena de polipirimidines i la forma de minimitzar els seus efectes. Així mateix, hem estudiat l'estabilitat d'aquestes molècules, la seva especificitat i l'activació de la resposta immune. S'ha analitzat l'aplicació terapèutica a partir de l'utilització de PPRHs contra la dhfr en cèl·lules de càncer de mama resistents al MTX, així com el disseny de diferents PPRHs dirigits contra gens amb importància terapèutica en càncer.- TesiMolecular characterization of carnitine palmitoyltransferase 1C(Universitat de Barcelona, 2010-12-16) Gratacòs i Batlle, Esther; Clotet Erra, Josep; Casals i Farré, Núria; Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Divisió IV)Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of long chain fatty acyl-CoAs into acylcarnitines, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo β-oxidation. This reaction is not only central to the control of fatty acid oxidation, but it also determines the availability of long chain acyl-CoA for other processes. There are three different CPT1 isozymes: CPT1A (expressed in liver, pancreas, kidney, brain, blood, and embryonic tissues), CPT1B (expressed only in brown adipose tissue, muscle, and heart) and the recently described CPT1C. CPT1C protein sequence is highly similar to that of the other two isozymes. Expression studies indicate that CPT1C is localized exclusively in the central nervous system, with homogeneous distribution in all areas (hippocampus, cortex, hypothalamus, and others). It has also been reported that CPT1c is localized in neurons but not in astrocytes of adult brain. 1. CPT1C strucutral model A 3-D structural model of the isozyme has been constructed by homology modeling. Residues contacting both substrates have been determined and compared to the same amino acid positions in CPT1A. The results obtained from the analysis show that the residues involved in the catalysis of the reaction in CPT1A and residues contacting both substrates are conserved mainly conserved in CPT1C or show semi-conservative substitutions. 2. CPT1 enzymatic activity Expression of rat CPT1C in Saccharomyces cerevisiae yields no catalytic activity when testing different conditions (longer periods of time, increased temperature, increased substrate concentration, testing of microsomal fraction or chimeric protein CPT1·ACA). Thus, the yeast expression system is not suitable for studying CPT1C enzymatic activity. 3. Subcellular localization Endogenous and overexpressed CPT1C is basically localized in the endoplasmic reticulum of mammalian cells (HEK293T, PC12, SH-SY5Y, primary cultures of fibroblasts and neurons). Some evidences indicated that CPT1C could also be found, in lower amounts, in mitochondrial associated membranes (MAMs). The specific sequence of CPT1C N-terminal domain (first 150 amino acids) drives the protein to the endoplasmic reticulum. 4. CPT1C N-terminus processing The N-terminal end of endogenous CPT1C in wild type mouse brain is processed (at least until Val27) and is not detected in mouse brain cortex lysates. 5. CPT1C membrane topology The N- and C-terminal domains of CPT1C are facing the cytosolic side of the endoplasmic reticulum membrane, whereas the loop domain is facing the endoplasmic reticulum lumen. 6. CPT1C interacting partners The data provided by the yeast two-hybrid assay do not indicate a unique binding partner of CPT1C. Instead the assay retrieved proteins involved in different functions: protein degradation, membrane trafficking, cell structure, signal transduction and metabolism. KEYWORDS: Carnitine palmitoyltransferase, Endoplasmatic reticulum, Subcelular localization, CPT1 activity, Structural model, Membrane topology
- TesiImplication of Long-Chain Fatty Acids in Glucose-Induced Insulin Secretion in the Pancreatic Beta-Cell(Universitat de Barcelona, 2004-11-24) Herrero Rodríguez, Laura; Asins Muñoz, Guillermina; García Hegardt, Fausto; Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Divisió IV)INTRODUCTION Carnitine palmitoyltransferase I, which is expressed in the pancreas as the liver isoform (LCPTI), catalyzes the rate-limiting step in the transport of fatty acids into the mitochondria for their oxidation. To directly examine whether the availability of long-chain fatty acyl-CoA affects the regulation of insulin secretion in the Beta-cell, we infected INS(832/13) cells and rat islets with an adenovirus encoding a mutant form of LCPTI (Ad-LCPTI M593S) that is insensitive to its inhibitor malonyl-CoA. C75 is described as a potential drug for treatment of obesity and type 2 diabetes. First known as a synthetic inhibitor of fatty acid synthase, it has been also described as an activator of CPTI, increasing peripheral energy utilization and fatty acid oxidation in mice. To further investigate the C75/CPTI interaction, we have characterized the effects of C75 on CPTI in vitro and in vivo. OBJECTIVES 1) Study of the malonyl-CoA/CPTI interaction in the pancreatic Beta-cell and its involvement in glucose-stimulated insulin secretion (GSIS). 2) Construction of an INS stable cell line overexpressing LCPTI wt and LCPTI M593S. 3) Determine the effect of C75 on the CPTI activity and palmitate oxidation in pancreatic Beta-cells. RESULTS. In Ad-LCPTI M593S infected INS(832/13) cells LCPTI activity increased six-fold. This was associated with enhanced fatty acid oxidation, at any glucose concentration, and a 60% suppression of GSIS. In isolated rat islets in which LCPTI M593S was overexpressed, GSIS decreased 40%. At high glucose concentration, overexpression of LCPTI M593S reduced partitioning of exogenous palmitate into lipid esterification products, and decreased PKC activation. Moreover, LCPTI M593S expression impaired KATP channel-independent GSIS in INS(832/13) cells. INS-1 stable clones of LCPTIwt and LCPTImut were constructed, however none of them resulted in an increase in LCPTI protein expression compared to endogenous LCPTI nor in CPTI activity. Therefore, slight basal overexpression of LCPTI could probably be toxic for the cells, as a result of which only those cells that do not contain the LCPTI plasmids survived throughout cell passages. When INS(823/13) cells are incubated with C75, CPTI activity is inhibited, as is fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 to mice produces a short-term inhibition of CPTI activity in mitochondria from liver and pancreas. DISCUSSION. The results with LCPTImut provide direct support for the hypothesis proposing that the malonyl-CoA/CPTI interaction is a component of a metabolic signalling network that controls insulin secretion. Overall, the findings with C75 provide compelling evidence that the drug is a potent inhibitor of CPTI.
- TesiRegulation of E2F1 Transcription Factor by Glycogen Synthase Kinase-3-Beta(Universitat de Barcelona, 2005-03-10) Garcia Alvarez, Gisela; Tauler Girona, Albert; Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Divisió IV)[eng] Whether a cell enters the cell cycle, undergoes apoptosis or survives is a consequence of the integration of several cellular and environmental signals. Growth factors, cell contact, and apoptotic inductors regulate a complex system of signal transduction pathways that trigger the activation of a large number of genes. One of the key proteins in the regulation of the cell cycle and the commitment to apoptosis is the transcription factor E2F1. It has been suggested that E2F1 activity levels could determine whether a cell enters the cell cycle, cell-cycle arrest or apoptosis. In these decisions, not only is transcriptional activity important, but also the synchronization of E2F1 activity with specific signal transduction pathways. In this context it has been suggested that activation of the PI 3-kinase pathway inhibits the apoptotic effect of E2F1 overexpression. Since GSK3-beta is a downstream effector of PI 3-kinase, we performed phosphorylation and binding analyses to examine the possible relationship between GSK3-beta and E2F1. The results obtained in this thesis demonstrate that GSK3-beta phosphorylates human E2F1 in vitro at serine 403 and threonine 433. In earlier studies it has already been shown that these two residues are also phosphorylated by the TFIIH kinase, cdk7. We did not detect phosphorylation by GSK3-beta in vivo . However, immunoprecipitation experiments revealed in vivo binding of these proteins. By transient transfection experiments with GSK3-beta and E2F1 constructs, GSK3-beta -RNAi assays, and the use of PI 3-kinase and GSK3-beta specific inhibitors, we demonstrate that GSK3-beta regulates E2F1 transcriptional activity through its interaction with E2F1 transactivation domain and that GSK3-beta kinase activity is not required for this regulation. Our data obtained here integrates into a model in which translocation of GSK3-beta to the nucleus modulates E2F1 activity and, as a consequence, determines whether a cell enters the cell cycle or undergoes apoptosis.