Myocardial tolerance to ischemia-reperfusion injury, training intensity and cessation.

Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% [Formula: see text]) or high (80% [Formula: see text]) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

The systemic control of circadian gene expression.

The mammalian circadian timing system consists of a central pacemaker in the brain's suprachiasmatic nucleus (SCN) and subsidiary oscillators in nearly all body cells. The SCN clock, which is adjusted to geophysical time by the photoperiod, synchronizes peripheral clocks through a wide variety of systemic cues. The latter include signals depending on feeding cycles, glucocorticoid hormones, rhythmic blood-borne signals eliciting daily changes in actin dynamics and serum response factor (SRF) activity, and sensors of body temperature rhythms, such as heat shock transcription factors and the cold-inducible RNA-binding protein CIRP. To study these systemic signalling pathways, we designed and engineered a novel, highly photosensitive apparatus, dubbed RT-Biolumicorder. This device enables us to record circadian luciferase reporter gene expression in the liver and other organs of freely moving mice over months in real time. Owing to the multitude of systemic signalling pathway involved in the phase resetting of peripheral clocks the disruption of any particular one has only minor effects on the steady state phase of circadian gene expression in organs such as the liver. Nonetheless, the implication of specific pathways in the synchronization of clock gene expression can readily be assessed by monitoring the phase-shifting kinetics using the RT-Biolumicorder.

Regulation of HSF2 and its function in mitosis

The cell is continuously subjected to various forms of external and intrinsic proteindamaging stresses, including hyperthermia, pathophysiological states, as well as cell differentiation and proliferation. Proteindamaging stresses result in denaturation and improper folding of proteins, leading to the formation of toxic aggregates that are detrimental for various pathological conditions, including Alzheimer’s and Huntington’s diseases. In order to maintain protein homeostasis, cells have developed different cytoprotective mechanisms, one of which is the evolutionary well-conserved heat shock response. The heat shock response results in the expression of heat shock proteins (Hsps), which act as molecular chaperones that bind to misfolded proteins, facilitate their refolding and prevent the formation of protein aggregates. Stress-induced expression of Hsps is mediated by a family of transcription factors, the heat shock factors, HSFs. Of the four HSFs found in vertebrates, HSF1-4, HSF1 is the major stress-responsive factor that is required for the induction of the heat shock response. HSF2 cannot alone induce Hsps, but modulates the heat shock response by forming heterotrimers with HSF1. HSFs are not only involved in the heat shock response, but they have also been found to have a function in development, neurodegenerative disorders, cancer, and longevity. Therefore, insight into how HSFs are regulated is important for the understanding of both normal physiological and disease processes. The activity of HSF1 is mainly regulated by intricate post-translational modifications, whereas the activity of HSF2 is concentrationdependent. However, there is only limited understanding of how the abundance of HSF2 is regulated. This study describes two different means of how HSF2 levels are regulated. In the first study it was shown that microRNA miR-18, a member of the miR-17~92 cluster, directly regulates Hsf2 mRNA stability and thus protein levels. HSF2 has earlier been shown to play a profound role in the regulation of male germ cell maturation during the spermatogenesis. The effect on miR-18 on HSF2 was examined in vivo by transfecting intact seminiferous tubules, and it was found that inhibition of miR-18 resulted in increased HSF2 levels and modified expression of the HSF2 targets Ssty2 and Speer4a. HSF2 has earlier been reported to modulate the heat shock response by forming heterotrimers with HSF1. In the second study, it was shown that HSF2 is cleared off the Hsp70 promoter and degraded by the ubiquitinproteasome pathway upon acute stress. By silencing components of the anaphase promoting complex/cyclosome (APC/C), including the co-activators Cdc20 and Cdh1, it was shown that APC/C mediates the heatinduced ubiquitylation of HSF2. Furthermore, down-regulation of Cdc20 was shown to alter the expression of heat shock-responsive genes. Next, we studied if APC/C-Cdc20, which controls cell cycle progression, also regulates HSF2 during the cell cycle. We found that both HSF2 mRNA and protein levels decreased during mitosis in several but not all human cell lines, indicating that HSF2 has a function in mitotic cells. Interestingly, although transcription is globally repressed during mitosis, mainly due to the displacement of RNA polymerase II and transcription factors, including HSF1, from the mitotic chromatin, HSF2 is capable of binding DNA during mitosis. Thus, during mitosis the heat shock response is impaired, leaving mitotic cells vulnerable to proteotoxic stress. However, in HSF2-deficient mitotic cells the Hsp70 promoter is accessible to both HSF1 and RNA polymerase II, allowing for stress-inducible Hsp expression to occur. As a consequence HSF2-deficient mitotic cells have a survival advantage upon acute heat stress. The results, presented in this thesis contribute to the understanding of the regulatory mechanisms of HSF2 and its function in the heat shock response in both interphase and mitotic cells.

Proteomic inventory of myocardial proteins from patients with chronic Chagas' cardiomyopathy

Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ß chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

Quantitative refinement of reaction-based biomodels

In the field of molecular biology, scientists adopted for decades a reductionist perspective in their inquiries, being predominantly concerned with the intricate mechanistic details of subcellular regulatory systems. However, integrative thinking was still applied at a smaller scale in molecular biology to understand the underlying processes of cellular behaviour for at least half a century. It was not until the genomic revolution at the end of the previous century that we required model building to account for systemic properties of cellular activity. Our system-level understanding of cellular function is to this day hindered by drastic limitations in our capability of predicting cellular behaviour to reflect system dynamics and system structures. To this end, systems biology aims for a system-level understanding of functional intraand inter-cellular activity. Modern biology brings about a high volume of data, whose comprehension we cannot even aim for in the absence of computational support. Computational modelling, hence, bridges modern biology to computer science, enabling a number of assets, which prove to be invaluable in the analysis of complex biological systems, such as: a rigorous characterization of the system structure, simulation techniques, perturbations analysis, etc. Computational biomodels augmented in size considerably in the past years, major contributions being made towards the simulation and analysis of large-scale models, starting with signalling pathways and culminating with whole-cell models, tissue-level models, organ models and full-scale patient models. The simulation and analysis of models of such complexity very often requires, in fact, the integration of various sub-models, entwined at different levels of resolution and whose organization spans over several levels of hierarchy. This thesis revolves around the concept of quantitative model refinement in relation to the process of model building in computational systems biology. The thesis proposes a sound computational framework for the stepwise augmentation of a biomodel. One starts with an abstract, high-level representation of a biological phenomenon, which is materialised into an initial model that is validated against a set of existing data. Consequently, the model is refined to include more details regarding its species and/or reactions. The framework is employed in the development of two models, one for the heat shock response in eukaryotes and the second for the ErbB signalling pathway. The thesis spans over several formalisms used in computational systems biology, inherently quantitative: reaction-network models, rule-based models and Petri net models, as well as a recent formalism intrinsically qualitative: reaction systems. The choice of modelling formalism is, however, determined by the nature of the question the modeler aims to answer. Quantitative model refinement turns out to be not only essential in the model development cycle, but also beneficial for the compilation of large-scale models, whose development requires the integration of several sub-models across various levels of resolution and underlying formal representations.

Reduction of carcass weight loss in swines

The carcass fast freezing is one of the aspects of great prominence to the final quality of pork. In order to reduce weight loss, two experiments were performed, in which the carcasses were monitored during 20 hours to evaluate the main variables involved during two different freezing processes (standard and proposed) as follows: microbiological quality, storage temperature, relative humidity (RH) and air velocity. In experiment I, the carcasses were submitted to a system using heat shock (2 hours in static tunnel at - 25 °C) and subsequently sent to the equalization chamber. In experiment II, the carcasses were submitted to the heat shock and stored in a chamber with RH between 80-85%. The chambers used in both experiments showed no change in the variables studied (internal temperature of 5 °C and air velocity of approximately 0.3 m/s). However, the relative humidity in the three chambers was evaluated and significant differences were found; as a consequence, high levels of weight loss were observed in both chambers In experiment II there was an increase of RH, which reduced the weight loss of the carcasses.

Mode of action of FMRFamide-like peptide on drosophila body wall muscle conractions

Neuropeptides are the largest group of signalling chemicals that can convey the information from the brain to the cells of all tissues. DPKQDFMRFamide, a member of one of the largest families of neuropeptides, FMRFamide-like peptides, has modulatory effects on nerve-evoked contractions of Drosophila body wall muscles (Hewes et aI.,1998) which are at least in part mediated by the ability of the peptide to enhance neurotransmitter release from the presynaptic terminal (Hewes et aI., 1998, Dunn & Mercier., 2005). However, DPKQDFMRFamide is also able to act directly on Drosophila body wall muscles by inducing contractions which require the influx of extracellular Ca 2+ (Clark et aI., 2008). The present study was aimed at identifying which proteins, including the membrane-bound receptor and second messenger molecules, are involved in mechanisms mediating this myotropic effect of the peptide. DPKQDFMRFamide induced contractions were reduced by 70% and 90%, respectively, in larvae in which FMRFamide G-protein coupled receptor gene (CG2114) was silenced either ubiquitously or specifically in muscle tissue, when compared to the response of the control larvae in which the expression of the same gene was not manipulated. Using an enzyme immunoassay (EIA) method, it was determined that at concentrations of 1 ~M- 0.01 ~M, the peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. In addition, the physiological effect of DPKQDFMRFamide at a threshold dose was not potentiated by 3-lsobutyl-1-methylxanthine, a phosphodiesterase inhibitor, nor was the response to 1 ~M peptide blocked or reduced by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. The response to DPKQDFMRFamide was not affected in the mutants of the phosholipase C-~ (PLC~) gene (norpA larvae) or IP3 receptor mutants, which suggested that the PLC-IP3 pathway is not involved in mediat ing the peptide's effects. Alatransgenic flies lacking activity of calcium/calmodul in-dependent protein kinase (CamKII showed an increase in muscle tonus following the application of 1 JlM DPKQDFMRFamide similar to the control larvae. Heat shock treatment potentiated the response to DPKQDFMRFamide in both ala1 and control flies by approximately 150 and 100 % from a non heat-shocked larvae, respectively. Furthermore, a CaMKII inhibitor, KN-93, did not affect the ability of peptide to increase muscle tonus. Thus, al though DPKQDFMRFamide acts through a G-protein coupled FMRFamide receptor, it does not appear to act via cAMP, cGMP, IP3, PLC or CaMKl1. The mechanism through which the FMRFamide receptor acts remains to be determined.

Investigation into the relationships between molecular chaperones, mitochondrial antioxidant enzymes, and endothermic vertebrate longevity

The maximum lifespan (MLSP) of endothermic vertebrates can range from as little as a year to over two centuries, yet the underlying phenotype of aging is very similar amongst this group of organisms. One organelle that may be important in the phenotype of aging is the mitochondrion. When damaged, this organelle is thought to contribute to many of the neurodegenerative diseases of aging. For this thesis, mitochondria from brain tissues of 7 mammalian and 2 avian species were isolated to assess whether the antioxidant glutathione system and major molecular chaperone, HSP60, is correlated to species MLSP. Furthermore, HSP60, and the major endoplasmic reticulum chaperone, GRP78, were measured under basal conditions, and following the introduction of an oxidative stress (hydrogen peroxide) in cultured mammalian myoblasts from 10 different species. My results indicate that the enzymes involved in the glutathione defense system are not correlated to species MLSP in brain mitochondria; however HSP60 levels are indeed higher in the longer-lived species. HSP60 levels are also higher at the basal level in cultured mammalian myoblasts and after 1 hour of hydrogen peroxide exposure. GRP78 induction is not correlated to species MLSP at the basal level or following hydrogen peroxide exposure. Therefore, these results suggest that HSP60 is a correlate of longevity in endothermic vertebrate species, but neither the glutathione antioxidant defense system, nor GRP78, correlates to species longevity.

Les R-loops et leurs conséquences sur l'expression génique chez Escherichia coli.

Des variations importantes du surenroulement de l’ADN peuvent être générées durant la phase d’élongation de la transcription selon le modèle du « twin supercoiled domain ». Selon ce modèle, le déplacement du complexe de transcription génère du surenroulement positif à l’avant, et du surenroulement négatif à l’arrière de l’ARN polymérase. Le rôle essentiel de la topoisomérase I chez Escherichia coli est de prévenir l’accumulation de ce surenroulement négatif générée durant la transcription. En absence de topoisomérase I, l’accumulation de ce surenroulement négatif favorise la formation de R-loops qui ont pour conséquence d’inhiber la croissance bactérienne. Les R-loops sont des hybrides ARN-ADN qui se forment entre l’ARN nouvellement synthétisé et le simple brin d’ADN complémentaire. Dans les cellules déficientes en topoisomérase I, des mutations compensatoires s’accumulent dans les gènes qui codent pour la gyrase, réduisant le niveau de surenroulement négatif du chromosome et favorisant la croissance. Une des ces mutations est une gyrase thermosensible qui s’exprime à 37 °C. La RNase HI, une enzyme qui dégrade la partie ARN d’un R-loop, peut aussi restaurer la croissance en absence de topoisomérase I lorsqu’elle est produite en très grande quantité par rapport à sa concentration physiologique. En présence de topoisomérase I, des R-loops peuvent aussi se former lorsque la RNase HI est inactive. Dans ces souches mutantes, les R-loops induisent la réponse SOS et la réplication constitutive de l’ADN (cSDR). Dans notre étude, nous montrons comment les R-loops formés en absence de topoisomérase I ou RNase HI peuvent affecter négativement la croissance des cellules. Lorsque la topoisomérase I est inactivée, l’accumulation d’hypersurenroulement négatif conduit à la formation de nombreux R-loops, ce qui déclenche la dégradation de l’ARN synthétisé. Issus de la dégradation de l’ARNm de pleine longueur, des ARNm incomplets et traductibles s’accumulent et causent l’inhibition de la synthèse protéique et de la croissance. Le processus par lequel l’ARN est dégradé n’est pas encore complètement élucidé, mais nos résultats soutiennent fortement que la RNase HI présente en concentration physiologique est responsable de ce phénotype. Chose importante, la RNase E qui est l’endoribonuclease majeure de la cellule n’est pas impliquée dans ce processus, et la dégradation de l’ARN survient avant son action. Nous montrons aussi qu’une corrélation parfaite existe entre la concentration de RNase HI, l’accumulation d’hypersurenroulement négatif et l’inhibition de la croissance bactérienne. Lorsque la RNase HI est en excès, l’accumulation de surenroulement négatif est inhibée et la croissance n’est pas affectée. L’inverse se produit Lorsque la RNase HI est en concentration physiologique. En limitant l’accumulation d’hypersurenroulement négatif, la surproduction de la RNase HI prévient alors la dégradation de l’ARN et permet la croissance. Quand la RNase HI est inactivée en présence de topoisomérase I, les R-loops réduisent le niveau d’expression de nombreux gènes, incluant des gènes de résistance aux stress comme rpoH et grpE. Cette inhibition de l’expression génique n’est pas accompagnée de la dégradation de l’ARN contrairement à ce qui se produit en absence de topoisomérase I. Dans le mutant déficient en RNase HI, la diminution de l’expression génique réduit la concentration cellulaire de différentes protéines, ce qui altère négativement le taux de croissance et affecte dramatiquement la survie des cellules exposées aux stress de hautes températures et oxydatifs. Une inactivation de RecA, le facteur essentiel qui déclenche la réponse SOS et le cSDR, ne restaure pas l’expression génique. Ceci démontre que la réponse SOS et le cSDR ne sont pas impliqués dans l’inhibition de l’expression génique en absence de RNase HI. La croissance bactérienne qui est inhibée en absence de topoisomérase I, reprend lorsque l’excès de surenroulement négatif est éliminé. En absence de RNase HI et de topoisomérase I, le surenroulement négatif est très relaxé. Il semble que la réponse cellulaire suite à la formation de R-loops, soit la relaxation du surenroulement négatif. Selon le même principe, des mutations compensatoires dans la gyrase apparaissent en absence de topoisomérase I et réduisent l’accumulation de surenroulement négatif. Ceci supporte fortement l’idée que le surenroulement négatif joue un rôle primordial dans la formation de R-loop. La régulation du surenroulement négatif de l’ADN est donc une tâche essentielle pour la cellule. Elle favorise notamment l’expression génique optimale durant la croissance et l’exposition aux stress, en limitant la formation de R-loops. La topoisomérase I et la RNase HI jouent un rôle important et complémentaire dans ce processus.

Génération de lignées de poissons zébrés exprimant le gène muté TARDBP

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative due à une dégénérescence des motoneurones. Plus de 40 mutations du gène TARDBP ont été identifiées chez des patients SLA. Les défauts biochimiques de ces mutations étant encore inconnus, les modèles animaux sont présentement la seule mesure possible d’un phénotype. Pour étudier les conséquences physiopathologiques d’une de ces mutations, nous avons développé deux lignées transgéniques de poisson zébré, exprimant le gène humain TARDBP soit de type sauvage, soit avec la mutation G348C liée à la SLA, sous le contrôle d’un promoteur de choc thermique. Ces lignées ont été étudiées sur trois générations, après avoir établi un protocole de choc thermique induisant une expression ubiquitaire du transgène. Les embryons transgéniques de la génération F2 de la lignée exprimant la mutation développent un phénotype moteur suite à un choc thermique de 38.5°C pendant 30 minutes lorsque les embryons sont à 18 heures post-fertilisation. 60% des embryons ont une réponse anormale au toucher. De plus, une réduction de 28% de la longueur de pré-branchement des axones des motoneurones est observée. Ces résultats indiquent que notre lignée exprimant la protéine mutante TDP-43 est un modèle génétique de la SLA prometteur, qui ouvre des perspectives pour la compréhension de la physiopathologie de la maladie et la découverte de molécules thérapeutiques.

Régulation transcriptionnelle du gène de la protéine de liaison de la chlorophylle-a et de la péridinine chez le dinoflagellé Lingulodinium polyedrum

Les dinoflagellés jouent un rôle très important dans l’écologie des océans en y réalisant une grande partie de la production primaire, en formant une association symbiotique avec les coraux et en ayant la capacité de produire des fleurs d’algues potentiellement toxiques pour les communautés côtières humaines et animales. Malgré tout, la biologie moléculaire des dinoflagellés n’a que très peu été étudiée dans les dernières années, les connaissances de processus de base comme la régulation de la transcription y étant fortement limitées. Une tentative pour élucider ce mécanisme a été réalisée chez les dinoflagellés photosynthétiques Lingulodinium polyedrum et Amphidinium carterae. Une expérience d’induction de la transcription du gène de la Peridinin chlorophyll-a binding protein, le complexe majeur de collecte de lumière, a été réalisée par une baisse de l’intensité lumineuse et a montré une faible augmentation (moins de 2 fois) du transcrit à court et long terme. Des expériences de simple-hybride et de retard sur gel (EMSA) ont été faits pour identifier de potentielles interactions protéine-ADN dans la région intergénique du gène PCP organisé en tandem. Ces essais ont été infructueux pour identifier de telles protéines. Une analyse du transcriptome de L. polyedrum a été effectuée, montrant une importante sous-représentation de domaines de liaison à l’ADN classique (comme Heat-shock factor, bZIP ou Myb) et une surreprésentation du domaine d’origine bactérienne Cold shock en comparaison avec d’autres eucaryotes unicellulaires. Ce travail suggère que les mécanismes de régulation transcriptionnelle des dinoflagellés pourraient différer substantiellement de ceux des autres eucaryotes.

Les sHsps en surera: capacitat protectora enfront l'estrés i variabilitat genètica

Els organismes responen a la temperatura i a molts altres estressos sintetitzant un grup de proteïnes anomenat proteïnes de xoc de calor (HSPs). En plantes les sHsps, d'entre 15 i 30 kDa formen el grup més abundant i divers, classificat en funció de la seva localització subcel.lular i homologia en: mitocondrials, cloroplàstiques, de reticle endoplasmàtic i citoplàsmiques de classe I i II. Les sHsps-CI s'ha descrit que s'indueixen per estrès tèrmic, hídric i oxidatiu (peròxid d'hidrògen, llum UV, ozó) i en resposta a algunes hormones. També s'expressen durant el desenvolupament, per exemple durant l'embriogènesi, on es creu que podrien tenir un paper protector de l'embrió enfront la dessecació. Tot i que hi ha abundants treballs que correlacionen la resistència a l'estrès i l'acumulació de sHsps-CI, els mecanismes moleculars d'aquesta activitat són poc conguts. Tot i això, per diverses sHsps-CI ha estat descrita una activitat xaperona in vitro i, més recentment, que la seva sobreexpressió augmenta la viabilitat de cèl.lules d'E.coli en condicions d'estrès tèrmic. L'estudi de l'acumulació de sHsps-CI en surera (Quercus suber) mitjançant immunodetecció en electroforesi bidimensional mostra uns patrons d'acumulació complexos i formats per dos grups d'espècies proteiques principals, a l'entorn dels 10 i 17 kDa respectivament, que mostren una inducció diferencial en funció del teixit i l'estrès. Mentre que les espècies proteiques de 17 kDa s'indueixen per temperatura però no per estrès oxidatiu, les de ca. 10 kDa ho fan per estrès oxidatiu i no per temperatura. Ambdós grups d'espècies proteiques s'acumulen conjuntament en fel.lema. Assajos de PCR i RT-PCR han permès clonar parcialment tres noves sHsps-CI en surera: Qshsp10-CI, QshspC-CI i QshspD-CI. Aquest fet confirma la multigeneïcitat de les sHsps-CI en surera que apuntava el patró bidimensional. Dels nous clons obtinguts destaca especialment Qshsp10-CI, un gen que presenta un codó stop enmig del domini -cristal.lí que fa que a la proteïna que se'n dedueix li manqui un 55% del domini -cristal.lí i tota l'extensió C-terminal. Es tractaria de la sHsp més petita i més truncada descrita fins al moment. L'anàlisi de l'expressió de Qshsp10-CI mitjançant RT-PCR mostra expressió en plantes tractades amb H2O2 però no en les que han estat sotmeses a un xoc de calor. Aprofitant l'oportunitat que oferia aquesta sHsp-CI de ser utilitzada com a model per l'estudi de la importància del domini -cristal.lí i l'extensió C-terminal en l'activitat protectora enfront l'estrès, es va voler determinar la capacitat que tenia d'augmentar la viabilitat de cèl.lules d'E. coli en condicions d'estrès tèrmic i oxidatiu. Els resultats mostren que la proteïna recombinant QsHsp10-CI, tot i la important truncació que té, és capaç de protegir cèl.lules d'E. coli en condicions d'estrès tèrmic i, remarcablement, en condicions d'estrès oxidatiu. Tots aquests resultats indiquen que les espècies proteiques de ca. 10 kDa podrien correspondre a Qshsp10-CI i tenir un paper en les cèl.lules del fel.lema en la protecció enfront l'estrès oxidatiu. L'estrès oxidatiu provoca lesions al DNA que poden produir errors en la replicació, transcripció o traducció i generar proteïnes aberrants. Donades les condicions d'estrès oxidatiu a les quals es troben sotmeses les cèl.lules del fel.lema, s'ha volgut estudiar la variabilitat dels seus àcids nucleics. La determinació de la taxa de mutació de la regió codificant del gen Qshsp17.4-CI en mRNA i DNA de fel.lema i àpex radicular, un teixit jove i en creixement actiu va mostrar unes taxes sorprenentment elevades en l'mRNA (1/1784 pb) i el DNA genòmic (1/1520 pb) del fel.lema. Aquestes taxes són les més altes descrites en un genoma nuclear eucariota i són similars a les dels virus d'RNA d'evolució ràpida com el virus de l'Hepatitis C. Amb aquestes taxes de mutació, un terç dels mRNAs del fel.lema de la surera contindrien missatges aberrants i la supervivència de les cel.lules es veuria compromesa. Això implica que el fel.lema hauria de ser considerat com un mosaic de cèl.lules genèticament heterogènies i, per tant, una sola seqüència no defineix en tota la seva amplitud un gen en aquest teixit. No es va detectar cap mutació en àpex de rel. Amb l'objectiu d'aprofundir en el coneixement de les mutacions que es donen en aquests dos teixits i per tal de poder fer una anàlisi qualitativa més completa que permetés especular sobre el seu origen, es va aplicar un mètode de selecció de seqüències mutants en base a la utilització d'enzims de restricció. Les mutacions detectades en fel.lema es corresponen amb les relacionades, en altres sistemes no nuclears (plasmidis, fags i DNA bacterià), amb l'estrès oxidatiu. En conseqüència, l'estrès oxidatiu al qual estan sotmeses les cèl.lules del fel.lema podria ser el causant de l'elevada taxa de mutació detectada. D'acord amb això, el tipus majoritari de productes d'oxidació de les bases del DNA que s'acumulen en brots de plàntules de surera en resposta al peròxid d'hidrògen produeixen el mateix tipus de mutacions detectades en l'mRNA del fel.lema de la surera. La major sensibilitat d'aquest nou mètode ha permès, a més, detectar mutacions en molècules d'mRNA de rel, un teixit en el qual no s'havia trobat cap mutació utilitzant el mètode de clonatge i seqüenciació directa. Tot i això, el tipus de mutacions predominants no estan relacionades amb l'estrès oxidatiu sinó amb erros en la reparació dels àcids nucleics.

New insights into boar sperm function and survival from integrated field and laboratory studies

En aquesta tesi s'han dut a terme dos tipus d'estudis diferents. L'objectiu del primer era la preservació del semen de porcí a 15ºC i el del segon eren els co-cultius homòlegs de cèl·lules epitelials de l'oviducte i espermatozoides de porcí. Pel que fa al primer estudi, s'ha observat que l'addició de la prostaglandina F2α i àcid hialurònic a les dosis seminals no malmena la qualitat espermàtica i que la tolerància dels espermatozoides als canvis d'osmolalitat del medi es pot correlacionar proves de fertilitat i prolificitat.. Respecte el segon, s'ha determinat que les cèl·lules oviductals afecten els paràmetres espermàtics i que la presència d'espermatozoides sobreexpressa els gens que codifiquen per les proteïnes de xoc tèrmic. Així, se suggereix que aquestes proteïnes tenen algun paper en els processos reproductius que tenen lloc a l'oviducte, malgrat que s'hagi observat, mitjançant la tècnica de la interferència de l'RNA, que la HSP90AA1 no està implicada en el perllongament de la viabilitat espermàtica.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.