No description available.

Las líneas de investigación de la presente tesis se encuentran enmarcadas en dos aspectos de la respuesta de los sistemas biológicos ante la presencia de metales. Los sujetos de investigación son tanto (1) elementos individuales (las proteínas denominadas Metalotioneínas), como (2) redes de respuesta global (Metalómica), y su relación e imbricación con el metabolismo celular (Metabolómica). Las metalotioneínas (MT), aunque ubicuas, son probablemente de origen polifilético, de modo que habrían aparecido numerosas “familias de homología” a lo largo de la evolución. El análisis computacional de los miembros de la familia de metalotioneínas en los genomas de Drosophila (5 formas parálogas), en doce especies (12 conjuntos de ortólogos) condujo a la identificación de 60 secuencias de isogenes/proteínas con secuencias de DNA/proteína altamente conservadas. La conservación en el número de isogenes del sistema MT en Drosophila sugiere que la multiplicidad de estos genes es debida a un evento evolutivo anterior a la especiación de este género. A pesar de exhibir una gran plasticidad, la región cromosómica donde se localizan los componentes del sistema de MT en Drosophila, preserva su localización en el elemento Müller E en las doce especies. La arquitectura de los genes, formados por dos exones y un intrón, también se conserva en el conjunto de ortólogos/parálogos. La reorganización identificada en la región cromosómica donde se localizan las isoformas de MT en Drosophila es debida principalmente a eventos de inversión de DNA que implican orientaciones diferentes de los cinco genes. El análisis de las propiedades de coordinación de metales de la isoforma MtnE la clasifican como una Cu-tioneína de carácter intermedio o no extremo, ya que a pesar de formar complejos homometálicos con el Cu, la capacidad de unión de cobre es menor que la observada para el resto de las isoformas. MtnE muestra la capacidad coordinante menos específica, en comparación con el resto de isoformas, lo que sugiere una función fisiológica relacionada con condiciones en las que se necesite un péptido con amplio espectro de coordinación de metales, por ejemplo condiciones de toxicidad mixta por varios metales, o elevado nivel de toxicidad de Cu en los que apoyaría la acción del resto de isoformas. Además se identificaron dos secuencias (BfMT1 y BfMT2) como potenciales MT en el genoma de Branchiostoma floridae, organismo procordado ubicado en la base evolutiva de los vertebrados. Las secuencias de ORFs, sus regiones reguladoras, y correspondientes productos proteicos, mostraron características que los reafirman como potenciales MT. Los genes BfMT1 y BfMT2 fueron identificados in vivo mediante RT-PCR sobre RNA total de organismos de Branchiostoma lanceolatum. Ambos genes se inducen in vivo en B. lanceolatum en condiciones de exceso de metales (Cd o Cu), mostrando BfMT1 un patrón correspondiente a un gen de expresión basal o constitutiva, mientras que la expresión de BfMT2 se corresponde con la de un gen inducible. El tratamiento de organismos vivos con distintas concentraciones de Cd demostró que en condiciones de exceso de este metal, se produce además una disrupción en la homoestasis del zinc. En cuanto a los elementos de respuesta global, desarrollamos el estudio genómico y transcriptómico de las redes de respuesta de S. cerevisiae como organismo modelo ante dosis excesivas de Zn que alteran sus equilibrios homeostáticos. Resultados previos de nuestro grupo mostraron una interrelación entre las vías metabólicas de respuesta a estrés de Zn y aquellas de distintos metales. Análisis de varios mutantes tolerantes a zinc nos permitió identificar una imbricación relevante entre procesos metabólicos vitales como la respiración, la respuesta a estrés oxidativo, la estabilidad del DNA mitocondrial y la tolerancia a zinc mediante redes de respuesta y señalización global que implican interconexiones de proceso / This thesis is framed in two aspects of the of biological systems responses to metals. The subjects of research are: (1) individual elements (proteins called Metalothioneins), and (2) networks of global response (Metallomics), and their relationship and overlap with cellular metabolism (metabolomics). Computational analysis of the members of the family of MTs in the genomes of Drosophila (5 forms parálogas), twelve species (12 sets of orthologous) led to the identification of 60 isogenes/proteins with highly conserved sequences and architecture of DNA/protein sequences. All the components of the MT system are located in the Müller E element in the twelve species. The MtnE isoform is classified as a not extreme Cu-thionein, despite forming homometallic complexes with the Cu. MtnE shows the less specific coordinating capacity compared with the rest of isoforms, suggesting a physiological function related to the need for a peptide with a broad spectrum of coordination of metals. We also identified two sequences (BfMT1 and BfMT2) as potential MTs in the genome of Branchiostoma floridae. ORFs sequences, their regulatory regions, and corresponding protein products, showed characteristics which confirm them as potential MT. The BfMT1 and BfMT2 genes were identified in vivo using RT-PCR on total RNA from Branchiostoma lanceolatum. Both genes are induced in vivo in B. lanceolatum in excess of metals (Cd or Cu) conditions, showing BfMT1 a pattern corresponding to a basal or constitutive expression gene, while the expression of BfMT2 corresponds with an inducible gene. The treatment of living organisms with different concentrations of Cd showed that under conditions of excess of this metal, there is also a disruption in zinc homoestasis. With respect to the elements of global response, we studied the metabolic responses of S. cerevisiae to high zinc. Previous results of our group showed an interconnection of zinc related metabolic pathways and those related to other metals. Analysis of several mutants tolerant to high zinc allowed us to identify a significant overlap between metabolic life processes such as respiration, oxidative stress response, stability of mitochondrial DNA and tolerance to high zinc through networks of response and global signalling involving interconnected processes.

Although inductively coupled plasma mass spectrometry (ICP-MS) is generally considered a reliable analytical technique, increasing demands on its capabilities require continued research and improvements. ICP-MS is susceptible to both matrix effects and drift, leading to a decline in accuracy and precision. A number of techniques are routinely used to compensate for these issues. Internal standardization is one such solution that requires relatively simple sample preparation and yet offers the possibility of improving both accuracy and precision. In order to be effective, an optimal analyte/internal standard pair must be chosen. Traditionally, analyte/internal standard pairs are chosen based on similarities in mass and/or ionization potential. The present studies sought to develop a program that determined standards based on the minimization of analytical error. 102 masses were monitored over 27 perturbations, i.e., changes to sample matrix and operating parameters. The standard deviations of the analyte/internal standard ratios were then used as a measure of internal standard performance. A thorough statistical analysis was conducted to determine trends between a good analyte/internal standard pair and similarities in chemical property. Similarities in mass offered the strongest relationship to a good internal standard choice, although many exceptions existed. The program was then tested over time and multiple instrument optimizations as well as on a completely different ICP-MS instrument. Results of these tests suggest that the data originally collected for the prediction program is not instrument-specific and thus provided a broader base of useful applications. Due to its unmatched sensitivity and multielement capabilities, ICP-MS is frequently utilized for biological samples. A more recent application, however, seeks to use ICPMS for the purpose of determining specific associations between metals and proteins. Such speciation requires a high resolution and reproducible separation prior to ICPMS analysis. Gel electrophoresis offers good separation and is well matched with the scanning properties of laser ablation sample introduction. The present study utilized native gel electrophoresis coupled with a uniquely modified electroblot system to improve sensitivity and to elucidate additional information. Chemically modified quartz fiber filters were successfully used as the transfer membrane to improve protein and metal capture efficiency. / text

Diabetes mellitus currently affects over 422 million people globally and over 80% of patients with diabetes will develop diabetic retinopathy. Patients with diabetic retinopathy initially develop background retinopathy, which does not cause significant deterioration of visual function; however, background retinopathy may progress and lead to proliferative diabetic retinopathy and diabetic macular oedema, both of which cause severe visual dysfunction if left untreated. Current therapies for diabetic retinopathy include invasive intravitreal injections and laser photocoagulation; however these treatments only attenuate the progression of proliferative diabetic retinopathy and diabetic macular oedema. Aside from prevention by maintaining good blood glucose and blood pressure control, there are currently no treatments to prevent progression to late-stage diabetic retinopathy and new innovations in the field have not significantly progressed. For this reason, we have used untargeted –omics approaches to identify previously unknown pathological pathways in diabetes. In this thesis, I have analysed a range of trace metals in donor retinas and found that total copper was increased in diabetic retinas compared with non-diabetic. This result was replicated in streptozotocin-induced diabetic rat retinas and further evidenced by upregulation of metallothioneins and caeruloplasmin in diabetic rat retinas compared with non-diabetic. Treatment with the copper chelator triethylenetetramine modulated these changes, the downstream effects of which require further study. This is the first description, to our knowledge, of dysregulated copper homeostasis in the diabetic retina. I have also mapped metabolic changes in streptozotocin-induced diabetic rat retinas and found previously undescribed metabolite changes such as diabetes-induced downregulation of scyllo inositol. This coincided with substantial changes to retinal lipids during diabetes and changes to individual lipids were consistent within their respective class. I have also found a pattern whereby regardless of the extent of change to a lipid class in diabetes, lipids containing docosahexaenoic acid (22:6 carbon chain) were consistently downregulated. This is thought to be the first study to describe this range of metabolite changes in the diabetic retina but also the first study to describe this range of metabolite analysis concomitantly within the same tissue sample. The data from this study provides new insights into metallomic and metabolic dysfunction in diabetic retinopathy and shown that these data are reproducible. We suggest that there is plenty of scope for further research to investigate mechanisms behind copper dysregulation, how this affects pathogenesis of diabetic retinopathy along with new insights into dysregulated metabolic pathways.

L’uranium (U) est naturellement présent à l’état de trace dans l’eau (µg.L-1), sa concentration pouvant atteindre localement quelques mg.L-1 du fait des activités anthropiques. Plusieurs études écotoxicologiques sur Danio rerio ont mis en évidence la toxicité, e.g. le stress oxydant, la génotoxicité mais aussi la reprotoxicité (i.e. moins de pontes et d’œufs pondus chez les poissons contaminés) de l’U dont les mécanismes ne sont pas connus.L’objectif de cette étude est de contribuer à la compréhension de la reprotoxicité de l’U par l’élucidation de mécanismes moléculaires perturbés après contamination. Pour cela, des investigations ont été menées sur les ovaires de poissons zèbre Danio rerio, reproduits (R) ou non (NR), après exposition par voie directe en condition de laboratoire à des concentrations représentatives d’environnements contaminés.Ce travail de thèse a été divisé en deux volets. Un premier volet analytique avait pour but la poursuite des développements de méthodes pour l’identification des complexes U-protéines en condition non dénaturante, autour du couplage de techniques de séparation (chromatographie d’exclusion stérique SEC, électrophorèse hors gel OGE) et de détection sensible par spectrométrie de masse élémentaire (ICP MS) et moléculaire (ESI MS). Le second volet a été dédié à l’étude de la reprotoxicité de l’U à l’échelle moléculaire, avec i) l’étude des complexes natifs U-protéines (approche métallomique), et ii) l’analyse différentielle de l’expression des protéines (approche protéomique).Les développements analytiques ont permis de garder le tampon physiologique et non dénaturant d’extraction pour l’étape de séparation OGE, améliorant le taux de recouvrement en U. En écotoxicologie, les principaux résultats montrent que l’ovaire est un organe accumulateur de l’U et que le statut de reproduction a une influence sur le niveau d’accumulation (R<NR). En revanche, cet état a peu d’influence sur sa distribution protéique pour laquelle 4 fractions (dont 1 principale) ont été identifiées, toutes contenant aussi du phosphore. L’identification des cibles potentielles de l’U et des protéines exprimées différentiellement (vtg, GST, GAPDH,…) a montré que les processus biologiques perturbés suite à la contamination sont de deux niveaux : 1/ générique (stress oxydant) et 2/ plus spécifique de la gonade (développement et maturation des ovocytes). En conclusion, ces deux approches complémentaires ont permis de mettre en évidence un effet direct (complexation) et indirect (expression protéique modulée) de l’U, et de proposer l’hypothèse d’un défaut de maturation des ovocytes après contamination. Ce défaut pourrait impacter le développement embryonnaire et in fine expliquer la reprotoxicité observée lors d’études écotoxicologiques précédentes. / Uranium (U) is naturally presents at trace level (µg.L-1) in aquatic environment; its concentration can increase up to a few mg.L-1 due to human activities. Several ecotoxicological studies have shown uranium toxicity in contaminated zebrafishes Danio rerio, e.g. oxidative stress, genotoxicity and reprotoxicity (i.e. lower number of spawn and eggs laid) but mechanisms are not well known.The objective of this study is to contribute to the understanding of uranium reprotoxicity by elucidating the disrupted molecular mechanisms after contamination. Therefore, investigations have been carried out on ovaries from reproduced (R) and non-reproduced (NR) zebrafishes after waterborne exposure in laboratory conditions at environmentally relevant concentrations.This project was divided into two parts. Firstly, analytical investigations were carried out to continue the development of non-denaturing methods for U-protein identification by coupling separative techniques (size exclusion chromatography SEC, off gel electrophoresis OGE) with elemental (ICP MS) and molecular (ESI MS) sensitive mass spectrometry detection. Secondly, studies of U reprotoxicity were investigated by studying i) native U-protein complexes (metallomics approach) and ii) differential analysis of protein expression (proteomics approach)Analytical developments allowed keeping the physiological and non-denaturing extraction buffer for OGE separation step, improving U recovery. In ecotoxicology, the major results showed that ovary is an U accumulating organ and that the reproduction status modifies the accumulation level (R<NR). However, this status is of little influence on its distribution on proteins with 4 fractions (including a major one) determined, all of them coeluting with phosphorus. The identification of U potential targets and of protein expression differences (vtg, GST, GAPDH…) showed that biological processes disrupted after contamination are at two levels: 1/ generic (oxidative stress) and 2/ more specific to gonad (oocyte development and maturation).As a conclusion, these two complementary approaches showed a direct (complexation) and indirect (modification of protein expression) effects of U, and enabled to hypothesize a lack of oocyte maturation after contamination. This defect could impact embryo development and in fine explain the reprotoxicity observed in previous ecotoxicological studies.