In fish, delayed spawning in nature, delayed egg collection in capture and delayed fertilization after egg stripping lead to excessive oocyte ageing and finally over-ripening. Until now there has been only poor understanding about the processes and underlying mechanism of oocyte ageing in fish as well as other vertebrates. Some studies on other vertebrates have suggested the oxidative stress as initiating factor for the progress of oocyte ageing. The present work investigated possible changes in the mRNA levels of transcripts involved in oxidative damage, mitochondrial function and stress response, with roles in fertilization, embryo development, transcriptional regulation and cell cycling as well as the ones related to the apoptosis during the egg over-ripening. This study investigated the oxidation status of oocytes during post-ovulatory ageing by measuring TBARs as the marker of lipid oxidation and carbonyls which shows the extension of protein oxidation. In addition the role of oxidative stress in the progress of oocyte ageing was assessed by evaluation the activity of antioxidant enzymes, CAT, SOD, GPX and GR. Possible changes in lipid class and fatty acid composition during fish oocyte aging were also examined. Egg viability parameters and larval ploidy levels were examined. Complete loss of egg viability for African catfish (Clarias gariepinus) occurred at 16 and 24 Hours Post Stripping (HPS) when eggs were stored at 25°C and 4°C respectively. Under both storage temperatures, the embryo mortality and larval malformation rates increased significantly over time and were the highest in the most aged oocytes. We determined the time in which the eggs retain their fertilizing ability 10 hours for tench (Tinca tinca) at 20 °C, 18 hours for goldfish (Carassius auratus) at 20 °C and 14 hours Post Ovulation (HPO) and more than 10 Hours Post Stripping (HPS) for common carp (Cyprinus carpio) at 20 °C. Our results demonstrated no significant changes in the mRNA levels of oxidative stress related genes and genes involved in cell cycling during the progress of oocyte ageing in any of species; African catfish, goldfish and common carp. Additionally, with elapsing time following ovulation the amount of TBARs and carbonyls, did not change in any of the oocytes from our experimented species; African catfish, tench, goldfish and common carp. However an increase in the mRNA abundance of apoptotic related genes were observed. Antioxidant enzyme analysis indicated no significant changes in the activity of CAT and SOD during post-ovulatory ageing of common carp, goldfish and tench oocytes. However, a significant decrease in the activity of GPX was observed during post-ovulatory ageing of tench oocytes which could play a major role in the overall drop of egg quality. This observation in GPX activity was not observed in common carp and goldfish oocytes. Besides the direct effects of oxidative stress on oocytes, also changes of fatty acid and lipid composition caused by oxidation could affect the functionality. We observed that post-ovulatory ageing of the oocytes does not change the fatty acids and lipid class composition of the oocytes. Therefore, according to our obtained results, oxidative stress is not the main initiator or promotor of the oocyte ageing process. However, complementary tests and analysis are required to clearly clarify its involvement. Increased mRNA levels of apoptotic related transcripts during oocyte ageing in this study demonstrates that apoptotic pathway might be involved in the molecular changes during the progress of oocyte ageing. Investigation of epigenetic changes associated with fish oocyte ageing seems to be interesting for future research works.

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Protein synthesis is frequently dysregulated in cancer cells; such conditions are known to favor aberrant cell growth and proliferation which lead to cancer. LARP1 is a novel target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, a circuitry often hyperactivated in cancer which regulates cell growth and proliferation primarily through the regulation of protein synthesis. I aimed to determine if LARP1 plays a role in cancer progression by comparing its expression in normal versus cancer tissues. My results demonstrate that LARP1 expression is altered (lost or overexpressed) in various cancers and correlates with cancer patients survival. My systematic bioinformatics assessment, the results of my functional assays assessing the effect of LARP1 knockdown on cancer cells, together with my antibody validation do not only provide new insights for its role in cancer progression and mRNA translation, but also emphasizes the potential of LARP1 as a cancer therapeutic target.

Intestinal diseases affect millions of people worldwide. Recently, a number of proteins have been shown to be upregulated in the intestinal cells of patients that contribute to disease progression. Therefore, these diseases could be amenable to RNA interference technology (RNAi). Utilizing RNAi to deliver short interfering ribonucleic acid (siRNA) to intestinal cells shows promise for the treatment of diseases by specifically suppressing the expression of disease relevant proteins. A class of lipid nanoparticles termed lipidoid nanoparticles (LNPs) have been shown previously to potently deliver siRNA to several cell types in vitro and in vivo. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of oral siRNA delivery to intestinal cells for the treatment of intestinal diseases. Initial in vitro studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal cells without inducing significant cytotoxicity or altering intestinal barrier function. LNP stability studies revealed that LNPs in an aqueous buffer remained stable for long periods of time when stored in the refrigerator (2 °C) compared to the freezer (-20 °C) or at room temperature. In addition, LNPs remained stable upon lyophilization with the addition of trehalose or sucrose to the LNP solution before freeze-drying. To determine potential for oral LNP delivery, we studied LNP stability under gastrointestinal (GI) tract conditions. LNPs remained potent and stable following exposure to solutions of varied pH, including pH values as low as 1.2. However, efficacy decreased following exposure to increasing concentrations of pepsin and bile salts. Mouse oral biodistribution studies indicated that siRNA-loaded lipid nanoparticles were retained in the GI tract for at least 8 hours. Confocal microscopy confirmed that nanoparticles entered the epithelial cells of the mouse small intestine and colon. Oral LNP therapeutic efficacy was measured in an inflammatory bowel disease (IBD) mouse model by targeting the upregulated genes myosin light chain kinase (MLCK) and Interleukin 18 receptor (IL18R) and were found to prevent some IBD disease progression. Lastly, a formulation for the co-delivery of siRNA and messenger RNA (mRNA) was developed and it was discovered that a negatively charged polymer can be used to improve LNP efficacy. Together, these studies have advanced our knowledge of lipid nanoparticle stability, and potential as an orally delivered intestinal therapeutic.

DNA and interfering RNA (RNAi) – short interfering RNA (siRNA) and micro RNA (miRNA) – are promising new cancer therapies, especially for drug resistant lines. However, they require a delivery system in vivo to prevent degradation and off target effects. Silica based nanoparticles, both solid and mesoporous, are a promising option due to their biocompatibility, ease of preparation and morphology control, reproducibility, and facile addition of functional groups including targeting ligands. After a brief introduction to cancer treatment and review of the current nanoparticle treatments undergoing clinical trials, this thesis details the many methods explored over the past ten years to fine-tune particle preparation, pore size, functionalization, and delivery strategies. The majority of both solid and mesoporous silica nanoparticles are synthesized using the sol-gel method and then various functionalization techniques are employed to load and protect the oligonucleotides. Externally loaded systems generally use a combination of polyethylenimine (PEI) and polyethylene glycol (PEG). Mesoporous silica nanoparticles internally load the DNA or RNAi, resulting in the added variable of pore size. Several groups have investigated how pore size alters loading and release kinetics to perfect this variable. Many groups have also tested ligands targeting for over expressed proteins on the intended cancer, triggered release techniques, cell-penetrating peptides in order to create a viable in vivo delivery system. By compiling the techniques employed by researchers over the past ten years, this thesis will elucidate which approaches are most promising for future research. Furthermore, overall strategies within the field are suggested to more easily compare studies and evaluate methods.

Le complexe EJC (Exon Junction Complex) joue un rôle central dans le couplage des processus post-transcriptionnels chez les métazoaires. Ce complexe multi-protéique est assemblé sur les ARN messagers (ARNm) par la machinerie d’épissage. Organisé autour d’un complexe cœur servant de plate-forme à de nombreux facteurs, les EJCs accompagnent les ARNm dans le cytoplasme et participent à leur transport, leur traduction et leur stabilité. L’importance physiologique de l’EJC est supportée par les nombreux défauts développementaux et les maladies génétiques associées aux composants de l’EJC. Les analyses transcriptomiques révélant un assemblage hétérogène des EJCs renforcent l’hypothèse que les EJCs participent à la régulation de l’expression des gènes. Cependant, malgré une connaissance précise de la structure de ce complexe, les liens fonctionnels entre l’assemblage de l’EJC et la régulation de transcrits spécifiques dans des conditions physiologiques doivent être établis puis caractérisés.Durant cette thèse, j’ai étudié l’expression d’eIF4A3, Y14 et MLN51, trois protéines du cœur de l’EJC, dans des cultures primaires de cellules souches neurales murines (CSN). Les CSN peuvent être différenciées en cellules épendymaires multi-ciliées qui tapissent les ventricules cérébraux et ont un rôle important dans le développement du cerveau. J’ai observé par immunofluorescence dans des CSN quiescentes que les 3 protéines sont concentrées autour du centrosome à la base du cil primaire. Ces localisations reflètent la présence d’EJC assemblés comme le prouve l’étude d’un mutant d’Y14 incapable de former l’EJC. / The Exon Junction Complex (EJC) plays a central role in coupling post-transcriptional processes in metazoans. This multi-protein complex is assembled onto messengers RNAs (mRNAs) by the splicing machinery. Organized around a core complex serving as a platform for numerous factors, EJCs accompany mRNAs to the cytoplasm and is involved in mRNA transport, translation and stability. The physiological importance of the EJC is supported by observations associating defects in EJC component expression to developmental defects and human genetic disorders. Transcriptomic studies revealing the non-ubiquitous deposition of EJCs strengthened the hypothesis that EJCs could participate to gene expression regulation. However, despite a precise picture of the structure of the EJC, functional links between EJC assembly and regulation of specific transcripts under physiological conditions is yet to be established.During this thesis, I studied the expression of eIF4A3, Y14 and MLN51 three core proteins of the EJC in primary cultures of mouse neural stem cells (NSCs). NSCs can be differentiated into multiciliated ependymal cells that line all brain ventricles and have important physiological functions in brain development. We observed by immunofluorescence that in quiescent NSCs, all three proteins are concentrated in the vicinity of the centrosome at the base of the primary cilia. This localization reflects the presence of fully assembled EJCs as proved by the study of Y14 mutant that prevent EJC core mounting.

Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized. RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives. RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

There is a growing interest in assessing the neurotoxic potential and endocrine disrupting properties of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, pre- and post- natal development and behaviour. PFC exposure altered hormone levels (e.g. thyroid hormone, estrogen, and testosterone) and the expression of hormone-responsive genes in mammalian and aquatic species. Hormone-mediated events are critical in central nervous system development and function, especially those controlled by thyroid hormones (THs). The studies presented in this thesis are the first to assess the effects of PFCs on primary cultures of neuronal cells in two avian species; the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following TH-responsive genes were examined using real-time RT-PCR: type II iodothyronine 5’-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif binding factor (Oct-1), and myelin basic protein (MBP). Several PFCs were shown to alter mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. It was determined that short-chained PFCs (<8 carbons) altered the expression of TH-responsive genes to a greater extent than long-chained PFCs (≥8 carbons). Although several significant changes in mRNA expression were observed in TH-responsive genes following PFC exposure in chicken embryonic neuronal (CEN) cells (Chapter 2), there were fewer changes in herring gull embryonic neuronal (HGEN) cells (Chapter 3). The mRNA levels of D2, D3, TTR, and RC3 were altered following treatment with several short-chained PFCs in CEN cells. Oct-1 and RC3 expression were induced following treatment with several short-chained PFCs in HGEN cells. These studies are the first to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and provide insight into the possible mechanisms of action of PFCs in the avian brain.

Nuclear export of messenger ribonucleic acid (mRNA) is a complex and essential process for a correct gene expression in all eukaryotic cells. The export of mRNA through the nuclear pore complex depends mostly on the crosstalk and coordination of several proteins forming what is known as mRNPs (messenger ribonucleoproteins) that play dynamic, interconnecting roles in the different mRNA biogenesis steps such as pre-mRNA processing, stability, and export. One key protein in this process is Mex67, conserved from yeast to humans, is the major messenger RNA exporter also involved in ribosomal RNA export. Mex67 interacts with Mtr2 to form an evolutionary conserved heterodimer essential for proper mRNA export and subsequently the survival of the cell. Mex67 have been studied for many years, however due to the complexity and interconnectivity of the different processes in mRNA biogenesis, there is yet to uncover many details on the dynamics of the process and the crosstalk between Mex67 and its many partners. In this study, using a combination of biochemical, biophysical, and structural analysis, we characterize the interaction between Mex67 and a novel partner protein called Mip6 (Mex67 interacting protein 6). We were able to reconstitute a stable complex in vitro, and extensively study the mechanism in which the two proteins interact. We also solved the crystal structure of the C-terminal region of Mex67 that interacts with Mip6 and identified the UBA domain of Mex67, known to bind FG nucleoporins and Hpr1 protein as also the site where Mip6 binds. However, little was known about the structure or function of Mip6 and its paralogue Pes4. Here we proved that Mip6 is an RNA binding protein with four RNA recognition motifs that binds RNA in vitro with high affinity. Additionally, its fourth RNA recognition motif was also the site of binding of Mex67. Furthermore, we showed that the Mex67 complex formation with Mip6 RRM4 compromises its ability to bind RNA or vice versa. We also designed a point mutation on Mip6 RRM4 that disrupts its interaction with Mex67 but not with RNA. Subsequent in vivo yeast assays led us to hypothesize a role of Mip6 as an adaptor protein for Mex67 in nuclear export especially upon stress. Additional function of Mip6 was the localization of its bound mRNA to cytoplasmic stress granules in cellular stress conditions. Moreover, the crystal structures of Mip6 RRM3, Pes4 RRM3, Pes4 RRM4, and Pes4 RRM3/4 were also solved. All RRMs adopted a canonical RRM fold with conserved RNP1 and RNP2 sequences normally involved in RNA binding, except Mip6 RRM3 that was missing the aromatic ring in RNP2. In the structure of RNA-free Pes4 RRM3/4, the tandem RRM domains were connected with a flexible disordered linker and no inter-domain contact between them. Finally, although Pes4 RRM4 was binding RNA in vitro, it did not have the ability to interact with Mex67 thus suggesting a separate evolutionary function for Mip6 and Pes4. / La exportación nuclear de ácido ribonucleico mensajero (ARNm) es un proceso complejo y esencial para una expresión correcta de los genes en todas las células eucariotas. La exportación de ARNm a través del complejo del poro nuclear depende principalmente de la interacción y coordinación de varias proteínas, que forman lo que se conoce como mRNPs (ribonucleoproteínas mensajeras), que tienen un papel dinámico e interconectado en las diferentes etapas de la biogénesis de ARNm, tales como el procesamiento del pre-ARNm, estabilidad, y exportación. Una proteína clave en este proceso es Mex67, conservada de levaduras a humanos, que es la principal exportadora de ARN mensajero y también está implicada en la exportación de ARN ribosomal. Mex67 interacciona con Mtr2 para formar un heterodímero conservado evolutivamente esencial para una exportación adecuada de ARNm y la consiguiente supervivencia de la célula. Se ha estudiado Mex67 durante muchos años, sin embargo, debido a la complejidad e interconectividad de los diferentes procesos de biogénesis de ARNm, todavía quedan por descubrir muchos detalles de la dinámica del proceso y las interacciones entre Mex67 y sus muchas proteínas asociadas. En este estudio, combinando un análisis bioquímico, biofísico y estructural, hemos caracterizado la interacción entre Mex67 y una nueva proteína asociada denominada Mip6 (proteína 6 que interacciona con Mex67). Hemos podido reconstituir un complejo estable in vitro y estudiar extensivamente el mecanismo por el cual interaccionan estas dos proteínas. También hemos resuelto la estructura cristalográfica de la región C-terminal de Mex67 que interacciona con Mip6 e identificado el dominio UBA de Mex67, conocido por unirse a nucleoporinas FG y a la proteína Hpr1, así como el sitio por el que se une Mip6. No obstante, se sabía muy poco sobre la estructura o la función de Mip6 y su parálogo Pes4. Hemos probado que Mip6 es una proteína de unión a ARN con cuatro motivos de reconocimiento de ARN que se unen a ARN in vitro con una afinidad alta. Además, su cuarto motivo de reconocimiento de ARN es también el sitio de unión a Mex67. Posteriormente, demostramos que la formación del complejo de Mex67 con el dominio RRM4 de Mip6 compromete su capacidad para unir ARN o viceversa. También diseñamos una mutación puntual en el RRM4 de Mip6 que rompe la interacción con Mex67 pero no con el ARN. Los ensayos posteriores in vivo en levaduras nos permitieron establecer una hipótesis sobre el papel de Mip6 como proteína adaptadora para Mex67 en la exportación nuclear, especialmente en condiciones de estrés. Una función adicional de Mip6 era la localización del ARNm que se unía a ella en gránulos de estrés en condiciones de estrés celular. Además, hemos resuelto las estructuras cristalográficas del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 y los RRM3 y 4 de Pes4. Todos los RRMs adoptaron una conformación canónica RRM con secuencias RNP1 y RNP2 conservadas generalmente implicadas en la unión a ARN, excepto el RRM3 de Mip6 que carecía del anillo aromático en RNP2. En la estructura sin ARN de los RRM3 y 4 de Pes4, los dominios RRM tándem estaban conectados por una región flexible desordenada y no había un contacto inter-dominio entre ellos. Finalmente, aunque el RRM4 de Pes4 se unía a ARN in vitro, no presentaba la capacidad de interaccionar con Mex67 lo cual sugiere una divergencia evolutiva de la función de Mip6 y Pes4. / L¿exportació nuclear d¿àcid ribonucleic missatger (mRNA) es un procés complex i essencial per a una correcta expresió gènica en totes cèl¿lules eucariotes. L¿exportació del mRNA a través del complex del porus nuclear depén principalment de la interacció i coordinació de diverses proteïnes, que formen el que es coneix com mRNPs (ribonucleoproteïnes missatgeres), que tenen un paper dinàmic i interconnectat en les diferents etapes de la biogènesi d¿ARNm, com el processament del pre-ARNm, estabilitat, localització i exportació. Una proteïna clau en aquest procés és MEX67, conservada de llevats fins a humans, que és la principal exportadora de ARN missatger i també està implicada en l¿exportació de ARN ribosomal. Mex67 interacciona amb Mtr2 per a formar un heterodímer conservat evolutivament essencial per a una exportació adequada d¿ARNm i la consegüent supervivència de la cèl¿lula. S¿ha estudiat Mex67 durant molts anys, però degut a la complexitat i interconectivitat dels diferents processos de biogènesi d¿ARNm, encara queden per descobrir molts detalls de la dinàmica del procés i les interaccions entre Mex67 i les seues moltes proteïnes associades. En aquest estudi, combinant l¿anàlisi bioquímic, biofísic i estructural, hem caracteritzat la interacció entre Mex67 i una nova proteïna associada anomenada Mip6 (proteïna 6 que interacciona amb Mex67). Hem pogut reconstituir un complex estable in vitro i estudiar extensivament el mecanisme pel qual interaccionen estes dos proteïnes. També hem resolt l¿estructura cristal¿logràfica de la regió C-terminal de Mex67 que interacciona amb Mip6 i identificat el domini UBA de Mex67, conegut per unir-se a nucleoporines FG i a la proteïna Hpr1, així com ser el lloc pel que s¿uneix Mip6. No obstant, se sabia molt poc sobre l¿estructura o la funció de Mip6 i el seu paràleg Pes4. Hem comprobat que Mip6 es una proteïna d¿unió a ARN amb quatre motius de reconeixement d¿ARN que s¿uneixen a ARN in vitro amb una afinitat alta. A més, el seu quart motiu de reconeixement d¿ARN és també el lloc d¿unió a Mex67. Posteriorment, demostràrem que la formació del complex de Mex67 amb el domini RRM4 de Mip6 compromet la seua capacitat per a unir ARN o viceversa. També vam dissenyar una mutació puntual en el RRM4 de Mip6 que trenca la interacció amb Mex67 però no amb l¿ARN. Els assajos posteriors in vivo en llevats ens van permetre establir una hipòtesi sobre el paper de Mip6 com a proteïna adaptadora per a Mex67 en l¿exportació nuclear, especialment en condicions d¿estrès. Una funció adicional de Mip6 era la localització de l¿ARNm que s¿unia a ella en grànuls d¿estrès en condicions d¿estrès cel¿lular. A més, hem resolt les estructures cristal¿logràfiques del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 i els RRM3 i 4 de Pes4. Tots els RRMs adoptaren una conformació canònica RRM amb seqüències RNP1 i RNP2 conservades generalment implicades en la unió a ARN, excepte el RRM3 de Mip6 que mancava del anell aromàtic en RNP2. En la estructura sense ARN dels RRM3 i 4 de Pes4, els dominis RRM tàndem estàven conectats per una regió flexible desordenada i no hi havia un contacte interdomini entre ells. Finalment, encara que el RRM4 de Pes4 es unia a ARN in vitro, no presentava la capacitat d¿interaccionar amb Mex67, la cual cosa sugerix una divergencia evolutiva de la funció de Mip6 y Pes4. / Mohamad, N. (2017). The RNA binding protein Mip6, a novel cellular partner of Mex67 export factor with implications in mRNA export [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90397 / TESIS

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22355号 / 医博第4596号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 岩田 想, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM