Non-coding RNAs (ncRNAs) are a subset of RNAs that do not code for protein. They are divided into a number of different groups based on their function and targets. Small nucleolar RNAs (snoRNAs) are ncRNAs that have long been known to function as guides for ribosomal RNA (rRNA) modifying enzymes. They are classified into two major groups: box C/D snoRNAs and box H/ACA snoRNAs. Most box C/D snoRNAs direct the 2'-O-methylation of rRNA substrates, but some lack known targets and are therefore termed 'orphan snoRNAs'. Studies have implicated orphan snoRNAs in pre-mRNA processing and stability, but the functional consequence of snoRNA binding to mRNAs has not been fully determined. Saccharomyces cerevisiae had two orphan snoRNAs, snR4 and snR45, with no known function in ribosome synthesis. This project aimed to determine the targets of these snoRNAs, and investigate the effects of snoRNA binding to non-canonical target RNAs, as well as the underlying mechanism. Synthetic gene array screens with deletions of the SNR4 and SNR45 genes identified multiple positive and negative genetic interactions. In particular, deletion of either snoRNA gene was synthetic-lethal with mutation of the snoRNA-associated methyltransferase, Nop1 (Fibrillarin in humans), demonstrating that both have important functions. CLASH analyses of RNA-RNA interactions showed that these snoRNAs bind multiple mRNAs, while RNA sequencing and RT-qPCR revealed that snoRNA deletion altered mRNA abundance. Both orphan snoRNAs were well conserved between fungi, with a region of high conservation indicating a potential binding site. Associations were identified between snR4 and snR45 and multiple sequences within rRNA, including two recently identified sites of 18S rRNA acetylation. Work elsewhere showed that snR4 and snR45 function as guides for the acetyltransferase Kre33 using the region of high conservation, removing their 'orphan' status. Orphan snoRNAs have been implicated in human diseases, such as Prader Willi Syndrome and cancers. The work discussed in this thesis helps to elucidate the RNA interactions of yeast orphan snoRNAs. It has provided a greater understanding of the mechanisms involved, and may inform future work in combatting human disease.

La biogénèse des mitochondries est un processus qui implique l’importation de plus de 98% des protéines constitutives de ces organites. Les signaux protéiques impliqués dans l’importation de ces protéines dans les mitochondries sont relativement bien caractérisés. Il y a une dizaine d’année, il a été montré chez la levure et les mammifères que d’autres signaux, présents au niveau des ARNm étaient également impliqués dans l’adressage et l’importation des protéines dans les mitochondries. Ce processus d’adressage d’ARNm à la surface des mitochondries s’est montré fondamental pour la biogénèse et la fonction des mitochondries chez la levure. Au cours de cette thèse, nous avons démontré que des ARNm étaient adressés à la surface des mitochondries chez trois espèces végétales. Par la combinaison d’approches in vitro et in vivo, nous avons également identifié des éléments cis permettant l’adressage d’ARNm à la surface des mitochondries à partir d’un messager candidat : AtVDAC3. Ces éléments cis sont localisés dans une séquence de 142 nt présente dans la région 3’UTR du messager AtVDAC3. Le rôle de ce processus chez les plantes est actuellement en cours d’étude. / Mitochondria biogenesis requires the import of more than 98 % of their constitutive proteins. Proteic signals involved in mitochondrial protein import are well known today. Ten years ago, it was shown in yeast and mammals that targeting signals are also present at the level of mRNAs. Cytosolic mRNA targeting to mitochondria is an extended process concerning half of the mRNAs encoded mitochondrial proteins in yeast. Furthermore, this process is fundamental for yeast mitochondria biogenesis and functions. During this PhD, we showed that some mRNAs are also targeting to mitochondria in three different plant species. These results highlighted the conservation of this process during evolution. By using in vivo and in vitro experimental strategies, we also identified a mitochondrial cis-targeting element in one candidate mRNA: AtVDAC3. This cis-element is 142 nt long and is located in the 3’UTR of the AtVDAC3 mRNA. We are now investigating the roles of this process in mitochondria biogenesis and functions in plants.

<p>This review article presents the splicing process during messenger RNA maturation and how it is regulated by different <em>Cis</em>-regulatory RNA-sequence elements and splicing factors. A more detailed description of the process alternative splicing and its importance to the function of genes from the model organism <em>Arabidopsis thaliana</em> is also given. A single eukaryotic gene can by the process alternative splicing (AS) give rise to a number of functionally mature mRNA-molecules, which in turn encodes for structurally and/or functionally different proteins. During the course of evolution, the process alternative splicing has thus shown to be effective in increasing transcriptome and proteome diversity of most eukaryotic organisms. This suggests therefore that the dominant theory in molecular biology, a gene encodes for a protein, needs to be corrected. A future challenge is to determine the function of the proteins obtained from a given gene by alternative splicing.</p>

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E₂ has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE₂ to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE₂ of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE₂ accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE₂ is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE₂ may reduce the hepatic gluconeogenic capacity via a Gᵢ-linked signal chain.

Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.

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.

Brominated flame retardants (BFR) such as hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO), bis(2-ethylhexyl)tetrabromophthalate (BEHTBP), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE) are contaminants of environmental concern. These BFRs are replacement alternatives for some of the major production BFRs, which have been restricted from the marketplace due to their adverse health effects. Their presence in environmental matrices, including wild birds, suggests they should be tested for possible toxic effects. BFR alternatives have been detected in the eggs of colonial fish-eating birds, suggesting maternal transfer during ovogenesis and the potential for these chemicals to bioaccumulate through the food chain. However, information regarding the toxicity of HCDBCO, BEHTBP, BTBPE and DBDPE exposure in birds is lacking. This thesis consisted of a combined in vitro/in ovo approach to determine: 1) the concentration-dependent effects of these four BFR alternatives in chicken embryonic hepatocytes (CEH), and 2) the dose-dependent effects of HCDBCO and BTBPE in chicken embryos following injection into the air cell of eggs prior to incubation. Changes in the mRNA expression levels of genes previously found to be responsive to other BFRs were assessed in CEH and liver tissue, in addition to examining overt toxicity (i.e. cytotoxicity, pipping success). None of the BFRs tested were cytotoxic up to 60 µM HCDBCO, 60 µM BEHTBP, 1.4 µM BTBPE or 0.2 µM DBDPE in CEH. Injection doses up to 50 µg/g egg HCDBCO and 10 µg/g egg BTBPE had no effect on embryonic pipping success. The accumulation of HCDBCO and BTBPE was variable in liver and did not follow a linear uptake pattern with respect to injection dose, due in part to difficulties with the solubility of these chemicals in the dimethyl sulfoxide (DMSO) vehicle. In, CEH, HCDBCO caused a decrease in CYP1A4/5 mRNA at all concentrations tested, while CYP2H1 and CYP3A37 were induced only at 10 µM. In contrast, only TTR mRNA was down-regulated in hepatic tissue at all injection concentrations of HCDBCO. The highest concentration of BTBPE induced CYP1A4/5 mRNA to 115- and 18-fold in CEH, and 6.5- and 1.8-fold in liver tissue. In vitro and in ovo exposure to BTBPE caused a concentration-dependent decrease in DIO3 mRNA, while CYP3A37 was down-regulated 2-fold at 10 µg/g in liver tissue. In CEH, DBDPE induced CYP1A4/5 mRNA to a maximum of 29- and 59-fold at 0.2 µM, and increases in DIO1 mRNA and decreases in CYP3A37 mRNA were also observed. None of the gene targets were responsive to BEHTBP exposure in CEH. This is the first study to report on the toxicological and molecular effects of HCDBCO, BEHTBP, BTBPE and DBDPE in an avian species. Using this combined in vitro/in ovo approach has permitted the characterization of these four BFR alternatives by defining possible mechanisms of biological action in a model avian species, the chicken.

Histone deacetylase inhibitors (HDIs) are a group of chemo-preventive and chemo-therapeutic agents that have generated significant attention in clinical trials, given their ability to selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Presently, these agents are proposed to function by altering gene expression levels, primarily by promoting histone hyperacetylation and gene transcription. However, in this thesis, HDIs are reported to control the expression of genes from the c-Src kinase family and p21WAF1 by means other than transcriptional activation. <p>Overexpression and activation of c-Src, a 60kDa non-receptor tyrosine kinase, has been implicated in the development, growth, progression, and metastasis of several human cancers, especially those of the colon. Butyrate and the more specific histone deacetylase inhibitor trichostatin A (TSA) were both found to effectively inhibit the expression of c-Src mRNA and protein in a number of tumor cell lines, including those of the colon, liver and breast. Expression of the SRC oncogene is alternatively regulated by the SRC1A and SRC1 promoters. HDIs were shown to repress c-Src expression by inhibiting transcription of both of these promoters, independent of any new protein synthesis. Furthermore, butyrate and TSA similarly regulated the expression of the c-Src family kinase (SFK) members Yes, Fyn, Lyn and Lck in human colon cancer cell lines. In addition, TATA binding protein (TBP) associated factor 1 (TAF1) was shown to be necessary for basal transcription of the SRC1A, YES and LYN promoters, but was not required for HDI mediated repression. <p>Induction of the potent cyclin dependent kinase inhibitor p21WAF1 has been identified to be a key feature of HDI mediated cell cycle arrest. The level of p21WAF1 expression has been extensively reported to be directly upregulated by HDIs in a p53 independent manner that requires Sp family binding sites in the p21WAF1 proximal promoter to induce transcription. However, HDIs were shown to be capable of inducing p21WAF1 gene expression, dependent on new protein synthesis, by increasing mRNA stability. To date, p21WAF1 mRNA stability has been extensively studied and a number of cis-acting elements in the 3 untranslated region (UTR) of the p21WAF1 mRNA have been implicated in the regulation of mRNA stability, such as AU rich elements (AREs) and a 42 nucleotide HuD/Elav binding element. Similarly, in this work, two novel cis-acting elements were identified in the 3 UTR of p21WAF1 and were shown to facilitate basal and HDI induced post-transcriptional regulation of p21WAF1 mRNA stability in HepG2 cells. Collectively, these studies highlight the intricacy of HDI mediated effects and challenge the preconceptions regarding the molecular mechanism of these anti-tumor agents.

Early embryonic development in vertebrates is directed in part by maternal mRNAs expressed in oocytes and stored in cytoplasmic messenger ribonucleoprotein particles (mRNPs). Abundant evidence demonstrates the importance of mRNPs in embryonic development and in post-embryonic cellular function; however their characterization has been hampered by lack of suitable methodologies. The Xenopus oocyte has been the primary model system for studies of mRNPs. YY1 is a well-studied transcriptional regulatory factor that is sequestered in the oocyte cytoplasm and present entirely in cytoplasmic oocyte mRNPs. The objective of this thesis was to examine the biochemistry of YY1 association with maternal mRNA molecules in order to shed light on the role of YY1 in development and the poorly understood biology of oocyte mRNPs. The initial working hypotheses were that association of YY1 with mRNPs is dependent on sequence-specific RNA-binding activity and, therefore, that YY1 associates with a definite subset of maternal mRNA. A number of unique methods were developed in this study to address these hypotheses. RNA immunoprecipitation-DNA microarray (RIP-CHIP) analysis establishes that YY1 associates with a subset of mRNAs in the oocyte pool. A novel sequence-specific RNA-binding activity of the YY1 protein is demonstrated, and the RNA-binding activity of YY1 is shown to be required for its association with oocyte mRNPs in vivo. The functional roles of YY1 mRNA substrates are discussed in the context of embryological development and the biological function of YY1 in oocyte mRNPs. Extension of the experimental approaches developed in this thesis to the entire set of mRNP proteins would significantly advance our understanding of mRNP composition and heterogeneity, as well as the biological function of maternal mRNAs and mRNPs in development.

The rapid evolutionary rate of HIV-1 has lead to the emergence of multi-drug resistant variants, emphasizing the need for novel inhibitory methods. One such method could be based upon inhibiting viral gene expression through disruption of HIV-1 RNA processing. A means of accomplishing this goal is through use of modified U1snRNA variants that target highly conserved regions of HIV-1 at its terminal exon and prevent 3’ end formation. The modification consists of a 10-nucleotide substitution at the 5’ end complementary to the conserved HIV-1 regions. When modified U1snRNA targeted to HIV-1 were cotransfected with replication deficient HIV-1 proviruses, western blot indicated a specific and significant reduction in the level of viral protein production (p24 and gp120), while U1 constructs that lacked complementary sequences had no effect on HIV-1 protein expression. To further investigate targeted U1snRNA inhibitory effects on HIV-1, efforts are currently underway to determine if this approach has the ability to suppress protein expression in a gene therapeutic model. To date, suppression of viral protein production has reached 50% when tested with a moderately inhibitory U1snRNA. If shown to be effective, such an inhibition would be increased with the use of combinatory modified U1snRNA constructs producing a synergistic effect.