Oxygenic photosynthesis is the major producer of both oxygen and organic compounds on earth and takes place in plants, green algae and cyanobacteria. The thylakoid membranes are the site of the photosynthetic light reactions that involve the concerted action of four major protein complexes known as photosystem II (PSII), cytochrome b6f complex, ATP synthase and photosystem I (PSI). The function of PSII is of particular interest as it performs the light–driven water splitting reaction driving the photosynthetic electron transport. My thesis addressed different aspects of PSII assembly and the functions of its low molecular weight PSII subunits PsbX and PsbW. Photosynthesis in green algae and higher plants is controlled by the nucleus. Many proteins of nuclear origin participate in the regulation of the efficient assembly of the photosynthetic protein complexes. In this investigation we have identified one of these nuclear encoded auxiliary proteins of photosystem II, REP27, which participates in the assembly of the D1 reaction center protein and repair of photodamaged PSII in the green algae Chlamydomonas reinhardtii. Interestingly, PSII is specially enriched in Low Molecular Weight (LMW) subunits that have masses less than 10kDa. These proteins account for more than the half of the PSII subunits. Several questions remains poorly understood regarding the LMW: Which is their evolutionary origin? What function do they perform in the protein complex? Where are they located in the protein structure? In this investigation the functions of two of these LMW subunits (PsbX and PsbW) have been studied using antisense inhibition and T-DNA knockout mutant plants in Arabidopsis thaliana. Deficiency of the PsbX protein leads to impaired accumulation and functionality of PSII. Characterization of PsbW knock-out plants show that PsbW participates in stabilization of the macro-organization of PSII and the peripheral antenna (Light Harvesting Complex, LHCII) in the grana stacks of the chloroplast, also known as PSII-LHCII supercomplexes.

Cleft palate is a common human birth defect caused by any process which interferes with palatogenesis. Studies in Hoxa2 mutant (Hoxa2-/-) mice which exhibit a secondary cleft palate were reported to be due to an abnormal positioning of the tongue which prevents normal palatal shelf fusion to occur. To obtain direct evidence for the importance of Hoxa2 in murine palate development, an in vitro whole organ palatal culture model was developed, eliminating any influences from the tongue. A retroviral gene delivery system was employed, containing either Hoxa2 sense or Hoxa2 antisense cDNA, to respectively enhance or knockdown the expression of Hoxa2 mRNA in the developing palate. <p>Our results show that palatal cultures infected with the lowest titer of Hoxa2 sense virus induce a fusion rate of 72.7%, which is similar to palatal cultures treated with the control virus (81.8%), although fusion rates of 41.2% to 50.0% were observed in palates infected with higher titers. With the antisense virus treated group, a more profound inhibition of the fusion rate was observed (27.7% - 46.1%), which is comparable with the frequency of palatal fusion in Hoxa2-/- mice (44.4%). Additionally, the palatal shelves in both sense and antisense virus treated groups appear to be relatively shorter in length, than those measured in the control group. Interestingly, in the antisense virus treated group, the ratio of the length of the fused portion to the length of palatal shelves appears to be relatively large compared to the control group. Verification and quantification of Hoxa2 mRNA in the developing palate between E12.5 and E15.5 was performed by real-time RT-PCR. Hoxa2 gene expression was observed at all stages studied, with expression being the highest at E12.5 and declining from E13.5. The expression level remained constant from E13.5 through E15.5. These findings demonstrate for the first time that Hoxa2 may play a direct role in murine palate development. Results suggest that both factors (the absence of Hoxa2 gene in the palate causing delayed palatal development, as well as the position of the tongue) appear to act in unison to produce cleft palate in Hoxa2 knockout mice.

Male rat copulation is mediated by estrogen-sensitive neurons in the medial preoptic area (MPO) and medial amygdala (MEA); however, the mechanisms through which estradiol (E2) acts are not fully understood. We hypothesized that E2 acts through estrogen receptor α (ERα) in the MPO and MEA to promote male mating behavior. Antisense oligodeoxyneucleotides (AS-ODN) complementary to ERα mRNA were bilaterally infused via minipumps into either brain area to block the synthesis of ERα, which we predicted would reduce mating. Western blot analysis and immunocytochemistry revealed a knockdown of ERα in each brain region; however, compared to saline controls, males receiving AS-ODN to the MPO showed significant reductions in all components of mating, whereas males receiving AS-ODN to the MEA continued to mate normally. These results suggest that E2 acts differently in these brain regions to express sexual behavior and that ERα in the MPO, but not in the MEA, promotes mating.

Cleft palate is a common human birth defect caused by any process which interferes with palatogenesis. Studies in Hoxa2 mutant (Hoxa2-/-) mice which exhibit a secondary cleft palate were reported to be due to an abnormal positioning of the tongue which prevents normal palatal shelf fusion to occur. To obtain direct evidence for the importance of Hoxa2 in murine palate development, an in vitro whole organ palatal culture model was developed, eliminating any influences from the tongue. A retroviral gene delivery system was employed, containing either Hoxa2 sense or Hoxa2 antisense cDNA, to respectively enhance or knockdown the expression of Hoxa2 mRNA in the developing palate. <p>Our results show that palatal cultures infected with the lowest titer of Hoxa2 sense virus induce a fusion rate of 72.7%, which is similar to palatal cultures treated with the control virus (81.8%), although fusion rates of 41.2% to 50.0% were observed in palates infected with higher titers. With the antisense virus treated group, a more profound inhibition of the fusion rate was observed (27.7% - 46.1%), which is comparable with the frequency of palatal fusion in Hoxa2-/- mice (44.4%). Additionally, the palatal shelves in both sense and antisense virus treated groups appear to be relatively shorter in length, than those measured in the control group. Interestingly, in the antisense virus treated group, the ratio of the length of the fused portion to the length of palatal shelves appears to be relatively large compared to the control group. Verification and quantification of Hoxa2 mRNA in the developing palate between E12.5 and E15.5 was performed by real-time RT-PCR. Hoxa2 gene expression was observed at all stages studied, with expression being the highest at E12.5 and declining from E13.5. The expression level remained constant from E13.5 through E15.5. These findings demonstrate for the first time that Hoxa2 may play a direct role in murine palate development. Results suggest that both factors (the absence of Hoxa2 gene in the palate causing delayed palatal development, as well as the position of the tongue) appear to act in unison to produce cleft palate in Hoxa2 knockout mice.

The discovery of RNA interference and the increasing understanding of disease genetics have created a new class of potential therapeutics based on oligonucleotides. This therapeutic class includes antisense molecules, small interfering RNA (siRNA), and microRNA modulators such as antagomirs (antisense directed against microRNA) and microRNA mimics, all of which function by altering gene expression at the translational level. While these molecules have the promise of treating a host of diseases from neurological disorders to cancer, a major hurdle is their inability to enter cells on their own, where they may render therapeutic effect. Nanotechnology is the engineering of materials at the nanometer scale and has gained significant interest for nucleic acid delivery due to its biologically relevant length-scale and amenability to multifunctionality. While a number of nanoparticle vehicles have shown promise for oligonucleotide delivery, there remains a lack of understanding of how nanoparticle coating and size affect these delivery processes. This dissertation seeks to elucidate some of these factors by evaluating oligonucleotide delivery efficiencies of a panel of iron oxide nanoparticles with varying cationic coatings and sizes. A panel of uniformly-sized nanoparticles was prepared with surface coatings comprised of various amine groups representing high and low pKas. A separate panel of nanoparticles with sizes of 40, 80, 150, and 200 nm but with the same cationic coating was also prepared. Results indicated that both nanoparticle surface coating and nanoparticle hydrodynamic size affect transfection efficiency. Specific particle coatings and sizes were identified that gave superior performance. The intracellular fate of iron oxide nanoparticles was also tracked by electron microscopy and suggests that they function via the proton sponge effect. The research presented in this dissertation may aid in the rational design of improved nanoparticle delivery vectors for nucleic acid-based therapy.

During photosynthesis, two multi-protein complexes, photosystems (PS) I and II work in tandem to convert the light-energy absorbed by the light-harvesting antennae into chemical energy, which is subsequently used to assimilate atmospheric carbon dioxide into organic carbon compounds. This is the main nutritional basis for life on Earth. The photosynthetic antenna of higher plants comprises at least ten different pigment-binding proteins (LHC), which play important roles in photosynthesis. Chlorophyll and carotenoid molecules associated with the LHC proteins are organised into an array, which can be modulated, thereby optimising light-harvesting processes and protection against oxidative damage under conditions of excessive light absorption. All ten LHC proteins have been conserved through eons of evolution, suggesting that there are strong evolutionary pressures to retain all ten proteins, and hence that each protein has a unique function. The light-harvesting antenna of higher plant PSI consists of at least four proteins, Lhca1-4, collectively called LHCI. By constructing transgenic Arabidopsis thaliana plants in which each Lhca gene has been individually repressed or knocked-out, a collection of plants with different Lhca protein contents was obtained. The objective was to use these plants to study the structure, function and regulation of the Lhca proteins in vivo. The major findings of this work are as follows. Removing single Lhca proteins influenced the stability of the other Lhca proteins, showing that there is a high degree of inter-dependency between the polypeptides in LHCI, and hence that a full set of Lhca proteins is important for maintaining the structural integrity of LHCI. This has provided insight into the organisation of LHCI by revealing clues about the relative positions of each Lhca protein in the antenna complex. The physiological consequences of removing individual Lhca proteins were dependent on the degree of antenna depletion. Plants with relatively small antenna changes could compensate, to some extent, for the loss of LHCI, while larger depletions had profound effects on whole plant resulting in growth reductions. The fitness of each Lhca plant was assessed by measuring their seed production in the harsh conditions in the field. We found that all Lhca-deficient plants produced fewer seeds under some conditions, with seed-production compared to wild type varying between 10-80% depending on the extent of LHCI reduction. Therefore, we conclude that each Lhca protein is important for plant fitness, and hence for the survival of the species. PSI is characterised by a pool of pigments absorbing light in the red end of the solar visible spectrum, thought to be especially important for plants in dense vegetation systems where the incident light is enriched in wavelengths higher than 690 nm. A majority of these pigments are situated on LHCI and, based on in-vitro studies, were thought to be mainly associated with Lhca4. Using our plants, we have established that red pigments are indeed present on all Lhca proteins and that these pigments become even more red upon association with PSI.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Ethylene (C2H4) is a very simple molecule, a gas, and has numerous effects on the growth, development and storage life of many fruits, vegetables and ornamental crops. In higher plants, ethylene is produced from L-methionine in essentially all tissues and ACC Synthase and ACC Oxidase are the two key enzymes in the biosynthesis of ethylene. The objective of the present study was to transform tobacco (Nicotiana tabacum L. cv. Samsun) plant with partial sequence of torenia acc oxidase gene in antisense and sense orientations via Agrobacterium-mediated gene transfer system, and to analyze its effect on ethylene production in transgenic plants. Six antisense and seven sense T0 putative transgenic lines were obtained and were further analyzed with several assays. Leaf disc assay and chlorophenol red assay under selection (75 mg/L kanamycin) revealed positive results compared to the non-transformed plant. T1 generations were obtained from all putative transgenic lines. PCR analysis and Northern Blot Hybridization results confirmed the transgenic nature of T1 progeny. Furthermore, ethylene amount produced by flowers were measured with gas chromatography, which resulted in an average of 77% reduction in S7 line and 72% reduction in A1 line compared with the control flowers. These results indicated that, transgenic tobacco plants carrying torenia acc oxidase transgene both in antisense and sense orientations showed reduced ethylene production thus a possibility of flower life extension.