Lung cancer arises from epithelial cells that line the air passages of the lungs. It is the second most common malignancy in the United States; trends suggest that over 228,000 new patients will be diagnosed with lung cancer in 2013. Due to the fact that lung cancer is highly aggressive, it has proven difficult to control. The 5-year survival rate has been shown to be only 15.9%, despite the advances made in terms of diagnosis and treatment. Therefore, we are faced with the problem of finding more effective methods that allow for an earlier diagnosis and the improved treatment of lung cancer. This study attempts to address these issues by investigating Tarsh, a novel molecule that is involved in the regulation of cellular senescence. Previous studies have shown that Tarsh is expressed in normal lung cells, but is significantly downregulated in lung tumors. These studies also determined that Tarsh is likely dependent upon the expression of p53, a tumor suppressor gene. The current study investigated these results, in addition to the biological effects of ectopically increasing Tarsh and/or knocking down p53 expression in two lung cancer cell lines: A549 and H1299 cell lines. It was determined that increasing the expression of Tarsh decreased the rate of proliferation in both cell lines. Additionally, it was shown that the knockdown of p53 increased proliferation in A549 cells. In regards to the migration rate of these cell lines, the overexpression of Tarsh decreased migration in A549 cells, but had no effect on H1299 cells. However, the role of p53 in migration is still unclear. The results of this study suggest that the knockdown of p53 decreases cell migration in A549 cells. This contradicts the fact that H1299 cells do not express p53, yet was found to have the highest migration rate. It is evident that a further investigation is needed to make more concrete conclusions. Nevertheless, the suppressive features of Tarsh on cell proliferation, and possibly migration, make it a promising target of research for lung cancer therapy.

RNA silencing with direct delivery of siRNA has been used to suppress ldhA gene expression in filamentous fungus Rhizopus oryzae. Here, for the first time we show that, introducing small interfering RNA which consequently forms silencing complexes can alter the gene expression and we report a significant reduction of lactic acid production for isolates containing short (25 nt) synthetic siRNA. In all samples lactic acid production was reduced comparing with wild types. The average concentration of lactic acid production by Rhizopus oryzae during batch fermentation process where glucose has been used as a sole carbon source, diminished from 2.06 g/l in wild types to 0.36 g/l in knockdown samples which signify 5.7 times decrease. Interestingly, the average concentration of ethanol production was increased from 0.38 g/l in wild types to 0.45 g/l in knockdown samples. In some samples we were able to report even a 10 fold decrease in lactic acid production. Since R.oryzae is capable to assimilate a wide range of carbohydrates hydrolysed from lignocellulosic material in order to produce many economically valuable bulk material such as ethanol, these results suggest that RNA silencing is a useful method for industrial biotechnology to be applied in fungus Rhizopus oryzae in order to trigger the metabolism and gene expression toward a desired product.

No description available.

No description available.

Le système génétique complexe des mitochondries de plantes supérieures n'a pu être étudié par des approches transgéniques car les méthodes conventionnelles ne permettent pas de transformer ces organites. Une approche alternative a été développée au laboratoire, grâce à l'existence d'un processus naturel assurant l'import d'ARN de transfert (ARNt) du cytosol dans les mitochondries. Il a été montré qu'un mime d'ARNt peut servir in vivo de navette pour importer dans les mitochondries de plante des ARN-passagers exprimés à partir de transgènes nucléaires. L'utilisation d'un transribozyme comme séquence-passagère a permis d'obtenir l'invalidation spécifique d'un ARN messager (ARNm) majeur dans les mitochondries de cellules végétales transformées. Nous avons mis en oeuvre cette stratégie pour développer des études de régulation mitochondriale. Cinq ARNm mitochondriaux (nad9, sdh3, cob, cox3 et atp9) ont été choisis comme cibles pour des transribozymes spécifiques à tête de marteau. Après validation de l'activité de ces ribozymes in vitro, les vecteurs d'expression portant les transgènes correspondants ont servi pour transformer des cultures cellulaires de Nicotiana tabacum, des plantes d'Arabidopsis thaliana (pour nad9, cob, cox3 et atp9) et des plantes de N. tabacum (pour sdh3). L'invalidation spécifique des ARN mitochondriaux ciblés par les ribozymes a été établie in vivo. La réponse, en termes de régulation, à l'invalidation des cibles individuelles a été analysée au niveau de l'ensemble du transcriptome. Alors qu'il a été généralement considéré jusqu'à présent que les processus de régulation mitochondriaux chez les plantes se passent essentiellement au stade post-transcriptionnel, nos résultats sont fortement en faveur de mécanismes de coordination des ARNm dans les mitochondries et entre les organites et le noyau.

Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.

Preimplantation embryo development in the mouse is a time of rapid cellular morphological and molecular changes leading to embryo implantation for the generation of offspring. The Mager lab studies these events occuring between fertilization and implantation in order to better understand the initial events which set the stage for all future aspects of development. The result of this research impacts many scientific disciplines including in-vitro based means of embryo culture, establishment of epigenetic marks, differentiation and cellular reprogramming and can be used in translational research for the improvement of in-vitro culture techniques and develop novel therapies such as cell replacement in the case of macular degeneration (Bin, L., 2009). Through the use of in-vitro embryo culture, RNA interference (RNAi) approaches and daily observations, gene function required in preimplantation embryo development can be determined. In the initial published body of work evaluating gene knockdown using our RNAi approach (Maserati M 2011), WDR74 was characterized in preimplantation embryo development. We now understand that WDR74 is implicated in RNA production and/or stability as gene knockdown at the 1 cell stage significantly depletes mRNA within the embryo by the morula stage. Furthermore, double knockdown of Trp53 and Wdr74 results in a partial rescue of blastocyst formation suggesting p53 mediated apoptosis in the failure to make a blastocyst phenotype. The initial characterization of 4 RNA processing genes (SF3b14, SF3b1/SAP155, Rpl7l1 and Rrp7a) required for blastocyst formation was later evaluated. The results of this work has been submitted for publication and will be published soon in the journal Zygote. SF3b14 and SF3b1, identified as being part of the splicesome complex, disproportionally contributes to gene transcription of those genes containing more than 1 exon verifying a role in RNA splicing. Rpl7l1, identified by GO terms as a possible ribosomal gene, was found to be present in the cytoplasm and, surprisingly, in the nucleus. It is surmised this gene influences polymerase 2 activity as Rpl7l1 gene knockdown embryos demonstrate reduced active polymerase 2 activity at the morula stage. Rrp7a was identified as being critical in blastocyst formation and is present in the cytoplasm while excluded from the nucleus. Based on location and GO terms, this suggests a role in translation. Taken together, these 4 genes act in 3 different ways impacting RNA production, splicing or translation promoting blastocyst formation in the mouse. The final gene evaluated in this work was Bcl-6 corepressor (Bcor). As opposed to our previous work with RNA processing factors, this gene knockdown does not result in a failure to make a blastocyst. Bcor knockdown increases the rate of physiologically normal blastocysts in both murine and bovine models. Although further characterization must be done, temporary Bcor gene knockdown might be a useful improvement of in-vitro embryo culture systems including murine, bovine, equine and possibly even human. This manuscript is divided into 4 chapters, the first of which is a review of preimplantation embryo development. This covers selected and relevant events between fertilization and just before implantation of the embryo into the uterus. I mainly focus on events after fertilization and the necessary changes required for zygotic genome transcription and lineage specification. The second chapter characterizes WDR74, a gene we identified as critical in the formation of a blastocyst in a reverse genetic screen. As state before, we assess WDR74 function with the developing embryo and conclude the protein plays a role in RNA production and/or stability of RNA transcripts. We also test to rescue blastocyst formation in WDR74 knockdown embryos in an attempt to further evaluate WDR74 function. We continue the characterization of genes whose temporary reduction causes the failure of blastocyst formation in the third chapter. Here we report on four additional RNA processing genes in a body of work which has been published in the journal Zygote. Since these genes contained similar GO terms, we assumed they may all function in a similar way so they were assayed together as a group. As function of these genes were unknown, we determined protein localization within the cell, function in RNA splicing, alternative splicing and to determine if the failure to make a blastocyst is due to lineage specification. In the final chapter, BCOR gene expression is characterized in preimplantation embryo development as in the former 2 chapters. However, the result of this gene knockdown does not lead to the failure to make a blastocyst, rather this improves the number of blastocysts formed during the correct physiological time; the same time that blastocysts form invivo. Undoubtedly, this could lead to possible commercial applications which are reviewed along with the preliminary data we have been able to collect thus far. Specifically, the continuation of the BCOR gene knockdown research in preimplantation embryo development is pitched in the form of academic and international business collaboration with InvitroBrasil for the production of cloned bovine, equine and ICSI in equine.

Insect Odorant-Binding Proteins (OBPs) are small, water-soluble molecules that solubilize hydrophobic odorant molecules in the sensillum lymph and transport them to their cognate receptors in the olfactory receptor neurons. With the availability of the genome sequence of the African malaria mosquito, Anopheles gambiae, there has been a profound interest in the characterization and functional analyses of Obp genes in order to understand the molecular basis of mosquito host-seeking behavior. However, no direct evidence has been found for specific functions of any mosquito OBPs. In this study, I describe the comparative genomics and expression analyses on two mosquito Obp genes (Obp1 and Obp7) as well as efforts to determine their functions. Both of these Obp genes were identified in Anopheles stephensi and only Obp7 gene was identified in Anopheles quadriannulatus by screening bacterial artificial chromosome (BAC) libraries of these species. Comparative analyses revealed several interesting features including segments of conserved non-coding sequences (CNSs) that contain potential regulatory elements relevant to olfactory tissue development and blood-feeding. The expression profiles of these genes were examined in detail in the Asian malaria mosquito An. stephensi. Obp1 and Obp7 transcripts were significantly higher in females than male mosquitoes and they were predominantly found in the antenna, which is the primary olfactory organ of mosquitoes. Twenty-four hours after a blood meal, mRNA levels of these two genes were significantly reduced in the maxillary palp and proboscis, referred to as secondary olfactory organs of mosquitoes. These findings collectively indicate that Obp1 and Obp7 genes in An. stephensi likely function in female olfactory response and may be involved in behaviors related to blood-feeding. To investigate the function of these Obp genes more directly, a Sindbis virus based expression system is established to knockdown the two Obp gene orthologs in Aedes aegypti. The effective knockdown of Obp1 and Obp7 genes (8 and 100-fold, respectively) is accomplished in female mosquito olfactory tissues. The potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique is discussed. Such analysis will provide the foundation for interfering with mosquito host-seeking behavior for the prevention of disease transmission. / Ph. D.

Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on microRNAs' role in zebrafish thrombopoiesis. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, I identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Knockdown of three microRNAs, mir-7148, let-7b, and mir-223, by the piggyback method in zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. I then verified these findings in zebrafish larvae after the knockdown of the above microRNAs followed by an arterial laser thrombosis assay. I concluded mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. Furthermore, I explored let-7b downstream genes in thrombocytes detected by RNA-seq analysis and chose 14 targets based on their role in cell differentiation (rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b) that are transcriptional regulators. The qRT-PCR analysis of expression levels the above genes following let-7b knockdown showed significant changes in the expression of 13 targets. I then studied the effect of the 14 targets on thrombocytes production and identified 5 genes (irf5, tgif1, irf8, cebpa, and rorca) that showed thrombocytosis and one gene ikzf1 that showed thrombocytopenia. Furthermore, I tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. I also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a opposite expression profiles following let-7b and mir-223 knockdowns (decreased and increased expression, respectively) and knockdown of ikzf1 resulted in thrombocytopenia I confirmed a definitive role for ikzf1 using an ikzf1 mutant obtained from the Zebrafish International Resource Center (ZIRC). The arterial laser thrombosis assay of ikzf1 mutant progeny confirmed our piggyback hybrid knockdown results. Taken together, these studies shed light on understanding the role and the regulatory effects of zebrafish microRNA on thrombopoiesis and identified novel downstream target transcription factors for let-7b and mir-223.