La localisation des ARNm dans différents compartiments subcellulaires est conservée dans un large éventail d'espèces et de divers types cellulaires. Le trafic est médié par l'interaction entre les protéines de liaison à l'ARN (RBP) et l'ARNm. Les RBP reconnaissent les éléments cis-régulateurs de l'ARNm, également appelés éléments de localisation. Ceux-ci sont définis par leur séquence et/ou leurs caractéristiques structurelles résidant dans la molécule d'ARNm. La localisation des ARNm est essentielle pour la résolution subcellulaire et temporelle. De plus, les ARNm se sont avérés enrichis dans de nombreux compartiments cellulaires, notamment les mitochondries, l'appareil mitotique, et le réticulum endoplasmique. En outre, des études ont démontré que les RBP et les ARNm sont associés aux structures de l'appareil mitotique. Cependant, le rôle que joue la localisation de l'ARNm au cours de la mitose reste largement inexploré. Ma thèse de doctorat vise à comprendre comment le trafic d'ARNm est impliqué lors de la mitose. La première partie de cette thèse porte sur l'interaction post-transcriptionnelle qui se produit entre les deux ARNm, cen et ik2. Les gènes qui se chevauchent sont une caractéristique frappante de la plupart des génomes. En fait, il a été constaté que le chevauchement des séquences génomiques module différents aspects de la régulation des gènes tels que l'empreinte génomique, la transcription, l'édition et la traduction de l'ARN. Cependant, la mesure dans laquelle cette organisation influence les événements réglementaires opérant au niveau post-transcriptionnel reste incertaine. En étudiant les gènes cen et ik2 de Drosophila melanogaster, qui sont transcrits de manière convergente avec des régions 3' non traduites qui se chevauchent, nous avons constaté que la liaison physique de ces gènes est un déterminant clé dans la co-localisation de leurs ARNm aux centrosomes cytoplasmiques. Le ciblage du transcrit ik2 dépend de la présence et de l'association physique avec l'ARNm de cen, qui est le principal moteur de la co-localisation centrosomale. En interrogeant les ensembles de données de séquençage de fractionnement, nous constatons que les ARNm codés par des gènes qui se chevauchent en 3' sont plus souvent co-localisés par rapport aux paires de transcrits aléatoires. Ce travail suggère que les interactions post-transcriptionnelles des ARNm avec des séquences complémentaires peuvent dicter leur destin de localisation dans le cytoplasme. La deuxième partie de cette thèse consiste à étudier le rôle que jouent les RBP au cours de la mitose. Auparavant, les RBP se sont avérés être associés au fuseau et aux centrosomes. Cependant, leur rôle fonctionnel au niveau de ces structures reste à étudier. Grâce à un criblage par imagerie avec plus de 300 anticorps, nous avons identifié 30 RBP localisés dans les structures mitotiques des cellules HeLa. Ensuite, pour évaluer les rôles fonctionnels de ces RBP, nous avons utilisé l'interférence ARN (ARNi) pour évaluer si la fidélité du cycle cellulaire était compromise dans les cellules HeLa et les embryons de Drosophila melanogaster. Fait intéressant, nous avons identifié plusieurs candidats RBP pour lesquels le knockdown perturbe la mitose et la localisation de l'ARNm dans les cellules HeLa. De plus, la perte des orthologues a entraîné des défauts de développement chez l'embryon de mouche. Grâce à ce travail, nous avons démontré que les RBP sont impliquées pour assurer une mitose sans erreur. En résumé, les travaux que j'ai menés mettent en lumière l'implication de la régulation post-transcriptionnelle au cours de la mitose. En définissant les fonctions et le mécanisme de localisation des ARNm en mitose, ce travail permettra de définir de nouvelles voies moléculaires impliquées dans la régulation de la mitose. Puisque la division cellulaire non contrôlée peut mener à des maladies tel le cancer, étudier le contrôle du cycle cellulaire sous cet angle « centré sur l'ARN » peut aider à développer de nouvelles approches thérapeutiques pour trouver des solutions aux problèmes de santé. / The localization of mRNAs to different subcellular compartments is conserved in a wide range of species and diverse cell types. Trafficking is mediated by the interaction between RNA binding proteins (RBPs) and mRNA. RBPs recognize mRNA cis regulatory motifs, otherwise known as localization elements. These are defined by their sequence and/or structural features residing within the mRNA molecule. Localization of mRNAs is essential for subcellular and temporal resolution. Furthermore, mRNAs have been found to be enriched in many cellular compartments including the mitochondria, mitotic apparatus, and endoplasmic reticulum. Moreover, studies have demonstrated that RBPs and mRNAs are associated with mitotic apparatus structures. However, the role that mRNA localization plays during mitosis remains largely unexplored. My PhD thesis aims to understand how the trafficking of mRNAs is implicated during mitosis. The first part of this thesis encompasses the post-transcriptional interaction that occurs between the two mRNAs, cen and ik2. Overlapping genes are a striking feature of most genomes. In fact, genomic sequence overlap has been found to modulate different aspects of gene regulation such as genomic imprinting, transcription, RNA editing and translation. However, the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. By studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed with overlapping 3’untranslated regions, we found that the physical linkage of these genes is a key determinant in co-localizing their mRNAs to cytoplasmic centrosomes. Targeting of the ik2 transcript is dependent on the presence and physical association with cen mRNA, which serves as the main driver of centrosomal colocalization. By interrogating global fractionation-sequencing datasets, we find that mRNAs encoded by 3’overlapping genes are more often co-localized as compared to random transcript pairs. This work suggests that post-transcriptional interactions of mRNAs with complementary sequences can dictate their localization fate in the cytoplasm. The second part of this thesis involves investigating the role that RBPs play during mitosis. Previously, RBPs have been found to be associated with the spindle and centrosomes. However, their functional role at these structures was yet to be investigated. Through an imaging screen with >300 antibodies, we identified 30 RBPs localized to mitotic structures in HeLa cells. Then, to assess the functional roles of these RBPs, we used RNA interference (RNAi) to assess whether cell cycle fidelity was compromised in HeLa cells and Drosophila melanogaster embryos. Interestingly, we identified several RBP candidates for which the knockdown disrupted mitosis and mRNA localization in HeLa cells. Furthermore, loss of the orthologs led to developmental defects in the fly embryo. Through this work, we demonstrated that RBPs are involved in ensuring an error-free mitosis. In summary, the work that I have conducted sheds light on the involvement of post-transcriptional regulation during mitosis. By defining the functions and mechanism of mRNA localization in mitosis, this work will help define new molecular pathways involved in mitosis regulation. As uncontrolled cell division can lead to diseases such as cancer, studying cell cycle control from this ‘RNA-centric’ angle may help to develop new therapeutic approaches to find solutions to health problems.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Nosso laboratório investiga os mecanismos moleculares que promovem o estabelecimento de padrões de expressão gênica regulados no desenvolvimento em eucariotos superiores. Como modelo, utilizamos o gene de pufe de DNA BhC4-1, que é amplificado e expresso de modo regulado na glândula salivar e na glândula protorácica no final do quarto estadio larval de B. hygida. Estudos funcionais em D. melanogaster resultaram na identificação de módulos cis-reguladores (MCRs) na região promotora do gene BhC4-1. O MCR de glândula anelar de 67 bp (-253/-187), promove a expressão de BhC4-1-lacZ na glândula anelar a partir do final do desenvolvimento embrionário. O MCR de glândula salivar de 129 bp (-186/-58), dirige a expressão do transgene nas glândulas salivares de prépupas. A glândula anelar é o principal órgão endócrino larval e em D. melanogaster é o resultado da fusão das glândulas protorácicas (responsáveis pela síntese de hormônios esteroides), corpus allatum (síntese de hormônio juvenil) e corpus cardiacum (glândula neuroendócrina). Neste trabalho foram obtidas 12 linhagens independentes transformadas com uma construção que contém o fragmento (-253/+40) do promotor do gene de pufe de DNA BhC4-1 clonado à montante do gene repórter GFP. O genótipo destas linhagens foi validado utilizando-se Southern blots. Inicialmente as 12 linhagens obtidas foram analisadas quanto ao padrão de expressão de GFP em larvas de terceiro estadio e em prépupas 2 horas. Em conjunto, esta análise revelou que o padrão de expressão de GFP é bastante variável nestas linhagens. A análise do padrão de expressão da proteína repórter foi estendida em duas linhagens representativas da série (- 253/+40)/GFP. Nestas linhagens a expressão de GFP é inicialmente detectada na glândula salivar durante o estágio de prépupa e na glândula anelar a partir do terceiro estadio larval. Diferentemente do anteriormente observado em linhagens (-253/+40)/lacZ, nestas linhagens não detectamos a expressão de GFP em tempos do desenvolvimento anteriores ao terceiro estadio larval. Experimentos de interação gênica revelaram que na ausência do fator de transcrição br, a expressão de GFP é mantida na glândula anelar e abolida na glândula salivar de larvas de terceiro estadio. Os resultados dos experimentos de interação gênica corroboram dados anteriores que indicavam que o conjunto de fatores de transcrição que regulam a expressão de BhC4-1-lacZ na glândula anelar é distinto daquele que promove a expressão do gene na glândula salivar. As linhagens obtidas neste trabalho constituem uma ferramenta a ser utilizada na caracterização de fatores de transcrição tecido-específicos que regulam o gene BhC4-1 na glândula anelar e/ou na glândula salivar a partir do final do desenvolvimento larval. / Our laboratory investigates the molecular mechanisms that promote the establishment of developmentally regulated gene expression patterns in metazoans. As a model, we employ the BhC4-1 DNA puff gene, which is amplified and expressed in a regulated manner in the salivary gland and in the prothoracic gland at the end of the fourth larval instar in B. hygida. Functional studies in D. melanogaster resulted in the identification of cis-regulatory modules (CRMs) in the BhC4-1 promoter region. The 67 bp (-253/-187) ring gland CRM drives BhC4-1-lacZ expression in the ring gland from late embryonic development. The 129 bp (-186/-58) CRM salivary gland drives transgene expression in the prepupal salivary glands. The ring gland is the major endocrine organ, and comprises the prothoracic glands (synthesis of ecdysteroid hormones), corpus allatum (synthesis of juvenile hormone) and corpus cardiacum (neuroendocrine gland). In this work, 12 independent lines transformed with a construct containing the BhC4-1 promoter fragment (- 253/+40) cloned upstream of the reporter gene GFP were obtained. The genotype of each line was validated using Southern blots. Initially, the 12 obtained lines were analyzed to investigate the pattern of GFP expression in the third instar larvae and in the 2 hours prepupae. This initial screening revealed that the pattern of GFP expression is highly variable in these lines. The developmental pattern of GFP expression was extended in two representative (- 253/+40)/GFP lines. In these lines, GFP expression is initially detected in the larval and prepupal salivary glands and in ring gland third instar. Differently from previously observed in (-253/+40)/lacZ lines, in these lines we did not detect GFP expression at developmental times prior to the third larval instar. Gene interaction experiments revealed that in the absence of the br transcription factor, GFP expression is maintained in the ring gland and abolished in the salivary gland of third instar larvae. The results of gene interaction experiments corroborate previous data indicating the set of transcription factors that regulate BhC4-1-lacZ expression in the ring gland is distinct from that which promotes gene expression in salivary glands. The lines obtained in this work constitute a tool to characterize the tissue-specific transcription factors that regulate BhC4-1 gene in the ring gland and/or in the salivary gland from the end of the larval development.

Submitted by Kelson Anthony de Menezes (kelson@ucb.br) on 2016-12-19T17:43:43Z No. of bitstreams: 1 PhilippedeCastroLinsDissertacao2015.pdf: 2365075 bytes, checksum: c5628aa72f1ff54c00d7f8617a9d41dd (MD5) / Made available in DSpace on 2016-12-19T17:43:43Z (GMT). No. of bitstreams: 1 PhilippedeCastroLinsDissertacao2015.pdf: 2365075 bytes, checksum: c5628aa72f1ff54c00d7f8617a9d41dd (MD5) Previous issue date: 2015-04-10 / Gene promoters regulate gene expression quantitatively and qualitatively. The regulatory sequences have cis elements and with trans acting that will direct and correctly position the RNA polymerase which is the process of DNA transcription. Genetic engineering of plants by transforming plants expressing genes of interest, use in most studies, constitutive CaMV35S promoter character. A new promoter isolated from soybeans, which is called uceS8.3 showing a constitutive promoter driving expression in different plant tissues. The analysis of the expression of the uidA gene, GUS, demonstrated that the expression profile uceS8.3 controlled by the promoter is comparable or superior to the CaMV35S promoter in plant tissues such as flower buds and roots. Were identified following the uceS8.3 promoter cis regulatory elements that may be responsible for gene expression profile controlled by this promoter. Modules of this promoter, when compared with the CaMV35S promoter and the uceS8.3 itself demonstrated a difference in expression in plant tissues. Cis regulatory elements as ROOTMOTIFPABOX1, POLLEN1LELAT52, MRE, Sp1 and Ibox. The set of specific cis elements located in the promoter uceS8.3 modules may be the minimum necessary elements to control expression in leaf and flower bud tissues. The quantitative expression analysis and fluorometric GUS assays leaf and root tissues have demonstrated a correlation between transcript levels and the specific activity levels of a ??-glucuronidase enzyme in module 2 (720pb), but there is a difference in the balance of these levels between uceS8.3 promoter and Module 4 (170pb). The studies presented here demonstrated that the promoter and its modules has high ability to drive expression in flower tissues and roots, may be used and applied to different types of biotechnologically biotic stresses, including insect pests and nematodes. / Promotores g??nicos regulam a express??o de genes, quantitativamente e qualitativamente. As sequ??ncias regulat??rias possuem elementos cis e trans atuantes que v??o direcionar e posicionar corretamente a RNA polimerase para que haja o processo de transcri????o do DNA. A engenharia gen??tica de plantas, por meio da transforma????o de plantas, expressando genes de interesse, vem utilizando, na maioria dos estudos, o promotor CaMV35S de car??ter constitutivo. Um novo promotor isolado de soja, denominado uceS8.3 ?? tamb??m um promotor constitutivo demonstrando conduzir a express??o em diferentes tecidos vegetais. A an??lise da express??o do gene uidA, GUS, demonstrou que o perfil de express??o controlada pelo promotor uceS8.3 ?? compar??vel ou superior ao promotor CaMV35S em tecidos vegetais, como bot??o floral e raiz. Foram identificados, na sequ??ncia do promotor uceS8.3, elementos cis regulat??rios que podem ser respons??veis pelo perfil de express??o g??nica controlada por esse promotor. Os m??dulos desse promotor, quando comparados com o promotor CaMV35S, e o pr??prio uceS8.3 demonstraram uma diferen??a de express??o nos tecidos vegetais. Elementos cis regulat??rios como ROOTMOTIFPABOX1, POLLEN1LELAT52, MRE, Sp1 e I-box. O conjunto de elementos cis espec??ficos, localizados nos m??dulos do promotor uceS8.3, podem ser os elementos m??nimos necess??rios para controlar a express??o em tecidos de folha e bot??o floral. A an??lise de express??o quantitativa e de ensaios fluorim??tricos de GUS nos tecidos folha e raiz, demonstraram uma correla????o entre os n??veis de transcrito e os n??veis de atividade espec??fica da enzima ??-glucuronidase no m??dulo 2 (720pb), por??m h?? uma diferen??a na correla????o destes n??veis entre o promotor uceS8.3 e o m??dulo 4 (170pb). Os estudos aqui apresentados demonstraram que o promotor uceS8.3 e seus m??dulos possuem alta capacidade de conduzir express??o em tecidos florais e ra??zes, podendo ser utilizado e aplicado biotecnologicamente para diferentes tipos de estresses bi??ticos, incluindo insetos-praga e nemat??ides.

Introdução: Obesidade central é um fator de risco para o diabetes e as doenças cardiovasculares. O estudo da sua associação com os transtornos mentais comuns pode ajudar a entender melhor a epidemia de obesidade que acontece no Brasil, e a relação entre saúde mental e doenças crônicas. Objetivo: Investigar a associação entre transtornos mentais comuns e obesidade central em uma coorte ocupacional- ELSA-Brasil. Métodos: Para a avaliação do transtorno mental comum (TMC) foi aplicado o questionário CIS-R em 15102 participantes entre 35 e 74 anos. A circunferência da cintura foi aferida junto com outras medidas antropométricas. Variáveis demográficas e comportamentais também foram coletadas através de questionários. Resultados: O transtorno mental comum mostrou associação com obesidade central (RP = 1,30; IC95% 1,25-1,36), e mesmo quando ajustada para sexo, idade, raça/cor da pele e centro de investigação ELSA, continuou significativa a associação (RP = 1,21; IC95% 1,16-1,27). Os transtornos específicos depressão (RP = 1,24, IC95% 1,14-1,34), ansiedade (RP = 1,18, IC95% 1,12-1,24) e misto de ansiedade e depressão (RP = 1,12; IC95% 1,06-1,18) também se mostraram associados, inclusive quando ajustados para as mesmas covariáveis. Conclusão: Participantes com transtorno mental comum e com os diagnósticos específicos de depressão e ansiedade apresentam maior prevalência de obesidade central comparados com os que não apresentam transtornos mentais. / Background: Central obesity is a risk factor for diabetes and cardiovascular disease, and the study of its association with common mental disorders can help understand the obesity epidemic in Brazil, and the relationship between mental health and chronic diseases. Objective: To investigate the association between common mental disorders and central obesity in an occupational cohort ELSA-Brasil. Methods: Waist circumference, among other anthropometric measures, was obtained, and the CIS-R questionnaire was applied in 15102 participants between 35 and 74 years old. Demographic and behavioral variables were also obtained. Results: Common mental disorder was significantly associated with central obesity in crude analysis (PR = 1,30, CI95%: 1,25-1,36), and when adjusted for gender, age, skin color and center study (PR = 1,21, CI95%: 1,16 – 1,27). The specific mental disorders depression and anxiety were also associated. Conclusion: Participants with common mental disorder, and with specific diagnoses of depression and anxiety, report a higher prevalence of central obesity than people without a mental disorder.

In molecules, electronic state transitions can occur via quantum coupling of the states. If the coupling is due to the kinetic energy of the molecular nuclei, then electronic transitions are best represented in the adiabatic frame. If the coupling is instead facilitated through the potential energy of the nuclei, then electronic transitions are better represented in the diabatic frame. In our study, we modeled these latter transitions, called ``nonadiabatic transitions.'' For one nuclear degree of freedom, we modeled the de-excitation of a diatomic molecule. For two nuclear degrees of freedom, we modeled the de-excitation of an ethane-like molecule undergoing cis-trans isomerization. For both cases, we studied the dependence of the de-excitation on the nuclear configuration and potential energy of the molecule. We constructed a numerical model to solve the time-dependent Schr\"{o}dinger Equation for two coupled wave functions. Our algorithm takes full advantage of the sparseness of the numerical system, leading to a final set of equations that is solved recursively using nothing more than the Tridiagonal Algorithm. We observed that the most effective de-excitation occurred when the molecule transitioned from a stable equilibrium configuration to an unstable equilibrium configuration. This same mechanism is known to drive fast electronic transitions in the adiabatic frame. We concluded that while the adiabatic and diabatic frames are strongly opposed physically, the mathematical mechanism driving electronic transitions in the two frames is in some sense the same.

La phosphorylation constitue un mécanisme de régulation de la fonction biologique des protéines lié au contrôle des associations inter-moléculaires, de l'activité enzymatique ou de la liaison de ligands. L'isomérisation des liaisons Ser/Thr-Pro après phosphorylation par des kinases spécifiques, souvent impliquées dans le contrôle du cycle cellulaire, se présente comme un nouveau mode de régulation. Ces deux mécanismes de signalisation sont étroitement liés par l'intermédiaire d'enzymes catalysant l'isomérisation cis/trans des prolines au niveau de motifs Ser/Thr-Pro phosphorylés telles que les peptidyl-prolyl cis/trans isomérases de la famille de Pin1. Elles jouent un rôle cellulaire essentiel mais leur rôle moléculaire exact est encore mal connu. Les interactions moléculaires entre Pin1 et de nombreuses phospho-protéines mitotiques indiquent un rôle dans la régulation du cycle cellulaire et dans l'oncogénèse, et font de Pin1 une cible pharmacologique émergente dans le traitement des cancers. Récemment, des interactions avec la protéine microtubulaire Tau dans sa forme pathologique hyperphosphorylée, au niveau d'un site unique centré autour du motif Thr231-Pro232, pourraient impliquer Pin1 dans la régulation de la liaison de Tau aux microtubules et dans les phénomènes de neurodégénérescence observés dans la maladie d'Alzheimer.<br /><br />Nous avons ciblé les interactions entre Pin1 et la protéine Tau comme modèle de substrats pour une étude détaillée des mécanismes intervenant à l'échelle moléculaire, sur base de substrats peptidiques, qui permettraient d'expliquer le rôle fonctionnel de Pin1. L'interaction avec les substrats au travers des motifs Ser/Thr-Pro phosphorylés est double : un domaine de liaison WW permet la liaison du substrat et un domaine catalytique PPIase (peptidyl-prolyl isomérase) catalyse l'isomérisation cis/trans des prolines. Un criblage par RMN des différents motifs phospho-Ser/Thr-Pro au sein de la protéine Tau a permis de déterminer un nouveau site d'interaction centré autour du motif Thr212-Pro213, phosphorylé uniquement dans la forme pathologique de Tau. <br /><br />Nous avons étendu l'investigation des interactions avec Pin1 à l'échelle de la protéine Tau entière. Comme pour la plupart des régions protéiques impliquées dans les interactions avec Pin1, la protéine Tau se caractérise par une absence de structure globale qui limite considérablement les études par RMN. Un fragment peptidique de 40 acides aminés comprenant les sites Thr231 et Thr212 phosphorylés a permis de montrer un rôle régulateur du domaine WW dans l'activité enzymatique. Une première étude avec une protéine mutante mimant l'état phosphorylé de Tau a montré une interaction avec le domaine catalytique de Pin1 et a nécessité la mise au point préalable d'une technique d'attribution de la protéine Tau par RMN que nous avons appelé « mapping peptidique ».<br /><br />La phosphorylation du domaine WW de Pin1 est associée à l'inhibition de la liaison des substrats et joue un rôle dans la régulation de l'activité de Pin1 in vivo. La forme non phosphorylée active de Pin1 est retrouvée majoritairement dans les cellules cancéreuses et la forme phosphorylée inactive dans les cellules saines. Nous avons envisagé de cibler les interactions entre Pin1 et les phospho-peptides avec la synthèse de molécules organiques mimant le dipeptide phosphoThr-Pro et la mise en œuvre d'un test de criblage par RMN pour l'obtention d'inhibiteurs ciblant le domaine WW de Pin1 qui pourraient mimer la forme inactive de la protéine.

RNA sequences and higher-order structures in the 5’ and 3’ untranslated regions (UTRs) of positive-strand RNA viruses are known to function as cis-acting elements for translation, replication, and transcription. In coronaviruses, these are best characterized in the group 2a bovine coronavirus (BCoV) and mouse hepatitis virus (MHV), yet their precise mechanistic features are largely undefined. Here, we use a reverse genetics system in MHV to exploit the ~30% nt sequence divergence between BCoV and MHV to establish structure/function relationships of 5’ UTR cis-replication elements. It had been previously shown that a precise replacement of the 391-nt MHV 3’ UTR with the 288-nt BCoV 3’ UTR yields wt-like MHV. Our attempts to replace the 209-nt MHV 5’ UTR with the 210-nt BCoV 5’ UTR, however, yielded a non-viable chimera. Therefore, a systematic analysis of individual 5’-terminal structures was made to identify compatible elements. By placing each of four putative cis-acting domains from the BCoV 5’ UTR into the MHV genome, we learned that (i) stem-loops (SLs) I & II and SLIII are functionally compatible, (ii) SLIV is compatible if it spans parts of the 5’ UTR and the nonstructural protein 1 (nsp1) cistron, thus identifying this part of ORF 1 as a component of the cis-replication signal, (iii) a relatively unstructured 32-nt region mapping between SLIII and SLIV defines a novel virus species-specific cis-replication element, (iv) spontaneous suppressor mutations within MHV SLI and nsp1 cistron compensated for growth defects arising from the BCoV 32-nt element in the MHV genome, (v) cross talk between the 32-nt element, SLI, and the nsp1 cistron appears essential for virus replication, (vi) the BCoV 5’ UTR and nsp1 cistron function together in the MHV genome to generate a wt-like MHV phenotype, and (vii) a functional 5’ UTR-nsp1 domain in group 2a coronaviruses cannot be substituted by the corresponding genomic element from the group 2b SARS-CoV. We postulate that the interaction between the 5’ UTR and nsp1 cistron (or possibly nsp1 protein) functions as a molecular switch between genome translation and ignition of negative-strand RNA synthesis.

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, β-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two β-1,3-endoglucanses while CA whole leaf protein extracts contain at least three β-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 β-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for β-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of β -1,3-endoglucanses.

In this paper, a heuristic backtracking search algorithm is proposed to adjust the phasing arrangement of primary feeders and laterals for phase balancing of distribution systems. The phase unbalance index of distribution feeders is calculated based on the phasing current magnitude of each line segment and branch, which has been solved by a 3-phase load flow program. The database of an automated mapping/facility management (AM/FM) system is used to retrieve the component attributes and topology process is executed to determine the electrical network configuration and to identify the customers served by each distribution transformer. By using the monthly energy consumption of the customers in customer information system (CIS) and the typical daily load patterns of customer classes, the hourly loading profiles of distribution transformers can be derived, which can be integrated to solve the load demand of each service zone. By this manner, the individual phase loadings of each primary feeder and lateral can be determined based on the phasing of distribution transformers and the power consumption served. The optimal phase balancing of distribution systems is performed by heuristic rule-based searching process to minimize the phase unbalance index so that the proper phasings of a primary feeder and its lateral can be assigned. To demonstrate the effectiveness of proposed methodology to enhance three-phase balancing of distribution systems, a practical distribution feeder with 2754 customers are selected for computer simulation. It is concluded that three phase balancing of distribution systems can be obtained by properly phasing design of primary feeders and laterals by considering customer load characteristics.