Consensus pattern problem (CPP) aims at finding conserved regions, or motifs, in unaligned sequences. This problem is NP-hard under various scoring schemes. To solve this problem for protein sequences more efficiently,a new scoring scheme and a randomized algorithm based on substitution matrix are proposed here. Any practical solutions to a bioinformatics problem must observe twoprinciples: (1) the problem that it solves accurately describes the real problem; in CPP, this requires the scoring scheme be able to distinguisha real motif from background; (2) it provides an efficient algorithmto solve the mathematical problem. A key question in protein motif-finding is how to determine the motif length. One problem in EM algorithms to solve CPP is how to find good startingpoints to reach the global optimum. These two questions were both well addressed under this scoring scheme,which made the randomized algorithm both fast and accurate in practice. A software, COPIA (COnsensus Pattern Identification and Analysis),has been developed implementing this algorithm. Experiments using sequences from the von Willebrand factor (vWF)familyshowed that it worked well on finding multiple motifs and repeats. COPIA's ability to find repeats makes it also useful in illustrating the internal structures of multidomain proteins. Comparative studies using several groups of protein sequences demonstrated that COPIA performed better than the commonly used motif-finding programs.

Reversion-inducing cysteine-rich with Kazal motif (RECK) is a cell surface anchoring protein, which known for the ability to inhibit matrix metalloproteinases (MMPs) and participate in angiogenesis regulation. The inhibition of membrane type-1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-7 and, MMP-9 by RECK has been demonstrated. Our previous studies show that RECK expression is suppressed by Ras and Her-2/neu oncogene. In addition, oncogenic Ras activates downstream ERK signaling pathway to increase Sp1/HDAC promoter binding affinity which results in reduction of RECK gene transcription and increase of tumor progression and metastasis. From the clinical investigation, RECK expression is down-regulated in a number of cancer types. In breast cancer, RECK expression is associated with the prognosis of the patients. Recently, single nucleotide polymorphisms (SNPs) of RECK promoter have been suggested to be linked with survival rate and prognosis of breast cancer patients. Whether SNP of the RECK promoter has any effect on RECK expression and its clinical significance is still unclear. . In this study, we investigate -402 SNP at RECK promoter and find this SNP directly affects RECK expression through progesterone receptor binding. Additionally, we also address the -402 SNP in the sample collected from patients and analyze its association with clinicopathological parameters to clarify its clinical significance. Our results suggest that RECK SNP may be an valuable prognosis factor for breast cancer.

Genome wide studies have uncovered the existence of large-scale copy number variation (CNV) in the human genome. The human genome of different individuals was initially estimated to be 99.9% similar, but population studies on CNV have revealed that it is 12-16% copy number variable. Abnormal genomic CNVs are frequently found in subtelomeres of patients with mental retardation (MR) and other neurological disorders. Rearrangements of chromosome subtelomeric regions represent a high proportion of cytogenetic abnormalities and account for approximately 30% of pathogenic CNVs. Although DNA double strand breaks (DSBs) are implicated as a major factor in chromosomal rearrangements, the causes of chromosome breakage in subtelomeric regions have not been elucidated. But due to the presence of repetitive sequences in subtelomeres, we hypothesized that chromosomal rearrangements in these regions are not stochastic but driven by specific sequence motifs. In a collaborative effort with Dr. Rudd (Department of human genetics at Emory University), we characterized subtelomeric breakpoints on different chromosome ends in search of common motifs that cause double-strand breaks. Using a yeast-based gross chromosomal rearrangement (GCR) system, we have identified a subtelomeric breakage motif from chromosome 2 (2q SBM) with a GCR rate that is 340 fold higher than background levels. To determine if the fragility of 2q SBM was driven by the formation of secondary structures, the helicase activities of Sgs1 and Pif1 were disrupted. These helicases have been shown to destabilize DNA secondary structures such as G-quadruplex structures. Disruption of these helicases augmented chromosomal rearrangements induced by 2q SBM, indicating that these helicases are required for maintenance of this sequence. We also donwregulated replication fork components to determine if 2q SBM was imposing any problems to the replication fork machinery. Downregulation of replication fork components increased chromosomal rearrangements, indicating that intact replication fork was a critical determinant of 2q SBM fragility. Using a yeast-based functional assay, these experiments have linked human subtelomeric repetitive sequences to chromosomal breakage that could give rise to human CNV in subtelomeric regions.

Lentiviruses can infect non-dividing cells, and various cellular nuclear transport proteins provide crucial functions for lentiviral nuclear entry and integration. Genome-wide small interfering RNA screens previously identified nuclear pore complex component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). We found that HIV-1 infection of NUP153 depleted cells resulted in normal levels of reverse transcription, a moderate reduction of 2-long terminal repeat circles, and a relatively large reduction in integrated proviruses, consistent with a role for NUP153 during nuclear entry of the HIV-1 pre-integration complex. We ascertained the capsid (CA) to be the major viral determinant for NUP153 dependence during infection, and accordingly observed a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5alpha restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified recombinant proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 function during infection. Mutagenesis experiments identified NUP153C and CA residues important for binding, and different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV CA proteins. HIV-1 CA binding mapped to residues that line a common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 each competed with NUP153C for binding to HIV-1 CA, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicated that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. We propose that HIV-1 CA binds NUP153 FG motifs to affect viral nuclear import, serving as a novel example of viral hijacking of a fundamental cellular process.

MYC is overexpressed in most types of tumors, but a means to selectively decrease its expression is yet to be found. Our recent findings on modulation of BCL2 gene expression through protein interactions with the BCL2 i-motif have provided a basis for further investigation of MYC gene control. It is proposed that the MYC i-motif could function by a similar molecular switch mechanism as in BCL2.Binding sites for heterogeneous nuclear ribonucleoprotein K (hnRNP K) within the MYC promoter also exist in the i-motif-forming sequence. Circular dichroism and bromine footprinting confirmed that this DNA sequence is able to form an i-motif, and systematic mutation of the cytosine residues in this sequence has revealed a 5:5:5 loop configuration. Indeed, all loops of the i-motif, when folded into a 5:5:5 loop configuration, contain the hnRNP K consensus sequence (CCCT). Previous studies show that hnRNP K binds to this i-motif-forming sequence, but it was assumed to be single-stranded. Binding studies revealed that hnRNP K has more binding affinity to its consensus sequence in the i-motif compared to a mutant sequence where the i-motif cannot form. Further investigation of the MYC promoter revealed an additional two runs of cytosine seven bases downstream of the MYC i-motif. Biophysical studies showed that the additional two runs were not involved in i-motif formation, however recent studies describe their importance for transcriptional activation. We found that hnRNP K preferred the longer 5CT sequence compared to the i-motif forming 4CT sequence when using a competitive binding assay. Utilizing luciferase reporters containing either the 4CT or 5CT sequence validated that hnRNP K required both the i-motif and 5th CT element for maximum transcriptional activation. Competition binding studies and bromine footprinting showed that hnRNP K bound to the downstream 5th CT element and the central and lateral loops of the i-motif.Additionally, we found that co-overexpression of Sp1 and hnRNP K induced a 10-fold increase in luciferase activity in the 5CT reporter only. We hypothesize that Sp1 continuously primes the promoter to initiate transcription inducing more negative superhelicity and increasing the melting of duplex DNA. This increased melting grants hnRNP K’s three KH domains access to the i-motif loops and the 5Th CT element. Confirmation by ChIP analysis validated that Sp1 overexpression causes an increase in hnRNP K occupancy at the MYC promoter. These findings provide new insight into the mechanisms of MYC transcriptional control by the i-motif and G-quadruplex.Recently, our group has demonstrated that two small molecules IMC-48 and IMC-76 can interact with the i-motif and can be an effective means to modulate BCL2 expression. Based on these results with the BCL2 i-motif, we employed a similar strategy and screened and identified small drug-like molecules that interact with MYC i-motif, using a FRET high-throughput assay. We then further validated that IMC-16 stabilizes the MYC i-motif through the interactions with the loops of the i-motif. No stabilization by IMC-16 treatment was observed with the MYC G-quadruplex and the BCL2 and PDGFRβi-motifs demonstrating selectivity for the MYC i-motif.Finally, we investigated the effects of IMC-16 on MYC expression in three lymphoma cell lines all expressing different levels of MYC. In the case of both Daudi and RAJI Burkitt’s lymphoma cell lines we demonstrated that selectively stabilizing the i-motif by IMC-16 could increase MYC expression. Furthermore, we demonstrated that the MYC G-quadruplex stabilizing compound GQC-05 and IMC-16, which stabilizes the MYC i-motif, have antagonistic effects on MYC expression, providing further evidence of a molecular switch mechanism in the NHEIII1. Directly targeting MYC expression through the i-motif offers advantages over targeting the G-quadruplex, because of the reduced stability and dynamic nature of the i-motif, additionally the i-motif is only found in DNA. The use of such i-motif interactive compounds is the first step into the development of new innovative approaches to treat cancers.

Herein we describe the regulatory significance and molecular targeting of novel non-B-DNA secondary structures formed from the PDGFR-Beta core promoter nuclease hypersensitivity element.

The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electronic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the dissertation is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the second sphere residue His228 is responsible for the water deprotonation through hydrogen bonding. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation. Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity.

Date stream mining techniques can be used in tracking user behaviors as they attempt to achieve their goals. Quality metrics over stream-mined models identify potential changes in user goal attainment. When the quality of some data mined models varies significantly from nearby models—as defined by quality metrics—then the user’s behavior is automatically flagged as a potentially significant behavioral change. Decision tree, sequence pattern and Hidden Markov modeling being used in this study. These three types of modeling can expose different aspect of user’s behavior. In case of decision tree modeling, the specific changes in user behavior can automatically characterized by differencing the data-mined decision-tree models. The sequence pattern modeling can shed light on how the user changes his sequence of actions and Hidden Markov modeling can identifies the learning transition points. This research describes how model-quality monitoring and these three types of modeling as a generic framework can aid recognition and diagnoses of behavioral changes in a case study of cognitive rehabilitation via emailing. The date stream mining techniques mentioned are used to monitor patient goals as part of a clinical plan to aid cognitive rehabilitation. In this context, real time data mining aids clinicians in tracking user behaviors as they attempt to achieve their goals. This generic framework can be widely applicable to other real-time data-intensive analysis problems. In order to illustrate this fact, the similar Hidden Markov modeling is being used for analyzing the transactional behavior of a telecommunication company for fraud detection. Fraud similarly can be considered as a potentially significant transaction behavioral change.

La science génétique tend de plus en plus à identifier des maladies génétiques et à associer des comportements humains au bagage génétique. Or, ces applications peuvent servir à exclure et stigmatiser des individus. Cela crée parfois ce qu'on appelle de la discrimination génétique. Le domaine de l'emploi est particulièrement propice à voir surgir cette forme de discrimination. Nous voulons ici déterminer dans quelle mesure les chartes des droits de la personne canadienne et québécoise protègent les travailleurs contre la discrimination génétique. Nous regardons d'abord si la lutte contre la discrimination génétique est compatible avec les objectifs de la règle anti-discrimination. Ensuite, nous examinons la prohibition de la discrimination basée sur le handicap afin de voir si celle-ci peut empêcher la discrimination génétique des travailleurs. Finalement, nous tentons de voir si les caractéristiques génétiques pourraient constituer un motif analogue de discrimination prohibé par la Charte canadienne. / Increasingly, the genetic science tends to identify sorne genetic diseases and to associate the human behaviors to the genetic code. This uses can serve to exclude and stigmatize the individuals. This sometimes creates what is called the genetic discrimination. The workplace is particularly favorable to see emerging this form of discrimination. We try to determine in what way the human rights charters protect the workers against the genetic discrimination. First, we scrutinize if the struggle against the genetic discrimination is well-suited with the objecti ves of the principles of the non-discrimination. Secondly, we examine if the prohibition of the disability discrimination could prevent the genetic discrimination against the workers. Finally, we try to establish if the genetic characteristics could constitute an analogous ground of discrimination prohibited by the Canadian Charter. / "Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de maître en droit option Droit des biotechnologies". Ce mémoire a été accepté à l'unanimité et classé parmi les 10% des mémoires de la discipline.

L'acide désoxyribonucléique (ADN) et l'acide ribonucléique (ARN) sont des polymères de nucléotides essentiels à la cellule. À l'inverse de l'ADN qui sert principalement à stocker l'information génétique, les ARN sont impliqués dans plusieurs processus métaboliques. Par exemple, ils transmettent l’information génétique codée dans l’ADN. Ils sont essentiels pour la maturation des autres ARN, la régulation de l’expression génétique, la prévention de la dégradation des chromosomes et le ciblage des protéines dans la cellule. La polyvalence fonctionnelle de l'ARN résulte de sa plus grande diversité structurale. Notre laboratoire a développé MC-Fold, un algorithme pour prédire la structure des ARN qu'on représente avec des graphes d'interactions inter-nucléotidiques. Les sommets de ces graphes représentent les nucléotides et les arêtes leurs interactions. Notre laboratoire a aussi observé qu'un petit ensemble de cycles d'interactions à lui seul définit la structure de n'importe quel motif d'ARN. La formation de ces cycles dépend de la séquence de nucléotides et MC-Fold détermine les cycles les plus probables étant donnée cette séquence. Mon projet de maîtrise a été, dans un premier temps, de définir une base de données des motifs structuraux et fonctionnels d'ARN, bdMotifs, en terme de ces cycles. Par la suite, j’ai implanté un algorithme, MC-Motifs, qui recherche ces motifs dans des graphes d'interactions et, entre autres, ceux générés par MC-Fold. Finalement, j’ai validé mon algorithme sur des ARN dont la structure est connue, tels que les ARN ribosomaux (ARNr) 5S, 16S et 23S, et l'ARN utilisé pour prédire la structure des riborégulateurs. Le mémoire est divisé en cinq chapitres. Le premier chapitre présente la structure chimique, les fonctions cellulaires de l'ARN et le repliement structural du polymère. Dans le deuxième chapitre, je décris la base de données bdMotifs. Dans le troisième chapitre, l’algorithme de recherche MC-Motifs est introduit. Le quatrième chapitre présente les résultats de la validation et des prédictions. Finalement, le dernier chapitre porte sur la discussion des résultats suivis d’une conclusion sur le travail. / Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are polymers of nucleotides essential for the survival of the cell. Contrary to DNA, whose main role is to store genetic information, RNA is involved in multiple metabolic processes. For example, RNA is involved in the transfer of information from DNA to protein, the processing and modification of other RNAs, the regulation of gene expression, the end-maintenance of chromosomes, and the sorting of proteins within the cell. This functional versatility of RNA comes from its structural diversity. Our laboratory developed MC-Fold, an algorithm that predicts RNA structures by representing them with nucleotide interaction graphs. The nodes in these graphs represent the nucleotides, and the edges the interactions between them. Our laboratory also observed that a limited number of interaction cycles can define the structure of any RNA motif. The formation of these cycles is determined by the nucleotide sequence and MC-Fold determines the most likely cycles based on that sequence. In this Master Degree project, I first built a database of structural and functional RNA motifs, bdMotifs, based on their constituent cycles. Then, I implemented an algorithm, MC-Motifs, which detects motifs within interaction graphs generated either by MC-Fold or by any other method. Finally, I validated my algorithm on known RNA structures such as the 5S, 16S and 23S ribosomal RNA (rRNA) and predicted structure of riboswitches. The Master thesis is divided into five chapters. The first chapter presents the chemical structure of RNA, its cellular functions and the structural folding of the polymer. In the second chapter, the database bdMotifs is described. In the third chapter, the MC-Motifs algorithm is introduced. In the fourth chapter, I present the results of MC-Motifs. Finally, in the last chapter, I discuss theses results and I give a conclusion on the project.