Copy Number Variants (CNVs) are defined as DNA segments of 1kb or more in length and present in a variable number of copies in the human genome. It has been recently shown that many human genetic diseases including organ malformations are caused by CNVs in a patient's genome. However, the genetic and molecular basis for Renal Agenesis (RA), which is a medical condition whereby unilateral or bilateral fetal kidneys fail to develop, has not yet been extended to CNV studies. By using array-based Comparative Genomic Hybridization, we are analyzing DNA from patients who have RA in order to identify CNVs that are causative for RA; genes within the CNVs will then be assessed for their potential involvement in RA by altering their dose in Xenopus embryos.

Sex determination in mammals is regulated by the sex-determining region on the Y chromosome (SRY); the presence of SRY activates the male developmental pathway and suppresses the gene network necessary for female gonad development. Mutations in sex determination genes lead to various abnormal sexual phenotypes, including sex reversal syndrome in which the genetic and phenotypic sex do not match. Sex reversal syndrome has been reported in humans, mouse, and several domestic species. In horses, SRY-negative XY sex reversal syndrome has been well described and is caused by deletions on the Y chromosome. However, the molecular causes of the SRY-positive condition in horses and other mammals are not known. This research investigated five horses affected with SRY-positive XY sex reversal syndrome. Sequencing of the coding exon region of the SRY gene in the five cases showed 99-100% alignment with the sequences of normal males. Genotyping of two closely related individuals with 46 normal male controls on an equine SNP50 Beadchip identified two statistically significant SNPs in a ~16 Mb region on the long arm of horse chromosome 3 (ECA3q). The region was analyzed using Gene Ontology (GO) and Gene Relationships Across Implicated Loci (GRAIL) to select functionally relevant candidate genes for sequencing. Further analysis of the entire horse genome was done through array comparative genomic hybridization (aCGH), which investigated possible structural rearrangements, such as copy number variants (CNVs). Deletions of olfactory receptor genes were detected on multiple chromosomes and confirmed through quantitative real-time PCR (qPCR). A homozygous deletion on ECA29 in a region containing genes of the aldo-keto reductase gene family, known to play a role in interconverting sex hormones between active forms and inactive forms, was discovered in two sex reversed animals. The findings were confirmed through qPCR and fluorescence in situ hybridization (FISH), and experiments to define the specific breakpoints of the deletion through PCR have been initiated. This research represents the first systematic search in the horse genome for mutations and CNVs related to sex determination. The findings contribute to better understanding of the molecular mechanisms of sex determination in horses and other mammals, including humans.

A endometriose é uma doença ginecológica benigna comum, mas agressiva, caracterizada pela presença de tecido endometrial ectópico. A teoria mais aceita para explicá-la é a teoria de Sampson, na qual o tecido endometrial descamado durante a menstruação sofre refluxo através das tubas uterinas, adere e se prolifera em sítios ectópicos da cavidade peritoneal. Por outro lado, apenas o refluxo tubário não é capaz de estabelecer a doença e vários estudos sugerem uma etiologia multidimensional incluindo fatores hereditários, hormonais e imunológicos. Várias metodologias têm sido propostas com o objetivo de identificar genes candidatos para a endometriose. A hibridação genômica comparativa (CGH) é uma técnica que permite que o genoma inteiro seja analisado em um só experimento, sem a necessidade de cromossomos metafásicos obtidos por cultura celular. Nossa proposta foi avaliar, por CGH, amostras de endometriomas ovarianos e de tecido endometrial eutópico de dez pacientes com diagnóstico firmado de endometriose, para screening do genoma. No grupo eutópico, 6/10 amostras apresentaram alterações caracterizadas por perdas ou ganhos de regiões cromossômicas e no grupo ectópico foram encontradas alterações em 7/10 casos. A presença de perdas e ganhos de regiões cromossômicas no endométrio eutópico, histologicamente normal, de mulheres com endometriose ovariana, pode ser considerada como alteração primária ao desenvolvimento da doença. A metodologia de CGH permitiu a detecção das regiões cromossômicas 11q12.3-q13.1, 17p11.1-p12 e 17q25.3-qter como regiões críticas, direcionando investigações futuras para identificação de genes associados à endometriose. / Endometriosis is a common benign gynecological disease, very aggressive, characterized by the presence of ectopic endometrial tissue. The most accepted theory to explain it is Sampson\'s implantation theory, which says that the endometrial tissue exfoliated during menstruation undergoes reflux through the uterine tubes, adheres and proliferates in ectopic sites of the peritoneal cavity. On the other hand, only reflux is not enough to the establishment of the disease and a number of studies suggest a multidimensional etiology including hereditary, hormonal and immunological factors. Several methodologies have been proposed for the identification of candidate genes for endometriosis. The comparative genomic hybridization (CGH) is a versatile technique that allows the entire genome to be analyzed in only one experiment without the necessity of metaphasic chromosomes from the sample, excluding the cell culture. We aimed to evaluate, by CGH, ovarian endometriomas and eutopic endometrial tissue samples from 10 patients with confirmed diagnosis of endometriosis, for a genomic screening. In the eutopic group, 6/10 samples presented genomic imbalances and 7/10 cases showed alterations in the ectopic group. The presence of losses and gains of chromosomic regions in the histologically normal eutopic endometrium from women with ovarian endometriosis can be considered as a primary alteration in the development of the disease. The CGH methodology allowed the detection of chromosomic regions 11q12.3-q13.1, 17p11.1-p12 and 17q25.3-qter as critical regions, leading to future investigations for the identification of genes associated to endometriosis.

Le cancer est désormais considéré comme une maladie génomique de la cellule. Les moyens d’étude de l’oncogénome étaient basés sur les différentes modalités du caryotype, peu résolutif. L’application des techniques de micromatrices d’oligonucléotides, notamment l’aCGH, a permis une avancée majeure dans la caractérisation des génomes des cancers.La première partie de notre travail a porté sur les lymphomes de Burkitt (LB), caractérisés par une translocation entre un gène d'immunoglobuline et MYC. L’étude portait sur 12 tumeurs primaires et 15 lignées cellulaires. L’aCGH (44K et 244K), concordait avec les cytogénétiques morphologique et moléculaire (FISH) sauf pour les translocations. Plus de la moitié des variations du nombre de copies (<2Mb) étaient des polymorphismes (CNV). Les anomalies pathologiques (CNA) (n=136) intéressaient les régions suivantes : gains 1q, 13q, 7q, 8q, 2p, 11q et 15q ; pertes 3p, 4p, 4q, 9p, 6p, 17p, 6q, 11pterp13 et 14q12q21.3. Vingt régions minimales critiques (MCR) d’une taille varie entre 0.07-71.36 Mb, étaient délimitées. Trois MCR étaient identifiées sur le 1q : 1q21.1q25.2, 1q32.1 et 1q44. La région proximale de 1q21.1q25.2 était le siège d’une amplification, contenant entre autres les gènes BCA2, PIAS, BCL9. L’étude par transcriptome, sur 15 lignées, a démontré la surexpression uniquement de BCL9, remanié dans les LAL B et faisant partie de la voie de signalisation de MYC. Sur la région 11q23.1, le gain intéressait le gène POU2AF1 dont le messager était élevé. La MCR 13q31.3q32.1 était le siège d’une amplification contenant ABCC4, et le polycistron miR17-92. La corrélation des résultats d’aCGH à ceux du transcriptome et du mirnome ont démontré une surexpression du miR17-92 qui contrôle le développement des lymphocytes B et intervient dans la voie de signalisation de MYC. Sur le 9p21.3, la perte emportait le locus p16INK4A/p15INK4B. Le transcriptome avait démontré une sous expression de p15INK4B. Le locus p16INK4A/p15INK4B contrôle les 2 voies majeures, pRB et p53.La seconde partie de notre travail a consisté à étudier 17 tumeurs congelées de carcinomes adénoïdes kystiques (CAK) par aCGH 44K. Les CNA étaient validées par FISH et/ou MLPA. L’expression protéique était étudiée par immunohistochimie. Les pertes excédaient les gains (41 versus 24). La t(6;9)(q23;p22) récurrente dans les CAK était indétectable car équilibrée. Dans un seul cas, le der(6)t(6;9) est probablement présent sur le profil aCGH. Les MCR les plus fréquentes (-6q22 et -6q24) n’incluaient pas 6q23. Treize MCR étaient identifiées. La MCR délétée en 8q impliquait le miR-124A2 qui régule les gènes CDK6 et MMP2. Sur le 9p21.3, le locus p16INK4A/p15INK4B était de nouveau perdu. Des gains isolés étaient observés au niveau des locus CCND1, KIT/PDGFRA/KDR, MDM2 et JAK2. Le gène MDM2, qui était amplifié sous forme de double minutes, est un élément clé de l’axe p16INK4A-ARF-p53.Pour la troisième partie de notre travail nous avons étudié 60 tumeurs primaires d’adénocarcinomes pulmonaires (AD) de non fumeur par aCGH (244K). Dans 50/60 tumeurs, le nombre de MCR était de 14. Cinq MCR contenaient un seul gène (MOCS2, NSUN3, KHDRBS2, SNTG1 et ST18). Une MCR gagnée, 5q35, contenait le gène NSD1. Une amplification, sous forme de HSR et mise en évidence par FISH, intéressait l’oncogène FUS. Une PCR quantitative avait permis de confirmer la surexpression FUS. A notre connaissance, c’est la première étude qui incrimine le gène FUS dans la carcinogenèse de l’AD du non-fumeur. D’autres gènes étaient également impliqués : ARNT, BCL9, CDK4, p15INK4B, EGFR, ERBB2, MDM2, MDM4, MET, MYC, NKX2-1 et KRAS. Un clustering non supervisé avait permis de dégager un groupe avec un gain de MYC ; un autre groupe caractérisé par la perte des gènes suppresseurs RB et WRN et un dernier groupe caractérisé par un gain 7p et 7q, et présentait une fréquence élevée de mutations de l’EGFR. Dans 10/60, le nombre de CNA était très rare et aucune MCR n’était détectée. / Much of our current understanding of cancer is based on the hypothesis that it is a genetic disease, arising as a clone of cells that expand in an unregulated fashion because of somatically acquired mutations. High-throughput tools for nucleic acid characterization, such as array comparative genomic hybridization (aCGH), now provide the means to conduct comprehensive analyses of somatic anomalies in the oncogenome.In the first part of our work we have carried out a fine mapping of additional chromosomal anomalies in Burkitt lymphoma (BL). The hallmark of this disease is the translocation t(MYC;IG). We have applied whole-genome 244K and 44k oligonucléotides aCGH to 15 cells lines and 12 primary tumors of BL respectively. Karyotype and FISH analysis were used to validate aCGH results. As expected, all translocations remained undetectable with aCGH. More than half of the copy number alterations (CNAs) < 2 Mb were mapped to Mendelian CNVs, including GSTT1, and BIRC6. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs, gains were found in 1q, 13q, 7q, 8q, 2p, 11q and 15q. Losses were found in 3p, 4p, 4q, 9p, 13q, 6p, 17p, 6q,11pterp13 and 14q12q21.3. Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q: 1q21.1q25.2, 1q32.1 et 1q44. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2, BCL9 and PIAS3. Only BCL9 high level transcrit was noted on oligonucleotide microarray gene expression that was done on 15 cells lines. BCL9, was implicated in a LAL B translocation t(1;14)(q21;q32) and it is a member of MYC pathway. The 13q31.3q32.1, 89.58–96.81 Mb MCR contained an amplicon with several genes. The miR-17-92 cluster, upregulated on mirnome analysis that was done on 15 cells lines, is the gene driver of 13q MCR. The miR-17-92 cluster is a member of MYC pathway. The 9p21.3 MCR harbored p16INK4A/p15INK4B locus which is downregulated. MYC activates ARF,a protein encodes by p16INK4A/p15INK4B locus. . On the second part of our work, a 44k aCGH was applied on 17 frozen adenoid cystic carcinoma (ACC) to delineate with a high resolution the CNA associated with ACC. aCGH results were validated with FISH and/or MLPA. Protein expression was screened with immunohistochemistry analysis. The translocation t(6;9)(q23;p23p24)/ MYB-NFIB recurrent in ACC, was not detected with aCGH. In one case, the der(6)t(6;9) was suspected in the aCGH pattern. There were recurrent gains at 7p15.2, 17q21–25, 22q11–13, and recurrent losses at 1p35, 6q22–25, 8q12–13, 9p21, 12q12–13, and 17p11–13. Thirteen MCR were detected. The recurrent deletion at 8q12.3–13.1 contained miRN124A2 gene, whose product regulates MMP2 and CDK6. The 9p21.3 MCR harbored p16INK4A/p15INK4B locus which was deleted. On 17p11p13, the MCR contained several genes and TP53 was deleted in 2 cases. The MDM2 gene, a member of p16INK4A-ARF-p53 pathway, was amplified and overexpressed in one case. Among the other unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, and JAK2. On the third part of this these, a high-resolution 244K aCGH was conducted on 60 frozen lung adenocarcinoma (AD) of never smokers patients in order to establish a catalog of CNA. In 50/60 tumors, fourteen new MCR of gain or loss was noted. One larger MCR of gain contained NSD1.One focal amplification and nine gains contained FUS. NSD1 and FUS are oncogenes hitherto not known to be associated with lung cancer. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. A FUS hsr was observed with FISH screening. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. Other cancer genes present in aberrations included ARNT, BCL9, CDK4, p15INK4B, EGFR, ERBB2, MDM2, MDM4, MET, MYC, NKX2-1 and KRAS.

Endometriosis is one of the most common gynecological diseases as it is thought to affect up to 15% of the female population. Characterized by the growth and proliferation of endometrial tissue outside of the uterine cavity, it is a complex condition with varying degrees of severity and can affect multiple regions of the body with symptoms ranging from a total lack of symptoms to debilitating pain and infertility. The most accepted theory of how endometriosis initiates is that of retrograde menstruation; however, approximately 90% of women with unobstructed fallopian tubes are thought to have some menstrual debris in the peritoneal cavity. Therefore, this theory does not explain in full why endometriosis occurs in some but not all women who experience retrograde bleeding. Genetic factors are thought to play a major role in the pathogenesis of endometriosis as women with a family history are 5 to 10 times more likely to develop the disease. The goal of this study was to determine if common chromosomal aberrations in the form of additions, deletions, or regions of loss of heterozygosity that may contribute to the establishment or progression of the disease are present in a population of endometriosis patients. DNA was isolated from the peripheral blood of endometriosis patients and endometriosis tissue biopsies, and it was analyzed using oligonucleotide based array comparative genomic hybridization. The results suggest that an addition on chromosome 17p13.3 may play a role in the biological mechanisms involved in endometriosis as it was identified in 75% of the DNA samples obtained from the peripheral blood and 100% of the DNA samples obtained from the tissue biopsies. This chromosomal imbalance is of particular interest as it is located in a region that harbors the tumor suppressor gene, hypermethylated in cancer-1 (HIC-1), whose aberrant expression has been reported in multiple cancers. Endometriosis has long been thought of as a benign disease despite its malignant characteristics, and individuals with endometriosis have been demonstrated to have an increased chance of developing ovarian cancer. This was the first study to examine the DNA from endometriosis patients using oligonucleotide based array comparative genomic hybridization to investigate genetic abnormalities in endometriosis. The findings may provide a novel target for future therapeutic options as well as indicate a link between endometriosis and cancer that has not been previously reported.

<p>Genetic variation is a natural occurrence in the genome that contributes to the phenotypic differences observed between individuals as well as the phenotypic outcomes of various diseases, including infectious disease and cancer. Single nucleotide polymorphisms (SNPs) have been identified as genetic factors influencing host susceptibility to infectious disease while the study of copy number variation (CNV) in various cancers has led to the identification of causal genetic factors influencing tumor formation and severity. In this work, we evaluated the association between genomic variation and disease phenotypes to identify SNPs contributing to host susceptibility in Staphylococcus aureus (<italic>S. aureus</italic>) infection and to characterize a nervous system brain tumor, known as oligodendroglioma (OD), using the CNV observed in tumors with varying degree of malignancy.</p><p>Using SNP data, we utilized a computational approach, known as in silico haplotype mapping (ISHM), to identify genomic regions significantly associated with susceptibility to <italic>S. aureus</italic> infection in inbred mouse strains. We conducted ISHM on four phenotypes collected from <italic>S. aureus</italic> infected mice and identified genes contained in the significant regions, which were considered to be potential candidate genes. Gene expression studies were then conducted on inbred mice considered to be resistant or susceptible to <italic>S. aureus</italic> infection to identify genes differentially expressed between the two groups, which provided biological validation of the genes identified in significant ISHM regions. Genes identified by both analyses were considered our top priority genes and known biological information about the genes was used to determine their function roles in susceptibility to <italic>S. aureus</italic> infection.</p><p> We then evaluated CNV in subtypes of ODs to characterize the tumors by their genomic aberrations. We conducted array-based comparative genomic hybridization (CGH) on 74 ODs to generate genomic profiles that were classified by tumor grade, providing insight about the genomic changes that typically occur in patients with OD ranging from the less to more severe tumor types. Additionally, smaller genomic intervals with substantial copy number differences between normal and OD DNA samples, known as minimal critical regions (MCRs), were identified among the tumors. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques. </p><p> The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques.</p><p> The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments.</p> / Dissertation

Fortschritte in der molekulargenetischen Charakterisierung von Tumorerkrankungen haben in den letzten Jahren die klinische Praxis zunehmend beeinflusst. Das Ziel dieser Arbeit war die Untersuchung von astrozytären Tumoren der WHO-Grade II bis IV und ihre Subtypisierung anhand der gefundenen chromosomalen Aberrationen. Ferner sollte der Einfluss der gefundenen Aberrationen auf klinische Parameter wie das Gesamtüberleben oder die rezidivfreie Zeit untersucht werden. Dazu wurden paraffinfixierte Proben von insgesamt 184 primären astrozytären Tumoren (28 low-grade Astrozytome, 6 low-grade Oligoastrozytome, 50 anaplastische Astrozytome, 4 anaplastische Oligoastrozytome, 96 Glioblastoma multiforme) mit der Comparativen Genomischen Hybridisierung (CGH) untersucht. Häufige Aberrationen in allen Malignitätsgruppen stellten chromosomale Zugewinne auf Chromosom 7 sowie Verluste von Chromosom 10 und 9p dar. High-grade Astrozytome zeigten ferner häufig Zugewinne von Chromosom 19 und 20 sowie Verluste von 13q, 14q und 15q. WHO-Grad-II-Astrozytome wiesen häufig Zugewinne auf Chromosom 8 sowie Verluste von Chromosom 4q und 6q auf. Eine kürzeres Gesamtüberleben zeigten high-grade Gliome mit Verlusten von Chromosom 10q und Zugewinnen auf 7p. In Glioblastomen verursachten zusätzlich Zugewinne auf 7q sowie Verluste von 14q, in anaplastischen Astrozytomen zusätzlich Verluste von 10p ein verringertes Gesamtüberleben. WHO-Grad-II-Astrozytome zeigten bei Verlusten von 3p ein schlechteres Gesamtüberleben. Chromosomale Aberrationen, die zu einem verlängerten Gesamtüberleben führten, waren Verluste von 1p und Zugewinne von 10p in WHO-Grad-III-Tumoren. Die rezidivfreie Zeit wurde in high-grade Gliomen durch Zugewinne auf 7p und Verluste von 10p verringert. Eine Verkürzung der rezidivfreien Zeit in Glioblastomen zeigten außerdem Tumoren mit Verlusten von 7q, 10q und 14q. In anaplastischen Astrozytomen führten Verluste von 1p und 19q sowie Zugewinne auf 8q und 10p, in WHO-Grad-II-Astrozytomen Verluste von Chromosom 6 zu einer verlängerten Zeit bis zum Rezidiv. Die Anfertigung onkogenetischer Baummodelle stellte verschiedene genetische Wege der Tumorgenese dar. Ein Cluster war gekennzeichnet durch einen Verlust von 6q, ein weiterer wurde initialisiert durch den Verlust von 13q, der dritte durch den Verlust von 9p. Der vierte Cluster wurde charakterisiert durch Zugewinne auf Chromosom 7 und Verluste von Chromosom 10, während der fünfte Cluster Zugewinne auf 8q sowie Verluste von 4q aufwies.

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Thesis--University of Oklahoma. / Includes bibliographical references.