Dissertations / Theses on the topic 'Medical genetics (excl. cancer genetics)'
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Peterson, Kristen N. "Investigating the Role of Bptf in Immunoediting in Breast Cancer and Melanoma." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3793.
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In this study, we explore the effects of NURF depletion on the growth of tumors in immune-competent mice. NURF depletion in tumors results in reduced tumor growth in immune-competent mice, suggesting enhanced anti-tumor immunity. Analysis of the tumor microenvironment by flow cytometry revealed a significantly elevated CD8 and progressively elevated activated CD8 phenotype in Bptf KD tumors, possibly contributing to the increase in cell death and decrease in tumor weight observed. Examination of antigen presentation was evaluated using the OT-1 and Pmel-17 models, though no significant difference in cytotoxicity was observed as measured by LDH and/or IFNγ assays. This indicates possible novel antigen presentation mechanisms in tumor cells, and not increased presentation of existing antigens, contributes to the decreased tumor weight observed in Bptf KD tumors.
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Whitmore, Scott Anthony. "Positional cloning of genes associated with human disease /." Title page, contents and summary only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phw616.pdf.
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Thesis (Ph.D.) -- University of Adelaide, Dept. of Cytogenetics and Molecular Genetics, 1999.Copies of author's previously published articles inserted. Amendments pasted onto back-end paper. Bibliography: leaves 255-286.
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Davis, Hayley Louise. "Functional analysis of cancer-causing FBXW7 mutations." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:9c1b7f72-0733-439f-919a-6c66f7f44bfc.
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FBXW7 encodes the substrate recognition component of an SCF E3 ubiquitin ligase complex. This complex regulates the degradation of multiple targets, such as Notch1, c-Jun, c-Myc and cyclin E, that function in critical developmental and cancer pathways. FBXW7 mutations are found in cancers of diverse tissue origins, with one of the highest mutation rates in the colorectrum. FBXW7 mutations are typically missense mutations that disrupt the substrate recognition domain at critical arginine propellor-tips. Mutations are often mono-allelic suggesting that FBXW7 is not a typical tumour supressor gene. Despite this, most of the evidence on FBXW7 function to date comes from null systems. Several Fbxw7 -null mouse models have been generated and suffer homozygous embryonic lethality due to disrupted vascular development. Conditional Fbxw7-null mice have been created but do not in general reflect the mutation spectrum found in human tumours. In order to analyse the functional effects of Fbxw7 propellor-tip missense mutations, mice carrying a commonly-occurring Fbxw7 R482Q mutation were generated. This propellor-tip mutation was knocked-in constitutively and whilst heterozygous mice developed normally in utero, they died perinatally due to defective lung development. Cleft palate and eyelid fusion defects were also observed with incomplete penetrance. Fbxw7 substrates were screened in embryonic lungs and significantly elevated protein levels of Klf5 and Tgif1 were observed. The Fbxw7 R482Q mutation was also conditionally knocked-in in the gut. In the heterozygous state, large adenomas in the small intestine were observed at a low multiplicity, in approximately 30% of mice at an age greater than 300 days. Upregulation of Wnt signalling and Ctnnb1 mutations have been identified in a selection of these tumours. Breeding the Fbxw7R482Q allele onto Apc-mutant backgrounds led to accelerated morbidity, in which compound R482Q/Apc-mutant mice exhibited polyps of increased number and size. Elevated protein levels of Fbxw7 substrates Klf5 and Tgif1 were observed in adenoma and normal intestinal tissue from these mice. In vitro work using epitope-tagged murine wildtype and propellor-tip mutant Fbxw7 proteins showed that they were capable of dimerising, opening the prospect of investigating a dominant negative mechanism of action. To conclude, an Fbxw7 propellor-tip mutation studied in vivo resulted in both disrupted embryonic development and intestinal tumorigenesis and was distinct from Fbxw7 -null alleles.
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Ivansson, Emma. "Contribution of Immunogenetic Factors in Susceptibility to Cervical Cancer." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9552.
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Cervical cancer is the second most common cancer in women worldwide. Persistent infection by an oncogenic type of human papillomavirus (HPV) is a necessary but not sufficient cause and there is also a genetic component. This thesis aims to identify host genetic risk factors for cervical cancer based on the hypothesis that susceptibility is affected by genetic variation in the immune response towards HPV infection. Paper I analyzed allergy in sons and cervical cancer in their mothers, and revealed an inverse association between cervical cancer and allergy across generations. Mothers of allergic sons have a lower incidence of cervical cancer, supporting the importance of immunogenetic factors. Paper II investigated the HPV type in 1079 women diagnosed 1965-1993. All women were from families with at least two affected. It appeared that HPV 16 was becoming less common with time. There was no evidence that related women were prone to infection by the same type, indicating that the immunogenetic factors act in a general, rather than an HPV type specific, manner. Paper III and IV analysed the association of candidate genes with susceptibility to cervical cancer in 1306 women with cervical cancer in situ and 288 unrelated controls. Paper III showed the association of variation in the two immune response genes chemokine receptor 2 (CCR-2) and interleukin 4 receptor (IL-4R) with cervical cancer. In paper IV variation at several loci in the MHC region was studied and the importance of the HLA class II locus DQB1 emphasized. This thesis work supports the contribution of genes of the immune system to cervical cancer susceptibility. The genetic risk factors so far identified account for only a part of the genetic susceptibility, which implies that other yet undiscovered variants of importance remain to be identified.
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Dursun, Ahmet. "The molecular pathologies of BRCA1 in ovarian cancer patients from the west of Scotland." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368585.
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Cheng, Timothy. "Genetic susceptibility to endometrial cancer." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:3a559ae0-156f-48a2-a64e-b03a13c562df.
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Endometrial cancer (EC) is the fourth most common cancer affecting women in the UK. Those with a family history of EC have an increased risk compared with the general population. Highly penetrant germline mutations in mismatch repair (MMR) genes and DNA polymerases account for only a small proportion of the familial aggregation. The aim of this thesis is to investigate the genetic susceptibility to EC in the general population using cases and controls of European ancestry. A GWAS meta-analysis totalling 7,737 EC cases and 37,144 controls yielded five novel EC risk loci of genome-wide significance (P < 5x10−8). In decreasing order of significance, these were at chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). A second independent EC signal was found in the 8q24 locus. The association found in a previous EC GWAS at HNF1B on chromosome 17 was replicated at a higher significance, with the most significant SNP being rs11263763. CYP19A1 SNPs have previously been associated with EC and higher circulating levels of oestrogen from candidate studies, but I confirmed this locus to be genome-wide significant for the first time. Functional annotation and in vitro studies for the EC risk loci at the intergenic region of chromosome 13q22 suggested that the functional SNP sits within a transcriptional repressor for KLF5, with the higher-risk allele reducing repressor activity. The propensity for germline MMR and DNA polymerase muations to cause both EC and colorectal cancer (CRC) prompted me to search for common variants associated with both cancer phenotypes. An EC CRC GWAS meta-analysis showed little evidence of shared susceptibility loci. However, this meta-analysis revealed a novel genome-wide significant risk locus: rs3184504, a missense SH2B3 SNP that has not previously been associated with either EC or CRC. This thesis has enhanced the understanding of genetic susceptibility to sporadic EC and increased the number of genome-wide EC-associated variants to seven.
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Quinn, Bridget A. "Novel Therapeutic Strategies for Pancreatic Cancer." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/4671.
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Pancreatic cancer is a devastating disease that leaves patients with a very poor prognosis and few therapeutic options. Many of the treatment options available are the same that have been used for almost 2 decades. There is a dire need for both novel treatments for this disease as well as novel strategies of treatment. This body of work will introduce and provide evidence in support of a novel combination therapy for pancreatic cancer treatment, a novel strategy of modifying currently used chemotherapeutics for pancreatic cancer therapy, and a novel transgenic preclinical mouse model of pancreatic cancer. Sabutoclax, an antagonist of the anti-apoptotic Bcl-2 proteins, and Minocycline, a commonly used antibiotic, show potent synergy when used in combination in both pancreatic cancer cells and in multiple immune-deficient and immune-competent mouse models of pancreatic cancer. Sabutoclax alone is capable of inducing cell cycle arrest and apoptosis in cells and its cytotoxicity is enhanced significantly when combined with Minocycline. This combination results in the loss of Stat3 activation both in vitro and in vivo, which is essential for its toxicity. It also inhibits tumor growth and prolongs survival in the KPC transgenic mouse model of pancreatic cancer. Also presented here are studies that demonstrate efficacy in vivo of modified versions of Gemcitabine and Paclitaxel. These drugs are linked to a peptide that shows specificity for the EphA2 receptor, which is overexpressed on the surface of pancreatic cancer cells and only minimally on normal cells. This peptide results in increased cellular uptake of drug, as it is bypassing its normal mechanism of entry. These normal mechanisms are often dysregulated in cancer, leading to decreased uptake and drug resistance. The use of these modified drugs show significantly increased tumor growth inhibition as compared to the parent drug alone. Finally, we provide data on the characterization of a novel transgenic mouse model of pancreatic cancer. This model, the Pan Met View (PMV) mouse, combines the commonly used KPC transgenic mouse model of pancreatic cancer and a mouse that expresses a Luciferase reporter gene under the control of the cancer-specific promoter, CCN1. Our data shows that double transgenic PMV mice can now be used to follow primary tumor and metastasis development in real time by Bioluminescent imaging (BLI) through disease progression and potentially therapy. This strategy will enhance the use of genetically engineered mouse models (GEMMS) to study cancer initiation and progression with potential to non-invasively monitor therapy. These chapters present novel and exciting data that have the potential to open multiple avenues of translational study and result in significant advances in pancreatic cancer therapy.
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Österberg, Lovisa. "Characterization of genetic alterations in ovarian cancer associated with chemotherapy response /." Göteborg : Department of Oncology, Institute of Clinical Sciences, The Sahlgrenska Academy at University of Gothenburg, 2009. http://hdl.handle.net/2077/20291.
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LI, CHAO. "HEAT SHOCK PROTEINS AS NOVEL CANCER THERAPEUTICS: TARGETING THE HALLMARKS OF CANCER." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2510.
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Molecular chaperones, commonly known as heat shock proteins (HSPs), are essential for mammalian cells to maintain homeostasis, and HSPs function by inducing an ATPase-coupled structural change, followed by interactions with diverse co-chaperones and over 200 client proteins implicated in many critical signaling networks. These highly expressed HSPs participate in the onset and progression of several human diseases including cancer, and their connection with tumorigenesis has facilitated research and clinical trials related to targeting HSPs as a novel anti-tumor therapy. The predominant mechanism of chaperone inhibition is through either disruption of the HSP association with client protein or an altered binding state that ultimately leads to proteasome-mediated degradation. Importantly, chaperone inhibition results in the degradation of several client proteins that play critical roles in many of the pathways known as the Hallmarks of Cancer, such as proliferation, angiogenesis, invasion, metastasis, and drug resistance. Here, we discuss: (1) the current knowledge of HSPs, particularly studies related to Hsp90-targeted cancer therapy, (2) the targeting of Hsp90-mediated signaling interactions to prevent emergence of core Hallmarks of Cancer, (3) the recent progression of Hsp90 inhibitors in clinical trials. Finally, we propose combinatorial therapy, additional inhibitor discovery, and location-specific inhibition of HSPs as necessary next steps in chaperone-targeted research relevant to cancer therapy.
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Zaro, Maren Lothyan. "Breast Cancer Risk Assessment: Evaluation of Screening Tools for Genetics Referral." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/8824.
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Purpose: This study assessed effectiveness of five tools recommended by the US Preventive Services Task Force (USPSTF), designed to help primary care clinicians determine which unaffected patients to refer to genetics specialists for breast cancer risk assessment based on concerning family history. Design: This descriptive secondary analysis included 85 women aged 40-74. All participants had a first-degree female relative previously diagnosed with breast cancer who also had uninformative negative BRCA1/2 tests. Methods: Each pedigree was evaluated using the five tools including the Family History Screen-7 (FHS-7), Pedigree Assessment Tool (PAT), Manchester Scoring System, Referral Screening Tool (RST), and Ontario-Family History Assessment Tool (Ontario-FHAT). All five tools were applied to each study participant. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated to describe each tool’s ability to identify women with elevated risk as calculated by the Claus model. Receiver operating curves (ROC) were also plotted. Differences between areas under the curve (AUCs) for all possible pairs of tools were estimated through logistic regression to assess for differences in tool performance. Results: Claus calculations identified 14 women out of 85 whose lifetime risk of breast cancer was elevated at > 15%. Only two tools, the Ontario-FHAT and FHS-7, identified all 14 women with elevated risk, a sensitivity of 100%. The FHS-7 tool flagged all 85 participants, meaning its specificity was zero. The Ontario-FHAT flagged 59 participants as needing referral (specificity 36.2%) and had a negative predictive value (NPV) of 100%, indicating that if a woman was not found to need a referral to a genetics professional, it is likely she did not have an elevated lifetime risk of developing breast cancer. AUC values were not significantly different between tools (all p values > .05), and thus were not helpful in discriminating between the tools. Conclusion: In this population, the Ontario-FHAT out-performed other tools in terms of sensitivity and negative predictive value; however, low specificity and positive predictive value must be balanced against these findings. Thus, the Ontario-FHAT can help determine which women would benefit from referral to a genetics specialist.
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Alhareeri, Areej. "Chromosome-Specific Telomere Length in Women with Breast Cancer: Their Relationship to Chemotherapy and Acquired Psychoneurological Symptoms." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/475.
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Breast cancer (BC) is one of the most common diagnosed malignancies in females. Although 90% of early diagnosed women are expected to survive for at least 5 years, their quality of life is adversely affected by a cluster of symptoms which we collectively named “psychoneurological symptoms’’ (PN). Given that acquired telomere attrition has been speculated to be a causal factor in chronic diseases and the lack in the literature of mechanisms giving rise to PN symptoms, this study was performed to assess telomere length using a chromosome-specific telomere assay before receiving chemotherapy and at the first chemotherapy. We showed significant telomere attrition on the short arm of chromosome 9. In addition, we showed a negative correlation between telomere length and depression. Furthermore, we evaluated several variables as predictors of the change in telomere length and showed that chemotherapy was predictive of shortened telomere length. Taken together, one can speculate that shortened telomeres could result in epigenetic alterations in the genes juxtaposed to the telomeric region, giving rise to the development and persistence of PN symptoms. Knowledge gained from this study will offer hope for the development of therapeutic interventions that could prevent undesirable side effects and ensure a better quality of life for patients with BC.
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Jones, Matthew. "MiR-215 regulates differentiation in colorectal cancer stem cells." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:5462857b-4cee-44a8-a299-63b798a67f3f.
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Since the initial description of cancer stem cells (CSCs) as a self-renewing subpopulation of malignant cells with tumor-initiating capacity, a growing body of evidence has supported the existence of CSCs in virtually every tumor type. Our previous work in colorectal cancer has identified the transcription factor CDX1 as a key regulator of colorectal CSC differentiation. CDX1 expression is frequently lost in colorectal cancer, resulting in more aggressive, poorly differentiated tumors with higher proportions of CSCs. Many miRNAs have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in colorectal cancer, remain poorly understood. We began by identifying miRNAs downstream of CDX1 by using high-throughput small-RNA sequencing to profile miRNA expression in two pairs of colorectal cancer cell lines with stable CDX1 overexpression or knockdown. Validation of candidates identified by RNAseq in a larger cell line panel revealed miR-215 to be most significantly correlated with CDX1 expression. ChIP-qPCR and promoter reporter assays confirmed that CDX1 directly transactivates miR-215 transcription. MiR-215 is depleted in FACS-enriched CSCs compared to unsorted samples. Overexpression of miR-215 in poorly-differentiated, highly clonogenic cell lines causes growth arrest and a dramatic decrease in colony formation. miR-215 knockdown using a miRNA sponge causes an increase in clonogenicity and impairs differentiation in CDX1-high cell lines. Indeed, the effects of CDX1 expression on both gene expression and colony morphology can be attenuated by miR-215 inhibition, indicating that miR-215 is a functional mediator of CDX1. Microarray studies following miR-215 overexpression indicate that miR-215 induces terminal differentiation-associated growth arrest, due in part to direct silencing of BMI1 expression and de-repression of BMI1 target genes including CDKN1A. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in colorectal cancer. We further characterize another miRNA-transcription factor axis in colorectal cancer, and we identify the novel miR-3189-3p as a potent effector of cell death with potential therapeutic implications.
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Zeron-Medina, Cuairan Jorge. "The identification and characterisation of germline genetic variants that affect human cancer." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:8942602e-c0f8-4793-8020-d2eadd41b252.
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Single nucleotide polymorphisms (SNPs) have great potential to serve as important biomarkers in the clinic to identify those at increased risk for developing cancer, progressing more rapidly, and not responding to therapies. However, the clinical application of cancer-associated SNPs has proven to be more complicated than expected. One of the necessary steps will certainly be the description of the molecular and cellular mechanisms behind the observed associations. The p53 tumour suppressor pathway harbours well-described SNPs that affect p53 signalling and cancer. The aim of the work presented in this thesis was to utilise this knowledge to more efficiently characterise cancer-associated SNPs. Firstly, cancer-associated SNPs in a p53 network gene, CD44, were studied. Specifically, based on CD44’s known roles in both p53-dependent and independent signalling, it was predicted that the cancer-associated SNPs could function as biomarkers for chronic lymphocytic leukaemia progression, and for the response to anti-EGFR therapy for colorectal cancer. Indeed, supportive data is presented. Next, a methodology is presented that aims to identify cancer-associated SNPs in functional p53 binding sites using genome-wide datasets. Interestingly, a SNP is identified that dramatically influences the ability of p53 to regulate transcription of the KITLG oncogene and that associates with one of the largest risks of cancer identified to date. Intriguingly, the SNP is also shown to have undergone positive selection throughout human evolution, signifying a selective advantage, but similar SNPs are demonstrated to be rare in the genome due to negative selection, indicating that polymorphisms in p53 binding sites have been primarily detrimental to humans. Lastly, and in order to begin to explore if other polymorphic transcription factor binding motifs could be found in cancer-associated SNPs, a methodology was designed to identify SNPs in E-box transcription factor binding motifs, as they are sensitive to single base pair changes and affect cancer. Taken together, the work presented in this thesis shows how the study of how SNPs associate with, and impact upon, cancer has great potential to improve both biological knowledge and clinical outcomes.
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Rosmarin, Daniel Norris. "Germline determinants of 5-fluorouracil drug toxicity and patient survival in colorectal cancer." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:d5e2c306-689c-4c53-b4c3-2c1001b04ec6.
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Despite a decade of publications investigating the effect of germline polymorphisms on both toxicity related to treatment with 5-fluorouracil-based (5-FU) chemotherapy and prognosis following diagnosis with colorectal cancer (CRC), few genetic biomarkers have been identified convincingly. For 5-FU toxicity and CRC prognosis, in four results chapters, this thesis aims to validate previously-reported genetic biomarkers, identify new markers, determine the mechanistic basis of associated polymorphisms, and expand upon methods in the field. The first three results chapters investigate genetic biomarkers for the prediction of toxicity caused by 5-FU-based treatment, particularly for the 5-FU prodrug capecitabine (Xeloda®, Roche). In the first, a systematic review and meta-analysis is performed for all variants that have been previously studied for an association with toxicity caused by any 5-FU-based drug regimen. 16 studies are analysed, including 36 previously-studied variants. Four variants show strong evidence of affecting a patient’s risk of global (any) 5-FU-related toxicity upon analysis of both the existing data and over 900 patients from the QUASAR2 trial of capecitabine +/- bevacizumab (Avastin®, Roche/Genentech): DPYD 2846, DPYD *2A, TYMS 5’VNTR and TYMS 3’UTR. Next, 1,456 polymorphisms in 25 genes involved in the activation, action or degradation of 5-FU are investigated in 1,046 patients from QUASAR2. At a Bonferroni-corrected p-value threshold of 3.43e-05, three novel associations with capecitabine-related toxicity are identified in DPYD (rs12132152, rs7548189, A551T) and the previously-identified TYMS 5’VNTR and 3’UTR toxicity polymorphisms are refined to a tagging SNP (rs2612091) downstream of TYMS and intronic to the adjacent ENOSF1, the latter of which appears to be functional. Finally, a genome-wide investigation of 4.77 million directly genotyped or imputed SNPs identifies one variant (rs2093152 on chr20) as significantly associated with capecitabine-related diarrhoea (p<5e-08), though no associations meet this threshold for global toxicity. In the study of CRC prognosis, a severe left truncation to the VICTOR trial is defined and shown to probably reduce statistical power but not bias effect estimates. Applying standard and novel genome-wide analysis approaches, a set of 43 SNPs are prioritised for future work. With over one million new CRC cases annually, this work helps define biomarkers that could become broadly applicable in the clinical setting.
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Pliuskys, Laurynas. "Epigenetic regulation of the myeloid cell lineage." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f4ee6659-ce0b-4730-ae5b-95c141f82e10.
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The myeloid cell lineage is a fundamental element of the immune system and it can give rise to a diverse set of terminally differentiated cells, such as macrophages or osteoclasts among many others. Mutations or misregulation of gene expression may lead to severe clinical conditions, such as arthritis, osteoporosis or cancers. Epigenetics, the regulation of gene expression and chromatin remodelling, is implicated in cell differentiation, function and disease, and hence it is a promising new area to explore in order to explain underlying cellular mechanisms. Firstly, human macrophage subtypes were studied. Chemokine (C-C motif) ligand (CCL) 1 and mannose receptor were validated to be granulocyte macrophage (GM) colony stimulating factor (CSF) induced macrophage markers, while CCL2 was specifically expressed in macrophage CSF (MCSF) macrophage population. By utilising publicly available high-throughput sequencing data, new biomarkers dehydrogenase/reductase (SDR family) member 2 and CCL26 were discovered to be MCSF-macrophage specific while guanylate binding protein 5 and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A were highly up-regulated in GMCSF cells. Secondly, a range of gene knock-down techniques for the myeloid cell lineage were optimised and established. Lentiviral short-hairpin RNA (shRNA) delivery methods were shown to induce an undesirable pro-inflammatory response in macrophages. Furthermore, the frequently utilised cytomegalovirus promoter for gene expression was shown to be completely silenced in macrophage populations. Locked nucleic acids were selected as a suitable alternative to shRNA knock-down and by employing this new tool it was shown that a histone demethylase lysine (K)-specific demethylase (KDM) 6B is fundamental for macrophage differentiation. Finally, a small molecule GSK-J4, a potent inhibitor of histone demethylases KDM6A, KDM6B and KDM5B specific for H3K27me3 and H3K4me3, respectively, was used to dissect epigenetic signalling in osteoclasts and multiple myeloma. In osteoclasts it was shown to act mainly by inhibiting transcriptional changes required for osteoclastogenesis when MCSF-macrophages are stimulated with Receptor Activator Of Nuclear Factor Kappa-B Ligand (RANKL), as indicated by the differential increase in H3K27me3 marks, leading to inhibition of c-Jun and potentially abolition of transcription factor AP-1, required for the transcriptional initiation of nuclear factor of activated T-cells 1 (NFATc1). In multiple myeloma cells, GSK-J4 causes a dramatic increase in expression, further supported by the build-up of global H3K4me3 marks, which results in the upregulation of the unfolded protein response pathway. In both cell systems, there is an early upregulation of metallothionein genes, which in multiple myeloma was shown to increase potentially due to rapid influx of zinc ions within the first 30 minutes, and as such may cause induction of apoptosis in multiple myeloma and may inhibit differentiation of osteoclasts.
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Glassberg, Andrea E. "Genetic testing for susceptibility to breast and ovarian cancer : a case study of clinical decision-making in medical genetics /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/10308.
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Diehl, Malissa. "CHAPERONE EXPRESSION AND EFFECTS OF ITS INHIBITION ON BREAST CANCER SENSITIZATION." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1897.
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Breast cancer is one of the most prevalent and deadly forms of cancer in women and is not restricted by race or ethnicity. Although a wealth of knowledge has been amassed on the biology of breast cancer, including its risk factors, diagnosis, prognosis, prevention, and treatment, it remains a serious health concern and active area of research. Initial response to standard chemotherapeutic and radiotherapeutic regimens is generally strong for many patients, yet breast tumors often recur, leading to more aggressive and resistant tumors. Because recurrence is such a clinical issue, more effective therapeutic approaches are needed to eliminate partial clinical responses and undesirable side effects. Molecular chaperones like the heat shock protein 90 (Hsp90) family are regarded as ubiquitous, highly conserved proteins that mainly respond upon induction of stress or disruption in cellular homeostasis. Chaperones are critically involved in controlling the conformation, stability, function, and degradation of many oncogenic client proteins by assisting in trafficking, remodeling of improperly folded client proteins, and suppression of protein aggregation. Hsp90-mediated folding events are an ATP-dependent process that involves the association with various co-chaperones and stimulators, including Hsp70, Hsp40, HOP, p23, and Aha1 for client stabilization and modification. Notably, Hsp90 seems to be particularly associated with cell signaling clientele, such as nuclear hormone receptors, protein kinases, and many other oncogenic proteins that directly influence the hallmarks of cancer. In order to define the role of chaperones in breast cancer progression, we assessed chaperone expression levels in normal and tumor lines. Based on our initial finding of increased expression of Hsp90 and p23 in immortal and cancer cell lines, it is possible that this phenomenon may be reflected in normal breast tissue as well as breast carcinoma specimens. Indeed, we observed high Hsp90 expression in invasive carcinomas, such that high nuclear expression correlates with a greater TNM stage, while high cytoplasmic Hsp90 correlates with ER negativity, suggesting that elevated Hsp90 may be an indicator or marker of advanced disease. p23 expression also increases appreciably in established breast cancer cell lines with invasive breast tissue specimens displaying an opposite trend. Although p23 does not appear to have any relationship with TNM stage, significant relationships with ER and PR emerged, with higher nuclear p23 correlating to ER positivity and higher total p23 being positively associated with PR presence. Due to the differential expression of Hsp90 in normal, DCIS, and invasive breast carcinomas, we determined the impact on Hsp90 function, finding that total Hsp90 in tumor cells is associated with an increase in both complexed and uncomplexed Hsp90, which is in contrast to a previously reported study. The intrinsic nature of heat shock proteins makes them especially relevant to a cell’s defense against cancer initiation. The preferential accumulation of Hsp90 in cancer cells also forms the basis for the unique sensitivity of tumor cells to Hsp90 inhibition. The Hsp90 specific inhibitor, radicicol, is more potent in terms of blocking ATPase activity than other Hsp90-related compounds like geldanamycin, which is much more toxic. All Hsp90 inhibitors prevent the association of the co-chaperone p23 with Hsp90, resulting in destabilization of the client protein. For these reasons, it may be possible that Hsp90 inhibition would sensitize breast cancer cells to be more responsive to standard chemotherapeutics. We determined that radicicol negatively alters cellular proliferation, and in combination with Adriamycin, elicits a more robust decline in growth and the expression of Hsp90 client proteins. This finding was associated with an increase in senescent cells without a detectable affect on apoptosis. Radicicol in combination with cisplatin or Taxol contributed to an increase in cell death (apoptosis) and differentially altered the expression of client proteins. Finally, ER negative breast cancer cells do not display altered p53 expression upon radicicol and Adriamycin treatment. Blockade of ER activity in ER positive cells with tamoxifen induced significant reductions in proliferation and decreased p53 expression without a corresponding decrease in p21 levels. In conclusion, these results point to the utility of Hsp90 inhibition as a valid form of targeted therapy for breast cancer, and the value of radicicol as a potential adjuvant treatment option in combination with standard chemotherapeutics.
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Alhazmi, Aiman. "Role of Nucleosome Remodeling Factor (NURF) in Tumorigenesis Using a Breast Cancer Mouse Model." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/379.
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Understanding the impact of epigenetic mechanisms on tumorigenesis is essential, as epigenetic alterations are associated with tumor initiation and progression. Because epigenetic changes are reversible, they are potential targets for cancer therapy. Nucleosome Remodeling Factor (NURF) is a chromatin-remodeling complex that regulates gene expression by changing nucleosome positioning along the DNA sequence. Previous studies have shown a role for NURF in embryonic development as well as regulating genes involved in tumor progression. In this work we investigated the impact of eliminating NURF function in tumorigenesis in vivo. BALB/c mice challenged with syngeneic 67NR breast cancer cell lines, injected into the mammary fat pad, lacking NURF, due to knockdown of its essential subunits Bptf, showed reduction in tumor growth comparing to control tumors. The observed reduction in tumor growth was abrogated in immunodeficient mice lacking a functional immune system. Bptf KD and control 67NR cells grew at similar rates in vitro. Similar findings were observed in our lab using 66cl4 breast cancer cell lines. Using immunofluorescence staining, no significant difference in CD8+, CD4+, NK and MDSC cells infiltrations into the tumor microenvironment was observed in 66cl4 tumors. Preliminary results from 67NR tumors suggested more CD4+ and CD8+ cells. Gene expression profile of tumor tissues from BALB/c mice injected with 67NR and 66cl4 cell lines showed enrichment of genes associated with immune response. Our findings suggested a role of the immune system in targeting tumor cells lacking Bptf in vivo.
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Grawenda, Anna Maria. "The identification and analysis of molecular biomarkers in the p53 tumour suppressor pathway that affect cancer progression in humans." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:5a76b7ca-22f6-4f49-b715-5ad43f916984.
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The tumour suppressor p53 is at the centre of the signalling pathway that controls cellular processes crucial in tumourogenesis, cancer progression and tumour clearance. Alterations in the p53 pathway that lead to cancer progression can be good candidates for molecular biomarkers that would assist in the identification of patients with different prognoses, but also serve as good predictors of appropriate targeted therapies. Patient cohorts and cancer cell panels are utilised to seek associations with the attenuation of the p53 pathway and cancer progression. Firstly, the alternatively spliced transcript of the p53 inhibitor HDMX, which is frequently found in tumours with poor prognosis, is studied. The high ratio of the alternatively spliced HDMX-S transcript over the full-length HDMX-FL transcript (HDMX-S/FL) is demonstrated to associate with p53 pathway attenuation in cancer cells and breast carcinomas, and with faster metastatic progression of osteosarcoma and breast cancer patients. Secondly, inherited polymorphism in the HDMX gene is investigated and demonstrated as a unique and highly reproducible eQTL, which identifies patients with different prognoses for metastatic disease in breast cancer and melanoma cohorts. Lastly, a screening approach to identify novel inherited polymorphisms in the p53 pathway genes that associate with metastatic progression of melanoma is developed and implemented, and subsequently in silico and in vitro functional analyses are performed to investigate a mechanism behind the FOXO3 SNP, identified as the strongest candidate, whereby the experimental evidence demonstrate that the causal SNP in the FOXO3 haplotype is controlled by the GATA3 transcription factor. Together, the work presented in this thesis provides strong support for the role of the p53 pathway in the metastatic progression of cancer, and suggests that molecular biomarkers that can detect changes in the activity of p53 pathway genes could offer a robust set of biomarkers for cancer progression applicable to different types of cancer.
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Nord, Helena. "Application of Genomic and Expression Arrays for Identification of new Cancer Genes." Doctoral thesis, Uppsala universitet, Genomik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-121957.
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Copy number variation (CNV) comprises a recently discovered kind of variation involving deletion and duplication of DNA segments of variable size, ranging from a few hundred basepairs to several million. By altering gene dosage levels or disrupting proximal or distant regulatory elements CNVs create human diversity. They represent also an important factor in human evolution and play a role in many disorders including cancer. Array-based comparative genomic hybridization as well as expression arrays are powerful and suitable methods for determination of copy number variations or gene expression changes in the human genome. In paper I we established a 32K clone-based genomic array, covering 99% of the current assembly of the human genome with high resolution and applied it in the profiling of 71 healthy individuals from three ethnic groups. Novel and previously reported CNVs, involving ~3.5% of the genome, were identified. Interestingly, 87% of the detected CNV regions overlapped with known genes indicating that they probably have phenotypic consequences. In papers II through IV we applied this platform to different tumor types, namely two collections of brain tumors, glioblastoma (paper II) and medulloblastoma (paper III), and a set of bladder carcinoma (paper IV) to identify chromosomal alterations at the level of DNA copy number that could be related to tumor initiation/progression. Tumors of the central nervous system represent a heterogeneous group of both benign and malignant neoplasms that affect both children and adults. Glioblastoma and medulloblastoma are two malignant forms. Glioblastoma often affects adults while the embryonal tumor medulloblastoma is the most common malignant brain tumor among children. The detailed profiling of 78 glioblastomas, allowed us to identify a complex pattern of aberrations including frequent and high copy number amplicons (detected in 79% of samples) as well as a number of homozygously deleted loci. These regions encompassed not only previously reported oncogenes and tumor suppressor genes but also numerous novel genes. In paper III, a subset of 26 medulloblastomas was analyzed using the same genomic array. We observed that alterations involving chromosome 17, especially isochromosome 17q, were the most common genomic aberrations in this tumor type, but copy number alterations involving other chromosomes: 1, 7 and 8 were also frequent. Focal amplifications, on chromosome 1 and 3, not previously described, were also detected. These loci may encompass novel genes involved in medulloblastoma development. In paper IV we examined for the presence of DNA copy number alterations and their effect on gene expression in a subset of 21 well-characterized Ta bladder carcinomas, selected for the presence or absence of recurrences. We identified a number of novel genes as well as a significant association between amplifications and high-grade and recurrent tumors which might be clinically useful. The results derived from these studies increase our understanding of the genetic alterations leading to the development of these tumor forms and point out candidate genes that may be used in future as targets for new diagnostic and therapeutic strategies.
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Gaynor, Katherine Ursula. "The role of the transcription factor GATA3 in calcium homeostasis and tumourigenesis." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:cc1cef5c-896d-438d-8b40-684ceb5804e7.
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Almanza, Deanna J. "Medical Decision Making among Individuals with a Variant of Uncertain Significance in a Hereditary Cancer Gene and those with a CHEK2 Pathogenic Variant." Scholar Commons, 2019. https://scholarcommons.usf.edu/etd/7726.
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Despite national guidelines, women with a BRCA VUS or CHEK2 pathogenic variant are choosing to have risk-reducing surgeries such as bilateral mastectomies which are not aligned with their level of cancer risk based on genetic test results alone. Semi-structured telephone interviews were conducted with 6 women with a BRCA VUS and 12 with a CHEK2 pathogenic variant exploring the factors influencing their decision-making process when considering medical management options. Patients from a cancer registry agreed to a recorded telephone interview. Coding was performed using the main constructs from the Ottawa Patient Decision Guide including: knowledge, uncertainty, values, and support. Iterative analysis was used to identify emerging themes.
Analysis of the interviews revealed overlapping of the four constructs in the decision-making process. The knowledge sought to make medical management decisions was driven by the uncertainty associated with the genetic test results. Participants often contextualized their risk by building on the risk associated with genetic test results with family history, variant re-interpretation, and the knowledge that the risks associated with other genes may be higher. Patients generally made the decision they thought was best for them, even though it was more difficult if that decision was not supported by healthcare providers, friends, or family. When faced with uncertain cancer risks and presented with options for medical management, values were weighed against the negatives of each option. Often mental health was prioritized over the negatives associated with ‘removing body parts’.
These findings offer a look into the decisional needs of patients such as accurate knowledge, certainty, decisional support, and attention to personal values. Better understanding of the unmet needs of these patients and working to rectify them through provider education, outreach, counseling strategies to mitigate uncertainty, and research on how to best address and identify each patient’s specific decisional needs can contribute to the goal of risk-appropriate and values-based decision-making. With a better understanding of patients’ decisional needs, healthcare providers can better advocate for tailored counseling sessions which explore and address specific patient needs to help them make informed, risk-appropriate, and value-based medical management decisions.
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Jensen, Keith Douglas Ostergaard. "Dual Regulation of Telomerase Activity By HSF1 And Its Role in Prostate Cancer Progression." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1630.
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Grochola, Lukasz Filip. "Identification and functional analysis of single nucleotide polymorphisms that affect human cancer." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:aacc7084-81a8-4e97-b1ac-024d9bed106e.
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Aims: The p53 regulatory network is crucial in directing the suppression of cancer formation and mediating the response to commonly used cancer therapies. Functional genetic variants in the genes comprising this network could help identify individuals at greater risk for cancer and patients with poorer responses to therapies, but few such variants have been identified as yet. Methods: We first develop and apply three different screens that utilize known characteristics of functional single nucleotide polymorphisms (SNPs) in the p53 network to search for variants that associate with allelic differences in (i) recent natural selection, (ii) chemosensitivity profiles, and (iii) the gender- and age- dependent incidence of soft-tissue sarcoma. Secondly, we study and explore the functional mechanisms associated with the identified variants. Results: We identify SNPs in the PPP2R5E, CD44, YWHAQ and ESR1 genes that associate with allelic differences in the age of tumour diagnosis (up to 32.5 years, p=0.031), cancer risk (up to 8.1 odds ratio, p=0.004) and overall survival (up to 2.85 relative risk, p=0.011) in sarcomas, ovarian and pancreatic cancers, and exhibit allelic differences in the cellular responses to cytotoxic chemotherapeutic agents (up to 5.4-fold, p=5.6x10-47). Lastly, we identify candidate causal SNPs in those genes and describe the regulatory mechanisms by which they might affect human cancer. Conclusions: Together, our work suggests that the inherited genetics of the p53 pathway have a great potential to further define populations in their abilities to react to stress, suppress tumor formation and respond to therapies.
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Henderson, Melissa. "Patient-physician Dialogue Matters: Factors that Impact Medical Management Decisions among Women with Pathogenic Variants in Moderate-penetrance Genes Associated with Hereditary Breast Cancer." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1554213725302437.
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Aisenberg, Jeremy Charles. "A Critical Review of Telomerase Biology and Model Systems for the Study of Telomerase." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/2120.
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Martinsson, Caroline. "Characterisation of EGFR and KRAS mutations in non-small cell lung cancer." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-126041.
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Background: Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with EGFR mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with KRAS mutations do not benefit of such treatment. Methods: This study investigates the frequency of EGFR and KRAS mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes. Results: Five EGFR mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. KRAS mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations. Conclusions: This study confirms previous observations regarding the frequency of EGFR and KRAS mutations in non-small cell lung cancer. Mutations in EGFR and KRAS were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.
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Zauri, Melania. "Tet2 and relevant potential intervention in cancer." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:09208267-5766-47b6-b9f4-5c97a6e6b5a2.
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Poynter, Kennon R. "Telomerase Inhibition and Sensitization of Breast Tumor Cells." VCU Scholars Compass, 2007. http://scholarscompass.vcu.edu/etd/774.
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Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation, as well as malignant progression of breast cancer cells. Telomerase is an attractive anticancer therapeutic target because its activity is present in over 90% of human cancers, including more than 95% of breast carcinomas, but undetectable in most somatic cells. Traditions chemo- and radio-therapies lack the ability to effectively control and cure breast cancer, in part because residual cells are or become resistant to DNA damaging modalities.While various telomerase inhibition strategies cause cancer cells to undergo apoptosis car senescence, there is often a lag period between administration and biologic effect (Corey, 2002). Our goal in this study was to compare the efficacy of different telomerase inhibition strategies in concert with standard chemotherapeutic agents at triggering senescence and/or apoptosis in cultures of breast cancer cells. We hypothesized that telomerase inhibition strategies will sensitize breast cancer cells to traditional chemotherapies, potentially reducing the lag phase, allowing for more potent anti-tumor effects at lower doses, and therefore ultimately imparting less toxicity to the patient.We blocked telomerase by targeting hTR and hTERT, individually and collectively utilizing synthetic short interfering RNA (siRNA), short hairpin RNA (siRNA), and a dominant negative form of hTERT (DN-hTERT) in MCF-7 breast cancer cells. We analyzed the efficiency of telomerase inhibition for each strategy alone and then treated the cells with two mainstay chemotherapeutic agents, Adriamycin (AdR) and Taxol. The most effective telomerase inhibition strategies were synthetic siRNA and DN-hTERT, individually. After treatment with various concentrations of AdR or Taxol, breast cancer cells with inhibited telomerase grew significantly slower and exhibited widespread senescence or apoptosis within a much shorter time period and at a dose that is insufficient to trigger cytostasis. In addition, we provide evidence that cells in which telomerase was inhibited were more sensitive to anti-cancer agents, whether the drug inhibited topoisomerase II resulting in DNA damage (AdR) or blocked mitosis via protracted microtubule stabilization (Taxol). Collectively, our data indicate that alone, anti-telomerase inhibition strategies differ in their efficacy. However, when used in the adjuvant setting with diverse acting chemotherapeutic agents, there is a potent synergy resulting in chemotherapeutic sensitization characterized in part by widespread senescence and/or apoptosis.
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Sumner, Evan T. "Characterizing the Oncogenic Properties of C-terminal Binding Protein." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4153.
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The paralogous C-terminal binding proteins (CtBP) 1 and 2 are evolutionarily conserved transcriptional coregulators that target and disrupt the expression of several genes essential for multiple cellular processes critical to regulating tumor formation. CtBP’s ability to govern the transcription of genes necessary for apoptosis, tumor suppression, invasion/migration and EMT gives rise to its oncogenic activities. Both isoforms of CtBP are found to be overexpressed in cancers including colorectal, pancreatic, ovarian, and breast, with higher levels correlating to lower overall median survival. Although multiple lines of evidence suggest CtBP plays a role in tumorigenesis, it has never been formally characterized as an oncogene. For this reason, the goal of this dissertation was to design a set of experiments to determine the transforming ability of CtBP2 in vitro using both murine and human fibroblast and in vivo using the Apcmin/+ mouse model of cancer. Specifically, we demonstrate that overexpression of CtBP2 alone can drive transformation of NIH3T3 cells leading to loss of contact inhibition, increased x invasion/migration, and anchorage independent growth. In addition, CtBP2 was found to cooperate with the large T-antigen (LT) component of the simian virus 40 (SV40) to lead to transformation of murine embryonic fibroblasts (MEFs) and with both LT and small T-antigen (ST) to induce migration/invasion and anchorage-independent growth in BJ human foreskin fibroblasts. To confirm the role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 heterozygous (Ctbp2+/-) mice, which otherwise live normal lifespans. CtBP is a known target of the APC tumor suppressor and is thus stabilized in APC mutated human colon cancers and is found in high levels in Apcmin/+ polyps. Remarkably, removing an allele of Ctbp2 doubled the median survival of Apcmin/+ mice (P <0.001) and reduced polyp formation to near undetectable levels. These data suggest the importance of CtBP2 in driving cellular transformation and identify it as a potential target for prevention or therapy in APC mutant backgrounds.
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Molloy, Doreen. "Saying ‘No’: A biographical analysis of the experiences of women with a genetic predisposition to developing breast/ovarian cancer who reject risk reducing surgery." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2015. https://ro.ecu.edu.au/theses/1713.
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Background: Genetic technologies have identified some of the genes implicated in cancer susceptibility. Women with mutations in breast/ovarian cancer-susceptibility genes (BRCA1 and 2) have a lifetime combined risk of breast/ovarian cancer of more than 80%. Risk reducing surgery (RRS) reduces cancer risk by as much as 90% in high risk populations. Despite this, some BRCA1/2 mutation-positive women say no to RRS.
Purpose: To illuminate an understanding of why women at high risk of developing breast/ovarian cancer say no to risk reducing surgery (RRS).
Design: Denzin’s (1989) interpretive biography was combined with Dolby-Stahl’s (1985) literary folkloristic methodology to provide a contextualised narrative of the life experiences of six high risk women who said no to RRS. The participants’ stories were captured through semi-structured interviews then read and interpreted through the lenses of three literary theories namely Marxist, Foucauldian and Feminist.
Findings: Different understandings of risk were central to the decision to say no to RRS. RRS was understood as a risk to body and self which superseded the genetic risk of cancer. However despite having the strength to keep their still-healthy bodies intact, the participants benchmarked their decisions to say no against the dominant discourse on cancer risk, leaving them in an unending state of flux as to whether they had made the right decision. The participants shared a genetic pessimism but there also existed an emergent private folklore which illuminated how they attempted to make sense of their experiences and negotiate the conflicts and contradictions thrown up by competing discourses.
Conclusions: The relationship between genetic testing and cancer prevention strategies is not straightforward and genetic information has the potential to harm as well as help high risk women. It is important health care providers approach this area from the viewpoints of those directly involved since without understanding; strategies to support these women may be ineffective.
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Ovtcharov, Slav. "Impact of TMPRSS2-ERG fusion gene on prostate cancer cell response to chemotherapy, radiotherapy and androgen deprivation therapy." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:f30bf48d-fff5-49e7-8258-107a500c8752.
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Many aspects of the mechanisms by which prostate cancer (PCa) progresses from being a confined tumour to advanced metastatic and castration-resistant disease remain unclear. The aim of this study is to evaluate in vitro the potential role of the fusion gene TMPRSS2-ERG in the response of PCa cells to ionising radiation (IR) and androgen deprivation therapy (ADT). This research focused on assessing the presence of the TMPRSS2-ERG transcript across various PCa cell lines and identifying any correlation between the TMPRSS2-ERG transcript and other genes, particularly genes related to DNA damage repair pathways. Several genes involved in cell metabolism and development were found to correlate with TMPRSS2-ERG but not genes involved in DNA repair. In accordance with previous reports, this research confirmed a proliferative advantage for cells expressing ERG. However this project also tested the role of ERG-status in response to chemotherapy, radiation and ADT. The data showed that VCaP and DuCaP cells exposed to low-dose radiation demonstrated decreased viability irrespective of their ERG-status. Similarly ADT decreased the viability of VCaP cells and seemed to neutralise the proliferative advantage of TMPRSS2-ERG positive cells. Stimulation with dihydrotestosterone caused increased radioresistance of TMPRSS2-ERG positive cells. Treatment with taxanes showed stronger effect on cells with lower ERG expression. This work suggests that the proliferative advantage conferred by ERG overexpression in in vitro models can be neutralised by castration and IR.
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Pfister, Anna. "Outcomes of Myosin 1C Gene Expression Depletion on Cancer-related Pathways, in Vitro and in Clinical Samples." Licentiate thesis, Sahlgrenska Academy at University of Gothenburg, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-12981.
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The unconventional myosin IC has previously been suggested to be a haploinsufficient tumour suppressor. The mechanism for this action has hitherto been unknown, however, and hence we decided to attempt to elucidate the genes involved. The first study involved knock-down of MYO1C using siRNA technology followed by whole transcriptiome microarray analysis performed on samples taken at different time points post transfection. This revealed a cornucopia of differential expressions compared to the negative control, among them we found an early up-regulation of the PI3K/AKT pathway and the pathway for prostate cancer. Among the down regulated pathways we found endometrial-, colorectal cancer and small cell lung cancer as well as the cell cycle pathway which was a little counter intuitive to the hypothesis that MYO1C suppresses cancer. For the next study six different genes (CCND1, CCND2, CDKN2B, CDKN2C, MYC, RBL1) important for the transitions into S-phase of the cell cycle were therefore chosen for validation using qPCR. These six genes and MYO1C were analysed on both the original time series and a new biological replicate as well as a well stratified set of endometrial carcinoma samples. We were able to verify the significant down-regulation of CCND2 in both time series indicating that this is caused by the depletion of MYO1C. In the tumour samples we saw a negative correlation between the expression of MYO1C and FIGO grade corroborating results previously found by our group when looking at protein expression.
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Schödel, Johannes. "Genome-wide mapping of the hypoxic response." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:5701c6b5-f397-4b21-a66b-cfc02043fe40.
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Hypoxia regulates many hundreds of genes with important roles in ischemic and neoplastic disorders. Central to this response are the hypoxia inducible transcription factors (HIF). This work aimed to better understand the direct transcriptional response to HIF by mapping HIF-binding sites across the genome using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). ChIP-seq for HIF in MCF-7 breast cancer cells under hypoxic conditions revealed more than 400 high-stringency HIF-binding sites genome-wide. Each member of the HIF heterodimer was present with near complete concordance. Binding of the two principle isoforms revealed a high degree of overlap with no differences in the DNA-binding motif. HIF-binding was associated with upregulation, but not downregulation of genes indicating that it functions as a transcriptional activator but not as a repressor. HIF-binding occurred preferentially at gene promoters, but was also present at promoter-distant sites, which were also associated with gene regulation, implicating long-range interactions in hypoxic gene activation. HIF-binding was associated with markers of open chromatin and active enhancers that were present in normoxia, indicating that HIF-binding sites are already “prepared” to bind HIF before the hypoxic stimulus. Analysis of normoxic and hypoxic RNA pol2 and H3K4me3 signals revealed distinctive hypoxia-inducible changes unique to HIF-binding genes. Comparable numbers of HIF-binding sites were observed in a second cell line (von Hippel-Lindau defective 786-O renal cancer cells) as in MCF-7 breast cancer cells, although approximately 65% were unique to 786-O cells. These unique sites were more frequently promoter-distant. Correlation with expression analyses from renal tumours indicated that many HIF-binding genes were upregulated in renal cancer. One such RCC unique promoter-distant HIF-binding site was identified at an intergenic locus on chromosome 11q13.3 that has been associated with renal cancer in Genome-Wide Association Studies. The HIF-binding site was in high linkage disequilibrium with the disease associated SNP and had the epigenetic hallmarks of an enhancer. Analysis of pan-genomic expression analyses identified the cell-cycle regulator cyclin D1 as highly HIF-regulated, and a physical association between the HIF-binding site and the CCND1 promoter could be determined. Furthermore, in a renal cancer cell line heterozygous at this locus, the RCC-protective allele disrupted HIF-binding leading to an allelic imbalance in cyclin D1 expression.
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Opat, Annette. "Exploring the experiences of people who have consented to tumour testing for a hereditary disposition to cancer." Connect to thesis, 2009. http://repository.unimelb.edu.au/10187/6962.
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Due to the costly and technically challenging nature of genetic testing, methods have been developed to target more specifically those who are at increased risk of carrying the Hereditary Non-Polyposis Colorectal Cancer (HNPCC) mutation. HNPCC is an inherited colorectal cancer syndrome. Testing of tumour material (which has previously been removed during surgery) for features of HNPCC has been found to be an effective and economic method of identifying those at higher risk of having a mutation. Only those at higher risk of having a mutation will undergo genetic testing. This practice of “tumour testing” has become widespread.There is currently no clarity about requirements for consent prior to testing of stored tumour tissue. The person giving consent to tumour testing does not always have an appointment with a genetics service prior to giving consent. This can be contrasted to genetic testing on blood samples where laws and guidelines state that informed consent is required prior to genetic testing and that comprehensive genetic counselling and support should be provided as part of this process. Protocols for genetic testing have been developed as a result of extensive research around the impact and implications of genetic testing.
Consumer opinion and participation through research is an important aspect of health policy and guideline development. Accordingly the purpose of this study was to contribute to such development by gaining insight into the experiences, understandings, decision making processes and opinions of those who had given consent to have their own or their relatives tumour tested. Seventeen people who had given consent for tumour testing either for themselves, or on behalf of a deceased relative were recruited through a Familial Cancer Centre and in-depth interviews conducted. The interviews were transcribed and analysed using thematic analysis.
Some participants had no memory of consenting to tumour testing. Others remembered basic concepts. Negative implications of testing were unknown or viewed as unimportant. Participants did not understand the difference between tumour testing and germline testing. Despite lack of memory or understanding participants did not want additional or more detailed pre-test information although they did want more follow-up and support after receipt of results. The decision to consent to testing was made as soon as participants were informed of the availability of tumour testing - the major reason being to provide information for the family that would aid in cancer prevention. Participants were more concerned with accessibility to testing than pre test information and counselling.
Findings in this study indicated participants made decisions heuristically rather than systematically and this as well as participants’ opinions and other decision-making research has implications for the traditional view of informed consent around genetic related decisions. This in turn has implications for policy and guidelines in the area. Implications for current practise as a result of findings from this study include ensuring participants understand negative implications of testing and follow up and support of those with negative as well as positive results to tumour testing.
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Alkhatib, Suehyb. "Characterizing the role of Nucleosome Remodeling Factor (NURF) in tumorigenesis and metastatic progression using mouse models of breast cancer." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/376.
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Increasingly the role of epigenetic machinery as a bridge between underlying DNA sequence and cellular phenotype is being discovered. The establishment of a myriad of unique cellular types sharing identical gene sequences in a multicellular organism gives a broad sense for the inherent role of epigenetic influence on cell differentiation. Importantly, the epigenetic mechanisms involved in establishing cell identity unsurprisingly contribute to diseased states, including cancer. Recent research continues to elucidate contributory roles of epigenetic mechanisms, such as DNA methylation, histone modification, and microRNA regulation, in human cancers. Additionally, chromatin remodelers, such as the Nucleosome Remodeling Factor (NURF), have been identified as important regulators for normal cell biology. While much has been done to identify and characterize the role of NURF chromatin remodeling complex as a key regulator of development in a number of model organisms, little has been published on the implications of NURF in diseases such as cancer. Our preliminary data shows dysregulation of E-cadherins, N-cadherins, and MHC-I genes in Bptf (an essential subunit of NURF) knocked down murine breast cancer cell lines. These proteins have well documented roles in the development and metastatic progression of cancers. To study the effect of Bptf knockdown on the development and progression of cancer we injected Bptf knocked down mouse breast cancer cell lines, 4T1, 66cl4, and 67NR, into syngenic BALB/c mice. Our findings reveal decreased tumor growth in 66cl4 and 67NR as measured by tumor weight at 3-4 weeks post injection. Tumor growth did not appear to be significantly affected in 4T1 challenged mice. However, mice inoculated with Bptf knockdown 4T1 cell lines have decreased metastasis to lungs as compared to control while metastasis of 66cl4 tumors to the lungs appear unaffected. To assess the role of the immune system in decreasing tumor growth in BALB/c mice, we injected 66cl4 tumors into NOD-SCID-Gamma (NSG) immune deficient mice. The tumors from these mice show no difference in tumor growth between Bptf knockdown and control tumors, implicating a role for the immune system regulating the decreased tumor weight in BALB/c mice. To delineate which immune cell effector may impede breast cancer carcinogenesis, we performed an in vitro natural killer (NK) cell cytotoxicity assay against 66cl4 tumors and found greater susceptibility to NK killing in Bptf knockdown tumors.
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Borgström, Annelie. "Analysis of tumour infiltrating leukocytes in colon cancer carcinoma in a syngeneic rat model." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-56910.
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Tumour immunity is a balance between immune mediators that promote tumor progression versus mediators that promote tumor rejection. Infiltrating lymphocytes in human colorectal cancer tissues are independent prognostic factors for a better survival and a high number of cytotoxic CD8+ T-cells have been associated with a better prognosis in terms of a longer and disease free survival for the patient. In our syngeneic rat model we induce colon carcinoma subperitoneally by injecting a colon cancer cell line BN7005, a cell line expressing the epitope (Lewis Y) for the BR96 antibody. Tumours are dissected out and treated with different fixatives and then either frozen, snap-frozen or embedded in paraffin followed by sectioning. Immunohistochemistry using monoclonal antibodies against the tumour infiltrating leukocytes was performed on the tissue. The results were seen as an infiltration of different leukocytes in the tumours.
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DeLigio, James T., and James Thomas DeLigio. "ALTERNATIVE SPLICING OF CYTOPLASMIC POLYADENYLATION ELEMENT BINDING PROTEIN 2 IS MODULATED VIA SERINE ARGININE SPLICING FACTOR 3 IN CANCER METASTASIS." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5660.
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Our laboratory delineated a role for alternative pre-mRNA splicing (AS) in triple negative breast cancer (TNBC). We found the translational regulator cytosolic polyadenylation element binding protein 2 (CPEB2) which has two isoforms, CPEB2A and CPEB2B, is alternatively spliced during acquisition of anoikis resistance (AnR) and metastasis. The splicing event which determines the CPEB2 isoform is via inclusion/ exclusion of exon four in the mature mRNA transcript. The loss of CPEB2A with a concomitant increase in CPEB2B is required for TNBC cells to metastasize in vivo. We examined RNAseq profiles of TNBC cells which had CPEB2 isoforms specifically downregulated to examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes. Downregulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition (EMT) and hypoxic response, whereas downregulation of the CPEB2A isoform did not have this effect. Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1a. Functional studies showed that specific downregulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce AnR and drive metastasis. The mechanism governing inclusion/ exclusion of exon 4 was determined to be serine/ arginine-rich splicing factor 3 (SRSF3). Binding of SRSF3 to a consensus sequence within CPEB2 exon 4 promoted its inclusion in the mature mRNA, and mutation of this sequence abolished association of SRSF3 with exon 4. SRSF3 expression was upregulated in TNBC cells upon acquisition of AnR correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 by siRNA in these cells induced the exclusion of exon 4. Downregulation of SRSF3 also reversed the CPEB2A/B ratio in a wild-type CPEB2 exon 4 minigene construct, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. Physiologic studies demonstrated SRSF3 downregulation ablated AnR in TNBC cells, and was “rescued” by ectopic expression of CPEB2B. Importantly, biostatistical analysis of The Cancer Genome Atlas database showed a positive relationship between alterations in SRSF3 expression and lower overall survival in TNBC. Overall, this study demonstrates that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer.
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González, Acosta María Isabel. "Desarrollo de nuevas aproximaciones para el diagnóstico molecular de los síndromes de predisposición hereditaria al cáncer asociados a deficiencia del sistema de reparación de apareamientos erróneos." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/668559.
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La función principal del sistema de reparación de apareamientos erróneos (MisMatch Repair, MMR) es corregir los errores principalmente introducidos por las DNA polimerasas durante la replicación del genoma. Las mutaciones germinales deletéreas en alguno de los cuatro genes principales del sistema MMR (MLH1, MSH2, MSH6 y PMS2) son las responsables de los síndromes de predisposición hereditaria a cáncer asociados a deficiencia de este sistema: el síndrome de Lynch (SL), causado por mutaciones monoalélicas en estos genes, y el síndrome de Deficiencia Constitucional de Reparación de Apareamientos Erróneos (CMMRD), causado por mutaciones bialélicas. A consecuencia de la deficiencia de este tipo de reparación, los tumores asociados a estos dos síndromes exhiben pérdida de expresión de las proteínas MMR y/o inestabilidad de microsatélites (MSI) y, aunque en menor grado, estas características también se observan en tejido normal.
El diagnóstico de estos síndromes se basa en la identificación de mutaciones patogénicas en los genes MMR en línea germinal. Sin embargo, el diagnóstico no siempre es posible. La presencia de mutaciones crípticas, la identificación de variantes de significado desconocido (VUS) (que representan el 30% de las variantes que se encuentran en la rutina de diagnóstico) y la existencia de fenotipos intermedios o solapantes con otros síndromes, dificultan el diagnóstico, lo que impacta en el manejo clínico del paciente y sus familiares.
El objetivo de esta tesis doctoral es mejorar el diagnóstico de los síndromes de predisposición hereditaria al cáncer asociados a deficiencia del sistema MMR. Con este objetivo global nos hemos planteado dos objetivos específicos. El primer objetivo es mejorar la evaluación de la patogenicidad de las variantes aplicando modelos multifactoriales, que integran múltiples líneas de evidencias tanto cualitativas como cuantitativas. También se ha estandarizado el ensayo in vitro de actividad reparadora, dirigido a testar la función más importante de una proteína MMR, con el fin de que pueda ser utilizado en la determinación de la patogenicidad de una VUS. El segundo objetivo es desarrollar una nueva metodología, basada en la detección con alta sensibilidad de la MSI en los tejidos normales de los portadores de mutaciones MMR, para poder diagnosticar de estos síndromes a pesar de no encontrar mutación o de la presencia de VUS.
En relación al primer objetivo, se han reclasificado a patogénicas o benignas el 89% de las variantes estudiadas en esta tesis doctoral gracias a la integración del cálculo multifactorial de probabilidad, la frecuencia poblacional, las predicciones in silico y los ensayos funcionales a nivel de RNA y proteína en un nuevo algoritmo de clasificación, lo que apoya su utilidad. Además, el ensayo in vitro de actividad reparadora ha sido optimizado a nivel de reactivos y validado a nivel analítico demostrando robustez y reproducibilidad. A destacar, se han establecido protocolos estándar para su realización, lo que representa el primer paso para su implementación en el diagnóstico
En cuanto al segundo objetivo, la metodología desarrollada para la detección con alta sensibilidad de la MSI en sangre periférica discrimina con una sensibilidad y especificidad del 100% a los pacientes CMMRD del resto de grupos (pacientes SL y de otros síndromes con fenotipo solapante), aunque no ha demostrado suficiente sensibilidad para detectar MSI en los pacientes SL. Esta herramienta, por lo tanto, podría ser especialmente útil para el diagnóstico de CMMRD, sobre todo en los casos con ausencia de mutaciones patogénicas identificadas en los genes MMR. Será necesario testar otros tejidos, como la mucosa colónica normal, para profundizar en la dinámica de MSI en tejidos diana de pacientes afectos de SL antes del desarrollo de la neoplasia.
El trabajo realizado en esta tesis doctoral representa una mejora de las actuales estrategias para el diagnóstico molecular de los síndromes de síndromes de predisposición hereditaria al cáncer asociados a deficiencia del sistema MMR.
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Smith, Jordan L. "Reversing Cancer Cell Fate: Driving Therapeutic Differentiation of Hepatoblastoma to Functional Hepatocyte-Like Cells." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1067.
Full textAbstract:
Background & Aims: Despite advances in surgical care and chemotherapeutic regimens, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and β-Catenin co-activation occurs in 80% of children’s HB; however, a lack of conditional genetic models precludes exploration of tumor maintenance and therapeutic targets. Thus, the clinical need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in children’s tumors, I sought to evaluate YAP1 as a therapeutic target in HB.
Approach & Results: Herein, I engineered the first conditional murine model of HB using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A, constitutive β-CateninDelN90, and a luciferase reporter to murine liver. Tumor regression was evaluated using in vivo bioluminescent imaging, and tumor landscape characterized using RNA sequencing, ATAC sequencing and DNA foot-printing. Here I show that YAP1 withdrawal in mice mediates >90% tumor regression with survival for 230+ days. Mechanistically, YAP1 withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative “hbHep cells.” hbHep cells have hepatocyte-like morphology and partially restored mature hepatocyte gene expression. YAP1 withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and can rescue liver damage in mice.
Conclusions: YAP1 withdrawal, without modulation of oncogenic β-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. Modulating YAP1 expression alone is sufficient to drive long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.
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Díaz, Gay Marcos. "Identification of new candidate genes for germline predisposition to familial colorectal cancer using somatic mutational profiling." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/668900.
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Colorectal cancer (CRC) is one of the malignant neoplasms with higher incidence and mortality in Spain, Europe and worldwide. As a complex disease, both environmental and genetic factors influence CRC predisposition. Up to 35% of CRC patients present familial aggregation for the disease, whereas only around 2-8% of cases are linked to a well-known hereditary syndrome associated to pathogenic germline alterations in specific genes, namely APC, MUTYH, POLE, POLD1 or the DNA mismatch repair genes. During last years, next generation sequencing (NGS) techniques such as whole exome sequencing (WES) have been used to address this gap of missing heritability. Characterization of somatic mutational profiles, performed by the application of NGS to both germline and tumor DNA, has also been recently established as a powerful tool to identify novel genes linked to CRC predisposition. However, although some bioinformatic packages have been developed to address this analysis, it remains inaccessible for a substantial proportion of the scientific community. Accordingly, the main purpose of this doctoral thesis was to identify new genes involved in germline predisposition to familial CRC, by using an integrated germline-tumor WES analysis and somatic mutational profiling, as well as facilitating the application of these genomic analyses to the scientific community.
As a first step, a bioinformatic tool to deal with somatic mutational profiling was developed. Shiny framework was used to build MuSiCa, a user-friendly web application freely accessible and potentially useful for non-specialized researchers. Tumor mutational burden calculation and mutational signature refitting analysis according to the information present in COSMIC database is available, as well as different options for sample classification through clustering and principal component analysis.
Subsequently, an integrated germline-tumor analysis was implemented in a cohort of 18 familial CRC unrelated patients. WES data of both germline and tumor DNA was available, allowing the identification of new potential tumor suppressor genes according to Knudson’s two-hit hypothesis. Benefitting from the development of MuSiCa application, somatic mutational profiling was also analyzed, uncovering five hypermutated samples. An enrichment of DNA repair-associated genes was found, as well as some genes previously linked to predisposition syndromes to other cancer types. BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were found as the most promising candidate genes for germline CRC predisposition. Interestingly, a germline mutation was found in the DNA repair gene RECQL in a patient with one of the hypermutated tumors, reinforcing the putative role of this gene in hereditary CRC. These findings could be helpful in clinical practice improving genetic counseling in the affected families.El cáncer colorrectal (CCR) es una de las neoplasias con mayor incidencia y mortalidad en España y el mundo. Aunque un 35% de los pacientes presentan agregación familiar, sólo un 2-8% se asocia con un síndrome hereditario conocido, causado por mutaciones germinales en genes como APC, MUTYH, POLE, POLD1 o los genes del sistema de reparación del ADN por mal apareamiento de bases. En los últimos años, las técnicas de secuenciación de nueva generación (SNG), como la secuenciación del exoma completo (SEC), han sido utilizadas para el descubrimiento de nuevos genes implicados en la predisposición al CCR. La caracterización de los perfiles mutacionales somáticos, aplicando SNG al ADN germinal y tumoral, también se ha utilizado recientemente en este proceso. Sin embargo, aunque se han desarrollado algunos paquetes bioinformáticos para su análisis, todavía permanece inaccesible para una gran parte de la comunidad científica. En consecuencia, el objetivo principal de esta tesis doctoral ha sido el de identificar nuevos genes implicados en la predisposición germinal al CCR familiar, utilizando un análisis de SEC germinal-tumoral y caracterización mutacional somática, así como facilitar la aplicación de estos análisis genómicos a la comunidad científica. En primer lugar, se llevó a cabo el desarrollo de una herramienta bioinformática denominada Mutational Signatures in Cancer (MuSiCa), una aplicación web de manejo sencillo y acceso libre desarrollada a través de la plataforma Shiny, que permite el cálculo de la carga mutacional tumoral y la caracterización de las firmas mutacionales según la información disponible en la base de datos COSMIC. Posteriormente, se implementó un análisis integrado de SEC germinal-tumoral en una cohorte de 18 pacientes de CCR familiar, complementado con una caracterización mutacional somática, gracias al desarrollo de MuSiCa. Se detectaron cinco tumores hipermutados, así como un enriquecimiento de mutaciones germinales en genes involucrados previamente en síndromes de predisposición a otros tipos de cáncer y a la reparación del ADN. Los genes BRCA2, BLM, ERCC2, RECQL, REV3L y RIF1 fueron priorizados como los más prometedores de cara a la predisposición al CCR. Estos descubrimientos podrían ser de utilidad en la práctica clínica, mejorando el consejo genético en las familias afectadas.
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López-Dóriga, Guerra Adriana. "Anàlisi de dades de seqüenciació de nova generació pel diagnòstic molecular del càncer hereditari i per la recerca de les bases genètiques del càncer colorectal esporàdic." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/401754.
Full textAbstract:
La seqüenciació de nova generació (Next Generation Sequencing, NGS) està canviant el diagnòstic genètic i la recerca genòmica gràcies a la gran capacitat de seqüenciació i el seu cost‐eficiència. En aquesta tesi s’ha utilitzat la NGS tant per al diagnòstic genètic del cáncer hereditari com per a la recerca de noves mutacions recurrents en el càncer colorectal (CCR) esporàdic.
En el primer article de la tesi es descriu el desenvolupament d’un protocol basat en la NGS per al diagnòstic rutinari de la síndrome de càncer de mama i ovari hereditari (Hereditary Breast and Ovarian Cancer Syndrome, HBOCS), per millorar el diagnòstic genètic dels gens BRCA1 i BRCA2. Es va dissenyar un protocol basat en NGS utilitzant les llibreries d’amplicons del MASTR kit de Multiplicom, seguit de la piroseqüenciació amb el GS Junior i l’anàlisi de les dades amb software lliure. El segon article presenta el desenvolupament d’una eina oberta executable via web, per a realitzar les anàlisis genètiques de la reseqüenciació per amplicons d’alguns gens específics d’alt risc per a cáncer hereditari, utilitzant el seqüenciador GS Junior. Concretament, l’anàlisi bioinformàtic està efocat a detectar i filtrar variants, i a proporcionar informació de la cobertura, permetent a l’usuari personalitzar alguns paràmetres bàsics. A més, la web està vinculada a la base de dades de mutacions de la nostra institució per ajudar a la classificació clínica de les variants identificades.
L’evolució del diagnòstic genètic ens porta cap a l’ús dels panells. En un breu apartat després del segon article, s’avalua el rendiment del panell comercial d’Illumina “Trusight Cancer Panel”per tal de valorar la seva efectivitat en la rutina diagnòstica del Càncer Hereditari. Aquest panell inclou 94 gens associats a càncer hereditari més un grup de 284 SNPs correlacionats amb càncer en diferents estudis d’associació. Els resultats mostren un bon rendiment del panell Trusight Cancer d’Illumina en la rutina del diagnòstic de càncer hereditari.
En el tercer i darrer article que forma la tesi, es presenten els resultats de la seqüenciació dels exomes de 42 mostres de tumors i normal aparellat de CCR esporàdic, per caracteritzar l’estat mutacional dels tumors i trobar noves mutacions recurrents que puguin estar implicades en la tumorigenesis del CCR. Els resultats mostren una gran heterogeneïtat mutacional, però tot i així, aquesta diversitat de mutacions convergeix en vies metabòliques comunes com el cicle cel∙lular o l’apoptosi. Enmig d’aquesta heterogeneïtat mutacional, ressalten les variants truncants al gen AMER1 (també anomenat FAM123 o WTX) que apareixen de forma recurrent en els casos de CCR. Validacions in silico i experimentals en grups de dades independents confirmen l’existència de mutacions amb alta probabilitat d’afectar la funció d’AMER1 en aproximadament el 10% dels tumors de CCR analitzats.Next Generation Sequencing, NGS, is changing genetic diagnosis and genomic research due to its huge sequencing capacity and cost-effectiveness. In this thesis, NGS has been used for the genetic diagnosis of hereditary cancer and for the research of new recurrent mutations in sporadic colorectal cancer. First paper describes the development of an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. The NGS-based workflow is designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing and data analysis using freely available software. Second paper presents the development of a free and user-friendly Web data analysis tool for the bioinformatics data analysis. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. Specifically, the tool detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. Moreover, the Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. The evolution of genetic diagnosis carries us to the use of multiple gene panels. In a brieve section after the second paper, we evaluate the performance of the commercial Illumina panel “Trusight Cancer Panel” which includes 94 genes and 285 SNPs associated to cancer, to evaluate its effectiveness in the diagnosis routine. Results show a good performance of the Trusight Cancer d’Illumina in the hereditary cancer diagnosis routine. In the third and last paper of the thesis, results of the exome sequencing for 42 colorectal tumors and their normal paired samples are presented. Results reveal tumor-specific mutational landscapes. Nevertheless, these diverse mutations converged into common cellular pathways, such as cell cycle or apoptosis. Among this mutational heterogeneity, variants resulting in early stop codons in the AMER1 (also known as FAM123B or WTX) gene emerge as recurrent mutations in colorectal cancer. In silico and experimental validation in independent datasets confirm the existence of functional mutations in AMER1 in approximately 10% of analyzed colorectal cancer tumors.
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Martínez, Fernández Alejandro. "Biomarcadores en cancer colorrectal: Metaloproteinasa 7 en pacientes intervenidos y mutaciones tras progresión a terapias anti-EGFR en enfermedad metastásica." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/291111.
Full textAbstract:
El cáncer colorrectal es uno de los principales problemas de salud en nuestro medio. Los pacientes presentan una evolución muy heterogénea y no es posible predecir de manera fidedigna la evolución clínica o el pronóstico de un paciente individual con los biomarcadores disponibles en la actualidad.
La presente tesis doctoral evaluó dos estrategias diferentes para determinar el valor de nuevos biomarcadores en cáncer colorrectal. La primera es la evaluación pronóstica de la determinación en sangre periférica de Metaloproteinasa de Matriz 7 (MMP-7). La segunda es una extensa descripción de eventos genéticos relacionados con la aparición de resistencia adquirida al tratamiento con el anticuerpo anti-EGFR cetuximab.
MMP-7 es un enzima que degrada proteínas extracelulares, y su expresión se ha relacionado con peor pronóstico en varias enfermedades oncológicas. Este sub-proyecto plateó como hipótesis que la concentración sanguínea de MMP-7 podría ser un biomarcador pronóstico en los pacientes diagnosticados de cáncer colorrectal que serán sometidos a cirugía curativa. Se determinó la concentración de MMP-7 mediante técnica de ELISA en 175 pacientes previa a la intervención quirúrgica. Los resultados mostraron que MMP-7 era un factor pronóstico independiente tanto para supervivencia libre de enfermedad (HR= 1.119, IC95% 1.055-2.0, p<0.001) como para supervivencia global (HR=1.113, IC95% 1.025-1.209, p=0.011). Utilizando el valor de la mediana para clasificar los pacientes en dos grupos, se determinó que los pacientes con MMP-7 elevada presentaban a cuatro años menor supervivencia libre de enfermedad (50.6% versus 78.4%, p<0.001) y supervivencia global (56.9% versus 92.6%, p<0.001). Asimismo, la elevación o no de MMP-7 ofrecía una información pronóstica complementaria a la afectación ganglionar por la enfermedad. Con estos resultados se concluyó que la determinación de MMP-7 en sangre periférica podría ser un biomarcador pronóstico y que puede realizarse mediante técnicas accesibles en la práctica clínica habitual.
El tratamiento con anticuerpos anti-EGFR es una estrategia terapéutica instaurada en el tratamiento del cáncer colorrectal metastásico. En la actualidad se dispone de dos anticuerpos contra EGFR: cetuximab y panitumumab. El segundo sub-proyecto estudió los posibles mecanismos genéticos de resistencia adquirida al tratamiento con cetuximab. La hipótesis del estudio fue que la comparación entre las mutaciones presentes en las células tumorales antes y después de la terapia basada en cetuximab podría revelar los mecanismos relevantes para la adquisición de resistencia al tratamiento. Se incluyeron pacientes con progresión al tratamiento con cetuximab tras una respuesta o una larga estabilidad. Mediante una biopsia de la lesión tumoral más accesible se determinó las mutaciones en los genes KRAS, NRAS, PIK3CA, BRAF y EGFR; y los resultados se compararon con las mutaciones de la muestra obtenida previo el inicio del tratamiento. De los 37 pacientes incluidos se pudo demostrar mutaciones tras el tratamiento con cetuximab en 22 de ellos. El 36% de los pacientes presentaban dos o más mutaciones simultáneas. La mayoría de eventos genéticos aparecían únicamente tras el tratamiento con cetuximab, siendo indetectables previo al tratamiento. Las mutaciones más frecuentes tras resistencia adquirida a cetuximab afectaban los genes KRAS y NRAS. El 14% de pacientes mostraban mutaciones de EGFR: el 8% de la mutación ya conocida S492R; pero además se detectó 2 nuevas mutaciones: K467T y R451C. Ninguna de las mutaciones de EGFR fue detectada previamente al inicio del tratamiento. En tres pacientes se analizó varias biopsias diferentes durante la evolución de la enfermedad, permitiendo concluir que los eventos moleculares son un fenómeno dinámico que varía tras cada tratamiento administrado. Se detectó además mutaciones en sangre periférica, logrando una correcta correlación con las mutaciones en tejido en el 62%. De esta manera, la detección de mutaciones en sangre podría tener aplicabilidad en la monitorización de los pacientes y diagnosticar la resistencia al tratamiento antes de su manifestación clínica o radiológica.INTRODUCTION: Colorectal cancer is a main health problem. New biomarkers to define prognosis or predict drug effectivity are warranted. The aim of this thesis is define new biomarkers using two different approaches: Evaluation of serum Matrilysine (MMP-7) as a prognostic marker in localized colorectal cancer and determination of the molecular events related with acquired resistance to the anti-EGFR antibody cetuximab in advanced disease. MATHERIAL AND METHODS: We evaluated MMP-7 blood concentration by ELISA in 175 patients before surgery with curative intention. On the other hand, 37 patients with acquired resistance to cetuximab were biopsied in order to analyze KRAS, NRAS, BRAF, PIK3CA and EGFR mutations. We compared these alterations with the mutational profile previous the cetuximab treatment RESULTS: MMP-7 blood concentration is an independent prognostic marker for overall survival and disease- free survival. Combining MMP-7 and nodal status we defined three different prognostic groups. Mutational analysis in tumors after acquired resistance to cetuximab shows that 66% of patients had almost one of the mutations analyzed and 63% of these mutations were not present before the treatment. KRAS and NRAS genes were the most frequently affected. 14% of patients presented mutations at EGFR gene: S492R, K467T and R451C. Biopsies form three patients treated with biological therapies after cetuximab failure were also available, detecting dynamic changes in the mutational profile. Mutations were also detected in 62% of serum circulating DNA. CONCLUSSION: MMP-7 is a potential prognostic biomarker for colorectal cancer and its determination is technically applicable in clinical practice. KRAS and NRAS are the genes most frequently mutated after resistance to cetuximab. We also define two new mutations affecting EGFR (K467T and R451C). Tumor mutational profile is dynamic, and changes after the acquired resistance to each therapy. Blood DNA-sequencing could effective to monitor resistance to cetuximab even before clinical and radiological progression.
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Stamatkin, Christopher W. "PHOSPHATIDYLINOSITOL 3-KINASE (PI3K) AS A THERAPEUTIC TARGET IN NSCLC." UKnowledge, 2014. http://uknowledge.uky.edu/pharmacy_etds/58.
Full textAbstract:
Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.
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Movassagh, Mercedeh J. "Comprehensive Computational Assessment And Evaluation of Epstein Barr virus (EBV) Variations, miRNAs, And EBERs in eBL, AML And Across Cancers." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1022.
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Viruses are known to be associated with 20% of human cancers. Epstein Barr virus (EBV) in particular is the first virus associated with human cancers. Here, we computationally detect EBV and explore the effects of this virus across cancers by taking advantage of the fact that EBV microRNAs (miRNAs) and Epstein Barr virus small RNAs (EBERs) are expressed at all viral latencies. We identify and characterize two sub-populations of EBV positive tumors: those with high levels of EBV miRNA and EBERS expression and those with medium levels of expression.
Based on principal component analysis (PCA) and hierarchical clustering of viral miRNAs across all samples we observe a pattern of expression for these EBV miRNAs which is correlated with both the tumor cell type (B cell versus epithelial cell) and with the overall levels of expression of these miRNAs.
We further investigated the effect of the levels of EBV miRNAs with the overall survival of patients across cancers. Through Kaplan Meier survival analysis we observe a significant correlation with levels of EBV miRNAs and lower survival in adult AML patients. We also designed a machine learning model for risk assessment of EBV in association with adult AML and other clinical factors.
Our next aim was to identify targets of EBV miRNAs, hence, we used a combination of previously known methodologies for miRNA target detection in addition to a multivariable regression approach to identify targets of these viral miRNAs in stomach cancer.
Finally, we investigate the variations across EBV subtype specific EBNA3C gene which interacts with the host immune system. Preliminary data suggests potential regional variations plus higher pathogenicity of subtype 1 in comparison to subtype 2 EBV.
Overall, these studies further our understanding of how EBV manipulates the tumor microenvironment across cancer subtypes.
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Jain, Jayati. "Engineering antibodies to study and improve immunomagnetic isolation of tumour cells." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:81355801-b331-4705-bfef-204a29ee0347.
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Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.
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Vice, President Research Office of the. "Newswire." Office of the Vice President Research, The University of British Columbia, 2008. http://hdl.handle.net/2429/2661.
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UBC's research community recently received a significant boost in financial support for five research hubs that will join the Centre for Brain Health as newly appointed national Centres of Excellence for Commercialization and Research (CECR).
Two UBC economics professors were recognized with separate Bank of Canada awards: the Research Fellowship 2008 and the Governor's Award.
UBC's Brain Research Centre has recevied $25 million from the Province of BC to establish a new facility focused on translational brain research.
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Whitmore, Scott Anthony. "Positional cloning of genes associated with human disease." Thesis, 1999. http://hdl.handle.net/2440/19353.
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Copies of author's previously published articles inserted.Amendments pasted onto back-end paper.
Bibliography: leaves 255-286.
ix, 286, [15] leaves, [5] leaves of plates : ill. (chiefly col.) ; 30 cm.
Aims to isolate the gene(s) responsible for Fancomi anaemia and breast cancer using a positional cloning strategy
Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and Molecular Genetics, 1999
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O'Doherty, Kieran Christian. "Risk communication in familial cancer : the discursive management of uncertainty in genetic counselling." 2005. http://hdl.handle.net/2440/37766.
Full textAbstract:
This thesis deals with the communication of cancer risk in genetic counselling sessions. There are two primary foci that form threads throughout both the theoretical and empirical chapters of the dissertation. The first concerns the meaning of risk as it manifests in familial cancer. In particular, there is a lack of a sound theoretical grounding for the probabilistic aspect of risk that is evident in many forms of risk communication. The thesis aims to illustrate the problem constituted by this lack of theoretical clarity. As most decisions faced by clients arise from attending genetic counselling, it is concluded that clients ' agency is highly constrained when genetic counselling is understood as a process of assisting decision - making. However, genetic counselling can be seen to enable agency when it is conceptualised as a process aimed at making available new medical technologies for the purpose of addressing clients ' own concerns.Thesis (Ph.D.)--Faculty of Health Sciences, Dept. of Psychology, 2005.
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Zaka, Masood-Ul-Hassan, Yonghong Peng, and Chris W. Sutton. "Integrated microarray analytics for the discovery of gene signatures for triple-negative breast cancer." 2014. http://hdl.handle.net/10454/10822.
Full textAbstract:
NoTriple-negative breast cancers (TNBC) are clinically heterogeneous, an aggressive form of breast cancer with poor diagnosis and highly therapeutic resistant. It is urgently needed for identifying novel biomarkers with increased sensitivity and specificity for early detection and personalised therapeutic intervention. Microarray profiling offered significant advances in molecular classification but sample scarcity and cohort heterogeneity remains challenging areas. Here, we investigated diagnostics signatures derived from human triple-negative tissue. We applied REMARK criteria for the selection of relevant studies and compared the signatures gene lists directly as well as assessed their classification performance in predicting diagnosis using leave-one-out cross-validation. The cross-validation results shows excellent classification accuracy ratios using all data sets. A subset signature (17-gene) extracted from the convergence of eligible signatures have also achieved excellent classification accuracy of 89.37% across all data sets. We also applied gene ontology functional enrichment analysis to extract potentially biological process, pathways and network involved in TNBC disease progression. Through functional analysis, we recognized that these independent signatures have displayed commonalities in functional pathways of cell signaling, which play important role in the development and progression of TNBC. We have also identified five unique TNBC pathways genes (SYNCRIP, NFIB, RGS4, UGCG, LOX and NNMT), which could be important for therapeutic interventions as indicated by their close association with known drivers of TNBC and previously published experimental studies.
Yorkshire Cancer Research for the Supplementary ort of CWS (BPP049 and B209PG)