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- All Subjects: Obesity
Description
Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity resulting in targeted therapy resistance and other challenges that make the management and treatment of these diseases rather difficult. Due to the presence of admixtures of non-neoplastic cells with polyclonal cell populations, it is difficult to define cancer genomes in patient samples. By isolating tumor cells from normal cells, and enriching distinct clonal populations, clinically relevant genomic aberrations that drive disease can be identified in patients in vivo. An in-depth analysis of clonal architecture and tumor heterogeneity was performed in a stage II chemoradiation-naïve breast cancer from a sixty-five year old patient. DAPI-based DNA content measurements and DNA content-based flow sorting was used to to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes of multiple biopsies from the breast cancer. The detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present within the tumor. The clonal analysis revealed driver events for tumor progression (a heterozygous germline BRCA2 mutation converted to homozygosity within the tumor by a copy number event and the constitutive activation of Notch and Akt signaling pathways. The highlighted approach has broad implications in the study of tumor heterogeneity by providing a unique ultra-high resolution of polyclonal tumors that can advance effective therapies and clinical management of patients with this disease.
ContributorsLaughlin, Brady Scott (Author) / Ankeny, Casey (Thesis director) / Barrett, Michael (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School for the Science of Health Care Delivery (Contributor)
Created2015-05
Description
To identify genes that can lead to obesity of Pima Native American heritage, an array of experiments can be conducted to determine possible candidate genes that can increase the likelihood of being obese in a set population. The studies available to identify these genes were (1) inspect follow-up genes identified by a previous genome wide associations studies, GWAS, previously conducted for the 1120 American Indian subjects data available, (2) to directly sequence candidate genes in literature, (3) to analyze whole sequence data from Native American subjects, and lastly (4) to perform functional studies on most promising variants associated with BMI. Analyzing the results presented from my work required the use of biological techniques such as: DNA sequencing, DNA large scale genotyping, PCR amplification, DNA transfections, DNA ligations, in vitro Luciferase assay and Cell culture. Inspecting the follow-up genes identified by the conducted GWAS showed the potential for the MAP2K3 gene to be a candidate to increase obesity in the set population, involve two single nucleotide polymorphisms (SNPs, rs12882548, rs11652094), to affect body weight through complex mechanisms involving food intake and hypothalamic inflammation. The follow-up genes identified in the GWAS that had an effect on obesity showed to affect it through the mechanism of reducing energy expenditure. Through the analysis of SNPs two variants (rs10507100 and rs17087518) were identified to test their roles in the reduction of energy expenditure. Rs17087518 showed to have a role in a relatively reduced EE resulting in weight gain. Directly sequencing a candidate gene known as MRAP2 showed that the SNP rs1928281 did not have a significant difference on obesity in the Native American subjects (p =.09). Analyzing whole genome sequencing SNPs gave rise to novel variants by association analyses with energy expenditure and BMI in 235 whole genomes, the most significant SNP, rs4984683, was examined to determine the variability in energy expenditures. With set quality control assessment a list of variants were received and were then later assessed with other data available to make a connection to EE. Performing functional studies showed the possibility for rs2001651 and rs1466314 to have an effect on MAP2K3 expression level. The initial functional studies gave way to a more in-depth study of this gene to predict BMI in Caucasians and Native Americans, which in turn showed an association with BMI. The use of these techniques have been an indicator for current research in the determination of candidate genes across many diseases. The works presented is an example of the current works in genetics and an exploration of new mechanism to detect, and possibly treat, disease through personalized sequencing.
ContributorsGale, Alex Mauricio Pompa (Author) / Ankeny, Casey (Thesis director) / Baier, Leslie (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05