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- All Subjects: Cancer
- Creators: School of Life Sciences
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Description
Due to artificial selection, dogs have high levels of phenotypic diversity, yet, there appears to be low genetic diversity within individual breeds. Through their domestication from wolves, dogs have gone through a series of population bottlenecks, which has resulted in a reduction in genetic diversity, with a large amount of linkage disequilibrium and the persistence of deleterious mutations. This has led to an increased susceptibility to a multitude of diseases, including cancer. To study the effects of artificial selection and life history characteristics on the risk of cancer mortality, we collected cancer mortality data from four studies as well as the percent of heterozygosity, body size, lifespan and breed group for 201 dog breeds. We also collected specific types of cancer breeds were susceptible to and compared the dog cancer mortality patterns to the patterns observed in other mammals. We found a relationship between cancer mortality rate and heterozygosity, body size, lifespan as well as breed group. Higher levels of heterozygosity were also associated with longer lifespan. These results indicate larger breeds, such as Irish Water Spaniels, Flat-coated Retrievers and Bernese Mountain Dogs, are more susceptible to cancer, with lower heterozygosity and lifespan. These breeds are also more susceptible to sarcomas, as opposed to carcinomas in smaller breeds, such as Miniature Pinschers, Chihuahuas, and Pekingese. Other mammals show that larger and long-lived animals have decreased cancer mortality, however, within dog breeds, the opposite relationship is observed. These relationships could be due to the trade-off between cellular maintenance and growing fast and large, with higher expression of growth factors, such as IGF-1. This study further demonstrates the relationships between cancer mortality, heterozygosity, and life history traits and exhibits dogs as an important model organism for understanding the relationship between genetics and health.
ContributorsBalsley, Cassandra Sierra (Author) / Maley, Carlo (Thesis director) / Wynne, Clive (Committee member) / Tollis, Marc (Committee member) / School of Life Sciences (Contributor) / School of Human Evolution and Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the USA and throughout the world. Two phenotypes that promote this deadly outcome are the invasive potential of NSCLC and the emergence of therapeutic resistance in this disease. There is an unmet clinical need to understand the mechanisms that govern NSCLC cell invasion and therapeutic resistance, and to target these phenotypes towards abating the dismal five-year survival of NSCLC. The expression of the tumor necrosis factor receptor superfamily, member 12A (TNFRSF12A; Fn14) correlates with poor patient survival and invasiveness in many tumor types including NSCLC. We hypothesize that suppression of Fn14 will inhibit NSCLC cell motility and reduce cell viability. Here we demonstrate that atorvastatin calcium treatment reduces Fn14 expression in NSCLC cell lines. Prior to Fn14 protein suppression, atorvastatin calcium modulated the expression of the Fn14 modulators P-ERK1/2 and P-NF-κβ. Atorvastatin calcium treatment inhibited the migratory capacity in H1975, H2030 and H1993 cells by at least 55%. When chemotactic migration in H2030 cells was induced by the Fn14 ligand TNF-like weak inducer of apoptosis (TWEAK) treatment, atorvastatin calcium successfully negated any stimulatory effects. Inversely, treatment of NSCLC cells with cholesterol resulted in a statistically significant increase in migration. Depletion of Fn14 expression via siRNA suppressed the migratory effect of cholesterol. Finally, atorvastatin calcium treatment sensitized cells to radiation treatment, reducing cell survival. These data suggest that atorvastatin calcium may inhibit NSCLC invasiveness through a mechanism involving Fn14, and may be a novel therapeutic target in NSCLC tumors expressing Fn14.
ContributorsCornes, Victoria Elisabeth (Author) / Stout, Valerie (Thesis director) / Whitsett, Timothy (Committee member) / Carson, Vashti (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Despite the 40-year war on cancer, very limited progress has been made in developing a cure for the disease. This failure has prompted the reevaluation of the causes and development of cancer. One resulting model, coined the atavistic model of cancer, posits that cancer is a default phenotype of the cells of multicellular organisms which arises when the cell is subjected to an unusual amount of stress. Since this default phenotype is similar across cell types and even organisms, it seems it must be an evolutionarily ancestral phenotype. We take a phylostratigraphical approach, but systematically add species divergence time data to estimate gene ages numerically and use these ages to investigate the ages of genes involved in cancer. We find that ancient disease-recessive cancer genes are significantly enriched for DNA repair and SOS activity, which seems to imply that a core component of cancer development is not the regulation of growth, but the regulation of mutation. Verification of this finding could drastically improve cancer treatment and prevention.
ContributorsOrr, Adam James (Author) / Davies, Paul (Thesis director) / Bussey, Kimberly (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis director) / Mason, Hugh (Committee member) / Hariadi, Hugh (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain on the cells. This study aimed to identify aromatic-sensitive native promoters and heterologous biosensors for construction of closed-loop control of efflux pump expression in E. coli. Using a promoter library constructed by Zaslaver et al., activation was measured through GFP output. Promoters were evaluated for their sensitivity to the addition of one of four aromatic compounds, their "leaking" of signal, and their induction threshold. Out of 43 targeted promoters, 4 promoters (cmr, mdtG, yahN, yajR) for styrene oxide, 2 promoters (mdtG, yahN) for styrene, 0 promoters for 2-phenylethanol, and 1 promoter for phenol (pheP) were identified as ideal control elements in aromatic bioproduction. In addition, a series of three biosensors (NahR, XylS, DmpR) known to be inducible by other aromatics were screened against styrene oxide, 2-phenylethanol, and phenol. The targeted application of these biosensors is aromatic-induced activation of linked efflux pumps. All three biosensors responded strongly in the presence of styrene oxide and 2-phenylethanol, with minor activation in the presence of phenol. Bioproduction of aromatics continues to gain traction in the biotechnology industry, and the continued discovery of aromatic-inducible elements will be essential to effective pathway control.
ContributorsXu, Jimmy (Author) / Nielsen, David (Thesis director) / Wang, Xuan (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Programmed cell death ligand-1 (PD-L1) is an overexpressed protein on many tumor cell types. PD-L1 is involved in normal immune regulation, playing an important role in self-tolerance and controlling autoimmunity. However, ligation of PD-L1 to PD-1 on activated T cells leads to tumor-mediated T cell suppression. Inhibiting the PD-1/PD-L1 pathway has emerged as an effective target for anti-tumor immunotherapies. Monoclonal antibodies (mAbs) targeting tumor-associated antigens such as PD-L1 have proven to be effective checkpoint blockades, improving therapeutic outcomes for cancer patients and receiving FDA approval as first line therapies for some cancers. A single chain variable fragment (scFv) is composed of the variable heavy and light chain regions of a mAb, connected by a flexible linker. We hypothesized that scFv proteins based on the published anti-PD-L1 monoclonal antibody sequences of atezolizumab and avelumab would bind to cell surface PD-L1. Four single chain variable fragments (scFvs) were constructed based on the sequences of these mAbs. PCR was used to assemble, construct, and amplify DNA fragments encoding the scFvs which were subsequently ligated into a eukaryotic expression vector. Mammalian cells were transfected with the scFv and scFv-IgG plasmids. The scFvs were tested for binding to PD-L1 on tumor cell lysates by western blot and to whole tumor cells by staining and flow cytometry analysis. DNA sequence analysis demonstrated that the scFv constructs were successfully amplified and cloned into the expression vectors and recombinant scFvs were produced. The binding capabilities of the scFvs constucts to PD-L1 protein were confirmed by western blot and flow cytometry analysis. This lead to the idea of constructing a CAR T cell engineered to target PD-L1, providing a possible adoptive T cell immunotherapy.
ContributorsPfeffer, Kirsten M. (Author) / Lake, Douglas (Thesis director) / Ho, Thai (Committee member) / Hastings, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for a plethora of genome engineering and transcriptional regulation applications. Here we employ engineered variants of CRISPR systems in proof-of-principle experiments demonstrating the ability of CRISPR-Cas derived single-DNA-strand cutting enzymes (nickases) to direct host-cell genomic recombination. E.coli is generally regarded as a poorly recombinogenic host with double-stranded DNA breaks being highly lethal. However, CRISPR-guided nickase systems can be easily programmed to make very precise, non-lethal, incisions in genomic regions directing both single reporter gene and larger-scale recombination events deleting up to 36 genes. Genome integrated repetitive elements of variable sizes can be employed as sites for CRISPR induced recombination. We project that single-stranded based editing methodologies can be employed alongside preexisting genome engineering techniques to assist and expedite metabolic engineering and minimalized genome research.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis director) / Haynes, Karmella (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05