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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

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
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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

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
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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

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
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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

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
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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,

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
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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

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
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Description
Cancer rates in our nearest relatives are largely unknown. Comparison of human cancer rates with other primates should help us to understand the nature of our susceptibilities to cancer. Data from deceased primates was gathered from 3 institutions, the Duke Lemur Center, San Diego Zoo, and Jungle Friends primate sanctuary.

Cancer rates in our nearest relatives are largely unknown. Comparison of human cancer rates with other primates should help us to understand the nature of our susceptibilities to cancer. Data from deceased primates was gathered from 3 institutions, the Duke Lemur Center, San Diego Zoo, and Jungle Friends primate sanctuary. This data contained over 400 unique individuals across 45 species with information on cancer incidence and mortality. Cancer incidence ranged from 0-71% and cancer mortality ranged from 0-67%. We used weighted phylogenetic regressions to test for an association between life history variables (specifically body mass and lifespan) and cancer incidence as well as mortality. Cancer incidence did not correlate with both body mass and lifespan (p>.05) however, cancer mortality did (p<.05). However, it is uncertain if the variables can be used as reliable predictors of cancer, because the data come from different organizations. This analysis presents cancer incidence rates and cancer mortality rates in species where it was previously unknown, and in some primate species, is surprisingly high. Microcebus murinus(grey mouse lemur) appear to be particularly vulnerable to cancer, mostly lymphomas. Further studies will be required to determine the causes of these vulnerabilities.
ContributorsWalker, William Charles (Author) / Maley, Carlo (Thesis director) / Boddy, Amy (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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Description
The human hairless gene (HR) encodes a 130 kDa transcription factor that is primarily expressed in the brain and skin. In the promoter and 5'-untranslated regions (5'-UTR) of HR, there are three putative consensus p53 responsive elements (p53RE). p53 is a tumor suppressor protein that regulates cell proliferation, apoptosis, and

The human hairless gene (HR) encodes a 130 kDa transcription factor that is primarily expressed in the brain and skin. In the promoter and 5'-untranslated regions (5'-UTR) of HR, there are three putative consensus p53 responsive elements (p53RE). p53 is a tumor suppressor protein that regulates cell proliferation, apoptosis, and other cell functions. The p53 protein, a known tumor suppressor, acts as a transcription factor and binds to DNA p53REs to activate or repress transcription of the target gene. In general, the p53 binding sequence is 5'-RRRCWWGYYY-3' where W is A or T, and R and Y are purines or pyrimidines, respectively. However, even if the p53 binding sequence does not match the consensus sequence, p53 protein might still be able to bind to the response element. The intent of this investigation was to identify and characterize the p53REs in the promoter and 5'-UTR of HR. If the three p53REs (p53RE1, p53RE2, and p53RE3) are functional, then p53 can bind there and might regulate HR gene expression. The first aim for this thesis was to clone the putative p53REs into a luciferase reporter and to characterize the transcription of these p53REs in glioblastoma (U87 MG) and human embryonic kidney (HEK293) cell lines. Through the transactivation assay, it was discovered that p53REs 2 and 3 were functional in HEK293, but none of the response elements were functional in U87 MG. Since p53 displayed a different regulatory capacity of HR expression in HEK293 and U87 MG cells, the second aim was to verify whether the p53REs are mutated in GBM U87 MG cells by genomic DNA sequencing.
ContributorsMaatough, Anas (Author) / Neisewander, Janet (Thesis director) / Hsieh, Jui-Cheng (Committee member) / Goldstein, Elliott (Committee member) / School of Life Sciences (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Laboratory animals represent an invaluable, yet controversial, resource in the field of biomedical research. Animal research has been behind many influential discoveries in the field of emerging therapeutics. They provide the link between the theory of the lab bench and the functional application of medicine to influence human health. The

Laboratory animals represent an invaluable, yet controversial, resource in the field of biomedical research. Animal research has been behind many influential discoveries in the field of emerging therapeutics. They provide the link between the theory of the lab bench and the functional application of medicine to influence human health. The use of animals in research is a consideration which must be heavily weighed, and the implementation must be carried out at a very high standard in order to retain research integrity and responsibility. We are in the process of conducting an experiment using laboratory mice to demonstrate cancer treatment using vaccinia (VACV) mutants as a possible oncolytic therapy for certain strains of melanoma. VACV is a double-stranded DNA poxvirus with a large and easily altered genome. This virus contains many genes dedicated to immune evasion, but has shown sensitivity to cell death by necroptosis in mouse studies (5). We have identified the absence of the kinase RIP3 which is vital in the necroptosis pathway as a potential target for oncolytic therapy using VACV mutants in specific strains of melanoma. Multiple groups of SCID Beige mice were inoculated with different melanoma cell lines and observed for tumor growth. Upon reaching 1 cm3 in volume, tumors were injected with either VACV- Δ83N, VACV- Δ54N, or PBS, and observed for regression. It was hypothesized that melanoma tumors that are RIP3-/- such as the MDA5 cell line will show regression, but melanoma tumors that are RIP3-positive and capable of necroptosis, such as the 2427 cell line, will resist viral replication and continue to proliferate. Our results so far tentatively support this hypothesis, but the data collection is ongoing. Strict and specific protocols with regard to the ethical and responsible use of mice have been implemented and upheld throughout the experiment. Animals are closely monitored, and if their quality of life becomes too poor to justify their continued use in the experiment, they are humanely euthanized, even at the expense of valuable data. The importance of commitment to a high ethical standard is pervasive throughout our work. Animals represent an invaluable contribution to research, and it is important to maintain high standards and transparency with regard to their use. Education and engagement in critical discussions about the use and care of animals in the laboratory contribute to the overall merit and legitimacy of biomedical research in the public and professional eye as a whole, and give legitimacy to the continued use of animals as models to advance science and health.
ContributorsBergamaschi, Julia (Author) / Kibler, Karen (Thesis director) / Jacobs, Bertram (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Bats (order Chiroptera) are the longest lived mammals for their size, with particularly extreme longevity evolving in the family Vespertilionidae, or vesper bats. Because of this, researchers have proposed using bats to study ageing and cancer suppression. Here, we study gene duplications across mammalian genomes and show that, similar to

Bats (order Chiroptera) are the longest lived mammals for their size, with particularly extreme longevity evolving in the family Vespertilionidae, or vesper bats. Because of this, researchers have proposed using bats to study ageing and cancer suppression. Here, we study gene duplications across mammalian genomes and show that, similar to previous findings in elephants, bats have experienced duplications of the tumor suppressor gene TP53, including five genomic copies in the genome of the little brown bat (Myotis lucifugus) and two copies in Brandt's bat (Myotis brandtii). These species can live 37 and 41 years, respectively, despite having an adult body mass of only ~7 grams. We use evolutionary genetics and next generation sequencing approaches to show that positive selection has acted on the TP53 locus across bats, and two recently duplicated TP53 gene copies in the little brown bat are both highly conserved and expressed, suggesting they are functional. We also report an extraordinary genomic copy number expansion of the tumor suppressor gene FBXO31 in the common ancestor of vesper bats which accelerated in the Myotis lineage, leading to 34\u201457 copies and the expression of 20 functional FBXO31 homologs in Brandt's bat. As FBXO31 directs the degradation of MDM2, which is a negative regulator of TP53, we suggest that increased expression of both FBXO31 and TP53 may be related to an enhanced DNA-damage response to genotoxic stress brought on by long lifespans and rapid metabolic rates in bats.
ContributorsSchneider-Utaka, Aika Kunigunda (Author) / Maley, Carlo (Thesis director) / Wilson Sayres, Melissa (Committee member) / Tollis, Marc (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2018-12