Matching Items (26)
Filtering by

Clear all filters

136542-Thumbnail Image.png
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
136024-Thumbnail Image.png
Description
Background: Human papillomavirus (HPV) is the cause of 99.7% of cervical cancers. Research of cervical cancer has made this disease mostly curable in the developing world. Head and neck cancer, which is increasingly caused by HPV, still is associated with a mortality rate of 50,000 in the US annually. This

Background: Human papillomavirus (HPV) is the cause of 99.7% of cervical cancers. Research of cervical cancer has made this disease mostly curable in the developing world. Head and neck cancer, which is increasingly caused by HPV, still is associated with a mortality rate of 50,000 in the US annually. This study proposed to evaluate the biology of HPV-16 in head and neck tumors by using RT-qPCR to measure the RNA expression and its relation to physical status of the virus. Methods: This study was to develop an assay that uses RT-qPCR to determine the quantitative expression of HPV-16 RNA coding for proteins E1, E2, E4, E5, E6, and E7 in tumor samples. The assay development started with creation of primers. It went on to test the primers on template DNA through traditional PCR and then on DNA from HPV-16 positive cell lines, SiHa and CaSki, using RT-qPCR. This paper also describes the troubleshooting methods taken for the PCR reaction. Once the primers are verified, the RT-qPCR process can be carried out on RNA purified from tumor samples. Results: No primer sets have been confirmed to produce a product through PCR or RT-qPCR. The primer sequences match up correctly with known sequences for HPV-16 E1, E2, E4, E5, E6, and E7. RT-qPCR showed results consistent with the hypothesis. Conclusion: The RT-qPCR protocol must be optimized to confirm the primer sequences work as desired. Then primers will be used to study physical status and RNA expression in HPV-positive and HPV-negative head and neck tumor samples. This assay can help shed light on which proteins are expressed most in tumors of the head and neck and will aid in the development of future screening and treatment options.
ContributorsKhazanovich, Jakob (Author) / Anderson, Karen (Thesis director) / Mangone, Marco (Committee member) / Sundaresan, Sri Krishna (Committee member) / Barrett, The Honors College (Contributor)
Created2015-05
133045-Thumbnail Image.png
Description
Human papillomavirus (HPV) is the causative agent of cervical cancer. Persistent infection with high-risk HPV 16, 18 or 45 species is associated with the development and progression of cervical cancer. HPV genotyping and Pap smear tests are the regular methods used to detect pre-invasive cervical lesions, but there is a

Human papillomavirus (HPV) is the causative agent of cervical cancer. Persistent infection with high-risk HPV 16, 18 or 45 species is associated with the development and progression of cervical cancer. HPV genotyping and Pap smear tests are the regular methods used to detect pre-invasive cervical lesions, but there is a need for developing a rapid biomarker to profile immunity to these viruses. The viral E7 oncogene is expressed in most HPV-associated cancers and anti-E7 antibodies can be detected in the blood of patients with cervical cancer. This research was focused on viral E7 oncogene expression to be used in development of low-cost point of care tests, enabling patients from low resource settings to detect the asymptotic stage of cervical cancer and be able to seek treatment early. In order to produce the E7 protein in vitro to measure antibody levels, GST tagged E7 genes from HPV 16, 18 and 45 species were inserted into the pDEST15 vector and expressed in E. coli BL21DE3 cells that were induced with 1mM of IPTG. The E7-GST fused expressed protein was then purified using glutathione beads and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein expression was 5.8 \u03bcg/ml for HPV 16E7 in 500 ml culture and for the 500 ml culture of HPV 18 E7 and 45 E7 were 10.5 \u03bcg/ml and 10.5 \u03bcg/ml for HPV 18E7 and 45E7 respectively. High yield values are showing high expression levels of GST-tagged E7 recombinant protein which can be used for serotyping a number of individuals. This shows that HPV E7 can be produced in large quantities that can potentially be used in point of care tests that can help identify women at risk of cervical cancer. In conclusion, the E7 protein produced in this study can potentially be used to induce humoral responses in patients\u2019 sera for understanding the immune response of cervical cancer.
ContributorsMakuyana, Ntombizodwa (Author) / Anderson, Karen (Thesis director) / Ewaisha, Radwa (Committee member) / Varsani, Arvind (Committee member) / Hou, Ching-Wen (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
137663-Thumbnail Image.png
Description
Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV strains, only “high-risk” types, may progress to cancer. Serum antibodies to HPV oncoproteins are stable and specific markers that may

Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV strains, only “high-risk” types, may progress to cancer. Serum antibodies to HPV oncoproteins are stable and specific markers that may be able to detect high-grade cervical intraepithelial neoplasia (CIN3). Biomarkers have potential as a rapid, point-of-care HPV screening tool for low resource areas in the way that traditional cytology cannot, and HPV DNA testing is not yet able to.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.
ContributorsResnik, Jack Isiah (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Purushothaman, Immanuel (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
173897-Thumbnail Image.png
Description

Henrietta Lacks, born Loretta Pleasant, had terminal cervical cancer in 1951, and was diagnosed at The Johns Hopkins University in Baltimore, Maryland, where researchers collected and stored her cancer cells. Those cells went on to become the first immortal human cell line, which the researchers named HeLa. An immortal cell

Henrietta Lacks, born Loretta Pleasant, had terminal cervical cancer in 1951, and was diagnosed at The Johns Hopkins University in Baltimore, Maryland, where researchers collected and stored her cancer cells. Those cells went on to become the first immortal human cell line, which the researchers named HeLa. An immortal cell line is an atypical cluster of cells that continuously multiply on their own outside of the organism from which they came, often due to a mutation. Lacks’s cancer cells enabled scientists to study human cells outside of the human body, though that was controversial since she did not voluntarily donate her cells for such research. Science writer Rebecca Skloot chronicled Lacks’s life in her book, The Immortal Life of Henrietta Lacks, which became a movie in 2017. Lacks’s HeLa cell line has contributed to numerous biomedical research advancements and discoveries and her story has prompted legal and ethical debates over the rights that an individual has to their genetic material and tissue.

Created2020-10-09
Description

The Human Papillomavirus (HPV) strains 16 and 18 are the two most common HPV strains that lead to cases of genital cancer. HPV is the most commonly sexually transmitted disease, resulting in more than fourteen million cases per year in the United States alone. When left untreated, HPV leads to

The Human Papillomavirus (HPV) strains 16 and 18 are the two most common HPV strains that lead to cases of genital cancer. HPV is the most commonly sexually transmitted disease, resulting in more than fourteen million cases per year in the United States alone. When left untreated, HPV leads to high risks of cervical, vaginal, vulvar, anal, and penile cancers. In 1983 and 1984 in Germany, physician Harald zur Hausen found that two HPV strains, HPV-16 and HPV-18, caused cervical cancer in women. In the early twenty first century, pharmaceutical companies Merck & Co. and GlaxoSmithKline created HPV vaccines protecting against HPV-16 and HPV-18, which have reduced the number of HPV infections by fifty-six percent in the US. Discovering HPV strains 16 and 18 allowed physicians to test for those cancer-causing cell populations using Pap smears, a diagnostic tool that collects cells from the woman's cervix to identify cancerous cases of HPV infection. By identifying the cancerous strains of HPV-16 and HPV-18 and utilizing preventative measures such as the Pap smear and HPV vaccines, the rates of cervical cancer and other HPV-related cancers have reduced.

Created2017-05-04
173235-Thumbnail Image.png
Description

From 1977 to 1987, Harald zur Hausen led a team of researchers across several institutions in Germany to investigate whether the human papillomavirus (HPV) caused cervical cancer. Zur Hausen's first experiment tested the hypothesis that HPV caused cervical cancer rather than herpes simplex virus type 2 (HSV-2), the then accepted

From 1977 to 1987, Harald zur Hausen led a team of researchers across several institutions in Germany to investigate whether the human papillomavirus (HPV) caused cervical cancer. Zur Hausen's first experiment tested the hypothesis that HPV caused cervical cancer rather than herpes simplex virus type 2 (HSV-2), the then accepted cause. His second and third experiments detailed methods to identify two previously unidentified HPV strains, HPV 16 and HPV 18, in cervical cancer tumor samples. The experiments showed that HPV 16 and 18 DNA were present in cervical tumor samples. Zur Hausen concluded that HPV, not HSV-2, caused cervical cancer, which enabled researchers to develop preventions, such as the HPV vaccine.

Created2017-03-09
173436-Thumbnail Image.png
Description

Richard Doll was an epidemiologist and public figure in the twentieth and early twenty-first centuries. Working primarily at the University of Oxford, in Oxford, England, Doll established a definitive correlation between cigarette smoking and lung cancer. Furthermore, Doll’s work helped legitimize epidemiology as a scientific discipline. Doll’s research also helped

Richard Doll was an epidemiologist and public figure in the twentieth and early twenty-first centuries. Working primarily at the University of Oxford, in Oxford, England, Doll established a definitive correlation between cigarette smoking and lung cancer. Furthermore, Doll’s work helped legitimize epidemiology as a scientific discipline. Doll’s research also helped establish modern guidelines for oncological studies, as well as for contemporary and future research on the effect of smoking on pregnancy and fetal development. In addition to studying the health effects of smoking, Doll also studied cervical cancer and contraceptives.

Created2017-09-20
173269-Thumbnail Image.png
Description

During the twentieth century, Austin Bradford Hill researched diseases and their causes in England and developed the Bradford Hill criteria, which comprise the minimal requirements that must be met for a causal relationship to be established between a factor and a disease. Hill also suggested that researchers should randomize clinical

During the twentieth century, Austin Bradford Hill researched diseases and their causes in England and developed the Bradford Hill criteria, which comprise the minimal requirements that must be met for a causal relationship to be established between a factor and a disease. Hill also suggested that researchers should randomize clinical trials to evaluate the effects of a drug or treatment by monitoring large groups of people. In addition, Hill advocated for case-control studies, in which researchers compare a group of people with a medical condition to a group without that condition to investigate the condition's possible causes. Hill's own work with clinical trials and case-control studies helped him prove that smoking caused lung cancer. The Bradford Hill criteria have also been used to establish causal links between factors and cancer, including reproductive cancers such as human papillomavirus that causes cervical cancer.

Created2017-06-15
173350-Thumbnail Image.png
Description

George Nicholas Papanicolaou developed the Pap test in the United States during the twentieth century. The Pap test is a diagnostic procedure used to test for cervical cancer in women. Papanicolaou’s work helped improve the reproductive health of women by providing an effective means of identifying precancerous cells and improving

George Nicholas Papanicolaou developed the Pap test in the United States during the twentieth century. The Pap test is a diagnostic procedure used to test for cervical cancer in women. Papanicolaou’s work helped improve the reproductive health of women by providing an effective means of identifying precancerous cells and improving the likelihood of early treatment and survival of cervical cancer.

Created2017-04-06