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- Creators: School of Molecular Sciences
Description
Background: Recent interests in continuous biomonitoring and the surge of wearable biotechnology demand a better understanding of sweat as a noninvasive biomarker resource. The ability to use sweat as a biofluid provides the opportunity for noninvasive early and continuous diagnostics. This thesis serves to help fill the existing knowledge gap in sweat biomarker discovery and applications.
Experimental Design: In part one of this study, exercise-induced eccrine sweat was collected from 50 healthy individuals and analyzed using mass spectrometry, protein microarrays, and quantitative ELISAs to identify a broad range of proteins, antibody isotypes, and cytokines in sweat. In part two of this study, cortisol and melatonin levels were analyzed in exercise-induced sweat and plasma samples collected from 22 individuals.
Results: 220 unique proteins were identified by shotgun analysis in pooled sweat samples. Detectable antibody isotypes include IgA (100% positive; median 1230 ± 28 700 pg/mL), IgD (18%; 22.0 ± 119 pg/mL), IgG1 (96%;1640 ± 6750 pg/mL), IgG2 (37%; 292 ± 6810 pg/mL), IgG3 (71%;74.0 ± 119 pg/mL), IgG4 (69%; 43.0 ± 42.0 pg/mL), and IgM (41%;69.0 ± 1630 pg/mL). Of 42 cytokines, three were readily detected in all sweat samples (p<0.01). The median concentration for interleukin-1α was 352 ± 521 pg/mL, epidermal growth factor was 86.5 ± 147 pg/mL, and angiogenin was 38.3 ± 96.3 pg/mL. Multiple other cytokines were detected at lower levels. The median and standard deviation of cortisol was determined to be 4.17 ± 11.1 ng/mL in sweat and 76.4 ± 28.8 ng/mL in plasma. The correlation between sweat and plasma cortisol levels had an R-squared value of 0.0802 (excluding the 2 highest sweat cortisol levels). The median and standard deviation of melatonin was determined to be 73.1 ± 198 pg/mL in sweat and 194 ± 93.4 pg/mL in plasma. Similar to cortisol, the correlation between sweat and plasma melatonin had an R-squared value of 0.117.
Conclusion: These studies suggest that sweat holds more proteomic and hormonal biomarkers than previously thought and may eventually serve as a noninvasive biomarker resource. These studies also highlight many of the challenges associated with monitoring sweat content including differences between collection devices and hydration, evaporation losses, and sweat rate.
Experimental Design: In part one of this study, exercise-induced eccrine sweat was collected from 50 healthy individuals and analyzed using mass spectrometry, protein microarrays, and quantitative ELISAs to identify a broad range of proteins, antibody isotypes, and cytokines in sweat. In part two of this study, cortisol and melatonin levels were analyzed in exercise-induced sweat and plasma samples collected from 22 individuals.
Results: 220 unique proteins were identified by shotgun analysis in pooled sweat samples. Detectable antibody isotypes include IgA (100% positive; median 1230 ± 28 700 pg/mL), IgD (18%; 22.0 ± 119 pg/mL), IgG1 (96%;1640 ± 6750 pg/mL), IgG2 (37%; 292 ± 6810 pg/mL), IgG3 (71%;74.0 ± 119 pg/mL), IgG4 (69%; 43.0 ± 42.0 pg/mL), and IgM (41%;69.0 ± 1630 pg/mL). Of 42 cytokines, three were readily detected in all sweat samples (p<0.01). The median concentration for interleukin-1α was 352 ± 521 pg/mL, epidermal growth factor was 86.5 ± 147 pg/mL, and angiogenin was 38.3 ± 96.3 pg/mL. Multiple other cytokines were detected at lower levels. The median and standard deviation of cortisol was determined to be 4.17 ± 11.1 ng/mL in sweat and 76.4 ± 28.8 ng/mL in plasma. The correlation between sweat and plasma cortisol levels had an R-squared value of 0.0802 (excluding the 2 highest sweat cortisol levels). The median and standard deviation of melatonin was determined to be 73.1 ± 198 pg/mL in sweat and 194 ± 93.4 pg/mL in plasma. Similar to cortisol, the correlation between sweat and plasma melatonin had an R-squared value of 0.117.
Conclusion: These studies suggest that sweat holds more proteomic and hormonal biomarkers than previously thought and may eventually serve as a noninvasive biomarker resource. These studies also highlight many of the challenges associated with monitoring sweat content including differences between collection devices and hydration, evaporation losses, and sweat rate.
ContributorsZhu, Meilin (Author) / Anderson, Karen (Thesis director) / Blain Christen, Jennifer (Committee member) / Gronowski, Ann (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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 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