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Sundaramurthy, GopinathA Probabilistic Approach for Automated Discovery of Biomarkers using Expression Data from Microarray or RNA-Seq Datasets
PhD, University of Cincinnati, 2016, Medicine: Systems Biology and Physiology
The response to perturbations in cellular systems is governed by a large number of molecular circuits that coalesce into a complex network. In complex diseases, the breakdown of cellular components is brought about by multiple molecular and environmental perturbations. While individual signatures of cellular components might vary significantly among clinical patients, commonality in signs and symptoms of disease progression is a compelling indicator that key cellular sub-processes follow similar trajectories? -. Our approach aims for an enhanced understanding of the effect of disease perturbations on the cell by developing an automated platform that assigns more significance to changes that occur at the sub-network level – focusing on genes that are “wired” together and change together. The platform that we have developed is motivated by the study of concomitant expression changes in sub-networks. The analysis by our platform produces a small subset of signaling and regulatory genes that are wired together and change together beyond random chance. In order to evaluate the effectiveness of our platform in producing subsets that can distinguish diseases and disease-subtypes, we used publicly available RNA-Seq and microarray breast cancer expression datasets. Each dataset was analyzed independently using our platform and the disease related sub-network perturbations among breast cancer subtypes were identified. The resulting subset was subjected to standard multi-way classification and predictions based on our approach were compared with PAM50 predictions. Biomarkers identified from the microarray and RNA-Seq dataset reproduced the PAM50 classification with 100% and 80% agreement respectively despite having only 10% of genes common with the PAM50. This proof-of-concept analysis using breast cancer datasets is indicative of the platform’s stable cross-validation results. This platform can potentially be used for automated and unbiased computational discovery of disease related genes. Our results suggest that probabilistic and automated approaches may offer a powerful complement to existing approaches by providing an unbiased initial screen.

Committee:

Steven Kleene, Ph.D. (Committee Chair); Judith| Heiny, Ph.D. (Committee Member); Anil Jegga, D.V.M. (Committee Member); Jaroslaw Melle, Ph.D. (Committee Member); Yana Zavros, Ph.D. (Committee Member)

Subjects:

Bioinformatics

Keywords:

Biomarker discovery;Probabilistic modeling;Network analysis;Expression Analysis;Complex disease;Genomics

Abulaban, Khalid MThe Role of Urinary Cell-free MicroRNA's as Biomarkers of Lupus Nephritis in Children
MS, University of Cincinnati, 2015, Medicine: Clinical and Translational Research
Background: There is a dire need of non-invasive biomarkers of lupus nephritis (LN) activity. MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs involved in the regulation of host genome expression at the post-transcriptional level that play a role in autoimmune disease. The objective of this study is to investigate the relationship of select urinary cell-free microRNA's in children with active LN vs. extra-renal systemic lupus erythematosus (SLE) vs. controls to the LN-Panel [neutrophil gelatinase associated lipocalin (NGAL), monocyte chemotactic protein 1(MCP1), transferrin (Tf), Beta-trace protein (BTP)] and traditional LN biomarkers. Methods: Select miRNA’s were measured using real-time polymerase chain reaction in the urine pellet (UP) and supernatant (SUP) in 14 patients with active LN ,10 with extra-renal SLE and 10 controls (five juvenile idiopathic arthritis and five fibromyalgia patients) prospectively every 3 months. Disease activity was measured by the systemic lupus erythematosus disease activity index (SLEDAI). Urine samples were assayed for the LN-Panel and traditional biomarkers were recorded. Results: MiR-125a, miR-150, and miR-155 in the SUP had moderate to good positive correlation with all LN-Panel (r = 0.35 – 0.68), but had weak correlation with SLEDAI and the traditional biomarkers (r-<0.3). On the other hand measurement of the miRNA’s in the UP did not correlate with any measure. Conclusion: The urinary supernatant of miRNAs miR-125a, miR-150, and miR-155 are differentially excreted in the urine with active LN. Our evaluation suggests that cell-free urinary miRNA complement, but not superior to the newer LN Panel in their ability to assess concurrent LN activity.

Committee:

Erin Nicole Haynes, Dr.P.H. (Committee Chair); Hermine Brunner, M.D. (Committee Member); Michael Bennett, Ph.D. (Committee Member); Jun Ying, Ph.D. (Committee Member)

Subjects:

Surgery

Keywords:

SLE;lupus nephritis;biomarker;microRNA

Grigsby, Claude CurtisA Comprehensive Tool and Analytical Pathway for Differential Molecular Profiling and Biomarker Discovery
Doctor of Philosophy (PhD), Wright State University, 2013, Biomedical Sciences PhD
The key requirements to any empirically based study are to: (1) accurately measure and then compare the collected results in determining the result of the hypothesis being tested; and (2) collect a sample representative of the entities being studied. To demonstrate that an informatics tool can be designed that provides spectral registration, spectral and chromatographic alignment, visualization, and comparative analysis for data generated from multiple analytical platforms, e.g., LC-MS and GC-MS, the results and data analysis of five unique sets of experiments using a suite of novel informatics tools are presented. Comprehensive and reproducible sample collection techniques were developed concomitantly with the informatics tool and used in the multiple, independent studies for the validation and further development of generated software tools and approaches. Data from a dose-response study examining an organ specific environmental toxicant exposure was analyzed using the prototype software tool for discovery of LC/MS based metabolomic biomarkers. This data set served as proof of concept in the development and illustration of the novel approach to spectral registration and visualization, and illustrates the rapid multi-sample analysis capability of the informatics tool. A variety of additional studies focused on volatile biomarker discovery, i.e., a murine model of infection to select agents, characterization of human and murine urine as it ages, human markers of age and ethnicity in axillary odors, and characterization of the binding between volatile ligands and murine major urinary proteins aided in algorithm and interface development for GC/MS functionality implemented in the developed software. The final phase of this work focused on utilization of these analysis tools in combination with novel sampling techniques to create an end-to-end discovery pipeline for large-scale small molecule and volatile organic compound biomarker and differential profiling studies. This combination of biologically and environmentally focused studies were successfully completed as final proof of concept for this work and demonstrate the universal utility of the approach.

Committee:

David Cool, PhD (Advisor); Mateen Rizki, PhD (Committee Member); Gerald Alter, PhD (Committee Member); Thomas Lamkin, PhD (Committee Member); Jeffery Gearhart, PhD (Committee Member)

Subjects:

Bioinformatics; Biomedical Research; Toxicology

Keywords:

biomarker; volatile organic compound, differential profiling, GCMS, mass spectral analysis

Kauffman, Ross M.Smoking and Tobacco in Ohio Prisons
Doctor of Philosophy, The Ohio State University, 2009, Public Health

The rate of incarceration in the United States has grown steadily since the mid-1970s to a point where 1% of American adults are currently being held in jails and prisons. Prisoners are sicker than the general population with a high prevalence of many negative health behaviors. Tobacco use is one such behavior, with past surveys finding that up to 85% of prisoners smoke. The Smoking and Tobacco in Ohio Prisons (STOP) Project was undertaken to examine the impact of incarceration on smoking behaviors and lay the groundwork for further research into tobacco use in prisons. A total of 200 recently-arrived male inmates, drawn from two Ohio prisons, were surveyed about their tobacco use.

Several measures of tobacco use were evaluated as part of the study. Self-reports were found to be a valid measure of tobacco use in the sample, based on biomarker confirmation (sensitivity = 98.5%, specificity = 88.9%). Carbon monoxide breath tests were less valid (sensitivity = 85.4%, specificity = 91.7%), especially among occasional smokers; however, as a non-invasive, inexpensive way of measuring short-term smoking they may still offer a useful measure in some research contexts. Using an enzyme immunoassay to measure salivary cotinine offers a less-expensive alternative to the gold standard liquid chromatography tandem mass spectrometry, however the current study found that economic savings are moderated by false-positive results (sensitivity = 94.2%, specificity = 100.0%).

The prevalence of current tobacco use was high prior to (77.5%) and during incarceration (82.0%). While entry into the prison was found to be associated with increases in the number of smokers, average cigarette consumption declined from 15.7/day to 8.6/day (p < 0.001). Smokeless tobacco use, including dual use of cigarettes and smokeless tobacco, increased following incarceration, especially among those sentenced from Appalachian counties. The tobacco use survey also exposed a gap between prison policy and prisoners' practice. A majority of smokers (51.2%) reported smoking indoors at least once since their arrival, and more than a third (34.1%) reported that they smoked inside on a daily basis. Despite widespread tobacco use among prisoners, a majority of users (70%) expressed a desire to quit smoking, indicating a high demand for effect cessation assistance.

Preliminary data were collected on participants' adverse childhood experiences (ACEs) using an audio computer-assisted self-interview system. Response rates for these questions, which covered sensitive topics including sexual abuse and household dysfunction, were high (>97%). Past studies in the general population have linked ACEs to smoking behavior. Non-significant trends in the current study suggest the existence of a similar relationship in incarcerated populations, however the limited sample size does not allow for the clear demonstration of an association.

Committee:

Amy Ferketich, PhD (Committee Chair); David Murray, PhD (Committee Member); Mary Ellen Wewers, PhD (Committee Member); Paul Bellair, PhD (Committee Member)

Subjects:

Epidemiology; Public Health

Keywords:

prison; tobacco; smoking; Ohio; biomarker; adverse childhood experiences; incarceration; epidemiology; tobacco policy

Fitzgerald, Sarah E.A Meta-Analysis of the Diagnostic Performance of Procalcitonin in the Diagnosis of Serious Bacterial Infection in Pediatric Febrile Neutropenia
MS, University of Cincinnati, 2012, Medicine: Clinical and Translational Research

Background:

Continued advancements and intensification of chemotherapy regimens have enhanced outcomes for children with cancer. With more aggressive therapy, however, infection rates due to immune system suppression have increased1. Although progress in supportive care strategies has improved management of such infections2, infection remains a major cause of morbidity and mortality among pediatric oncology patients3. Serious bacterial infections often present non-specifically with only fever and neutropenia; however, infection represents only one etiology of fever and neutropenia.

Objective:

Identification of a biomarker to more quickly diagnose serious infection in fever and neutropenia episodes would be advantageous. Prior small studies suggest that procalcitonin may be a feasible and reliable marker for bacterial infection in febrile neutropenic pediatric patients. Because the implementation of such a biomarker would be significant to pediatric oncology practice, stronger evidence is required before the assay can be meaningfully, reliably, and safely applied to patient care.

Design/Methods:

We conducted a systematic review and meta-analysis of the literature regarding the use of procalcitonin as a marker of serious infection in neutropenic febrile children. We searched several electronic databases that included records from 1966 to July 2011 using the keyword ¿¿¿¿¿¿¿procalcitonin¿¿¿¿¿¿¿ alone and in combination with several terms related to our topic.

Results:

The ten selected studies included 591 pediatric oncology patients with a total of 1161 febrile episodes. Regression analysis and the random effects model were used to estimate the common effect size for each measure of diagnostic performance for procalcitonin: pooled odds ratio was 39.29, with 95% confidence interval (12.61, 122.4); sensitivity was 0.709, or 70.9%, with p<0.0001 and 95% confidence interval (0.357, 1.06); specificity was 0.826, or 82.6%, with p<0.0001 and 95% confidence interval (0.629, 1.02).

Conclusions:

Procalcitonin is an appropriate candidate biomarker for serious bacterial infection in pediatric oncology fever and neutropenia patients, given that the odds of having an elevated procalcitonin assay and a serious bacterial infection are 39.29 times higher than the odds of having an elevated procalcitonin assay in the absence of serious bacterial infection. Furthermore, this meta-analysis produced evidence of modest to good sensitivity and better specificity with values of 71% and 83%, respectively.

References:

1. Finberg R. and Talcott J., New England Journal of Medicine, 1999. 2. Rubnitz J., Lensing S., Zhou Y., et al, Cancer, 2004. 3. Slats A., Egeler R., Van Der Does-van den Berg A., et al, Leukemia, 2005.

Committee:

Erin Nicole Haynes, PhD (Committee Chair); Paul Succop, PhD (Committee Member); Jeffrey Welge, PhD (Committee Member)

Subjects:

Oncology

Keywords:

procalcitonin;fever and neutropenia;pediatric oncology;biomarker;;;

Gu, JiayinBiomarkers for Age-Related Macular Degeneration
Doctor of Philosophy, Case Western Reserve University, 2009, Chemistry
The goal of this thesis research was to develop methods to predict susceptibility to age-related macular degeneration (AMD). AMD is the most common cause of legal blindness in the elderly in developed countries, and early identification of susceptibility is important for preventing this progressive disease. In the thesis, new evidence supporting the prognostic potential of plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies as biomarkers for AMD is presented. CEP adducts and autoantibodies were quantified by ELISA from 916 AMD and 488 control donors and mean levels were found to be elevated in AMD plasma by ~60% and ~30%, respectively. For individuals with both CEP markers elevated, the odds ratio for AMD risk based on marker concentrations alone was ~ 3 fold greater in AMD than in control donors. The AMD risk predicted for individuals carrying homozygous genotypes conferring major AMD risk and exhibiting elevated CEP marker concentrations was 2-3 fold greater than the risk based on genotype alone. Mass spectrometric analysis was performed on peptides immunocaptured from AMD and control plasma to identify peptidomic patterns as potential biomarkers for AMD. Peptide patterns differentiating AMD and control were obtained from bioinformatic analyses of MALDI-TOF spectra. These profiles provided 88% correct classification of the plasma as either AMD or control with 80-87% sensitivity and 80-90% specificity. In retinal light damage studies, levels of CEP adducts and autoantibodies in rat plasma and CEP immunoreactivity in rat retina and RPE were found to be significantly increased after intense light exposure. CEP adduct concentration and autoantibody titer were reduced by pretreatment with DMTU, a scavenger of reactive oxygen species, or experimental drugs prior to the light exposure. These observations support the application of CEP biomarkers for monitoring oxidative damage in animal models and for evaluating the efficacy of therapeutics. Overall, this thesis provided evidence that CEP plasma biomarkers offer a potential early warning system for predicting AMD susceptibility and monitoring the efficacy of AMD therapeutics.

Committee:

Robert G. Salomon, PhD (Advisor); John W. Crabb, PhD (Advisor); Mary D. Barkley, PhD (Committee Chair); Michael Zagorski, PhD (Committee Member); Alfred B. Anderson, PhD (Committee Member); Stephanie A. Hagstrom, PhD (Committee Member)

Subjects:

Biochemistry; Chemistry

Keywords:

age-related macular degeneration; biomarker; carboxyethylpyrrole; oxidative modification; light damage; peptidomic profiling

Zheng, QiaoyunStudy of Cancer Related Proteins: LRG-1 and PD-L1
Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2017, College of Sciences and Health Professions
PROJECT I: In this study, we used the proteomic method to identify a potent biomarker candidate as leucine-rich-a-2-glycoprotein-1 (LRG-1) for cancer from the urine of patients with hepatocellular carcinoma. Further screening revealed that LRG-1 was also present in the urine of patients with a wide range of cancer types and inflammation. We found that LRG-1 could be secreted outside cells as a glycosylated form. To characterize LRG-1, we systematically studied the structure and function of this protein by using chemical N-link and/or O-link glycosylation inhibitors and site-directed mutagenesis, and showed that glycosylation of LRG-1 was able to prevent its degradation by a protease although its secretion was independent on glycosylation. Interestingly, cells deficient LRG-1 migrated faster than wild types cells and the expression of Cyclooxygenase-2 (COX-2) was down-regulated in T cells after incubated with the glycosylated form of LRG-1, suggesting a role of LRG-1 in anti-inflammation. Our findings provide very useful information for developing LRG-1 as a noninvasive urinary biomarker for diagnosis of cancer and inflammatory diseases. PROJECT II: Programmed death-ligand 1 (PD-L1) on cancer cells interacts with programmed cell death protein 1(PD1) of immune cells, resulting in suppression of anti-tumor immunity. Various factors contribute to the high expression of PD-L1 in cancer cells and lead to the escaping from the immune system. Targeting the PD-L1 and PD1 pathway is a novel strategy to restore the immunity and re-activate the immune system to abolish the tumor. Currently, the main research focusing on the pathway uses antibody drugs to block the interaction between PD-L1 and PD1. There is yet no any research effort focusing on the blockade of the PD-L1 expression in cancer cells. Small molecule drugs that can downregulate the PD-L1 expression in cancer tissue have multiple advantages such as penetration of the blood brain barrier compared to antibody drugs. To search for such type of potential lead compounds, a luciferase-based reporter assay was designed, developed, and validated to identify small molecules that can manipulate the expression of PD-L1 in cancer cells. By screening 1680 small molecule compounds, several of them were found to affect the PD-L1 expression in cancer cells.

Committee:

Aimin Zhou, Ph.D. (Committee Chair); Michael Kalafatis, Ph.D. (Committee Member); Xue-Long Sun, Ph.D. (Committee Member); Nolan Holland, Ph.D. (Committee Member); Baochuan Guo, Ph.D. (Committee Member); Bin Su, Ph.D. (Other)

Subjects:

Biomedical Research; Health Sciences; Molecular Biology; Pharmacy Sciences

Keywords:

LRG-1, glycosylation, urinary biomarker, inflammation, PD-L1, reporter luciferase assay, cancer, immunotherapy

Allison, RyanThe Commercialization of a Novel Cell Retrieval Device and Diagnostic Biomarker for Barrett's Esophagus
Master of Sciences, Case Western Reserve University, 2015, Cell Biology
Barrett’s Esophagus (BE) is a metaplastic lesion in the distal esophagus that predisposes the esophageal lining to the development of esophageal adenocarcinoma (EAC). The tumorigenic risk, while low during the patient’s lifetime, has prompted the need for better screening technologies that are less invasive, less costly, and better at stratifying the risk of EAC. The commercialization of one such novel technology, including the different pathways and strategies available, has undergone a comprehensive assessment. The novel cell retrieval device incorporates a design that will aid in providing more accurate diagnostic capabilities, while methylation of the Vimentin (VIM) gene has shown promise as an indicator of epigenetic alterations indicating the conditions of BE and EAC. This thesis explores the basic science and epidemiology of the disease state associated with BE, and continues with a comprehensive analysis of the business-related aspects covering the addressable market, competition, regulatory and reimbursement strategies, and funding.

Committee:

Christopher Cullis (Advisor)

Subjects:

Biomedical Engineering; Cellular Biology; Molecular Biology; Oncology

Keywords:

barretts esophagus; methylation; epigenetics; medical device; commercialization; entrepreneurship; biomarker; assay; adenocarcinoma; gastroesophageal reflux disease

Harshman, Sean WilliamCharacterization of Histone H1 and Extracellular Vesicles by Mass Spectrometry
Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program
The use of mass spectrometry to analyze difficult samples and to identify large numbers of proteins has been used in many applications. Utilizing mass spectrometric approaches, I first developed a method for isolation and analysis of histone H1 from different cellular compartments. This study showed that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Utilizing the developed method, the soluble linker histones were profiled throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. To demonstrate the utility of the analysis, histone H1.2-H1.5 translocation to the cytosol was monitored in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell. Next, I characterized the histone H1 phosphorylation from breast cancer cells. The results from this study show the extent of H1 phosphorylation can distinguish between the different cell lines. The phosphorylation at threonine 146 was found to be a site of mitotic histone H1 phosphorylation with potential biological implications. To explore the prognostic value of H1 phosphorylation, extracellular stimulus, estradiol or kinase inhibitor LY294002, were applied to cells in vitro to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation (T146) can increase and decrease in response to the extracellular stimuli applied. Further exploration of this hypothesis was conducted by staining primary breast tumors for T146 phosphorylation. Variable staining patterns were observed across tumor grades and subtypes with pT146 labeling and associate with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer. Finally, I identify the protein content of extracellular vesicles isolated from multiple myeloma (MM) cell lines. These two studies show the protein content and relative abundance of vesicles derived from four distinct MM cell lines. Additionally the localization of serum CD44 is shown to be on the peripheral vesicles of MM patients. The utility of serum CD44 as a biomarker in MM is shown through positive correlation with the accepted MM biomarker beta 2-microglobulin. These results generate a foundation for hypothesis driven studies of MM biology and the potential use of serum CD44 as a novel serum biomarker of MM. Collectively, our results utilize mass spectrometric approaches to solve biological problems across two distinct disease states.

Committee:

Michael Freitas, Ph.D (Advisor); Jeffrey Parvin, M.D., Ph.D (Committee Member); Mark Parthun, Ph.D (Committee Member); Mitch Phelps, Ph.D (Committee Member)

Subjects:

Biomedical Research

Keywords:

Mass Spectrometry; Histone H1; Biomarker; Exosomes; Microvesicles; Extracellular Vesicles; Shotgun Proteomics

Telu, Kelly H.Development of Mass Spectrometric Methods for Biomarker Discovery
Doctor of Philosophy, The Ohio State University, 2012, Chemistry

The subject of this dissertation is the development of mass spectrometry-based methods for the discovery of proteomic biomarkers. Two different projects are presented herein. Chapters 2-4 describe the discovery of protein biomarkers for early detection and/or to monitor bladder cancer recurrence, progression, and response to treatment by the use of mass spectrometry-based proteomics. Chapter 5 describes a mass spectrometry workflow for the identification of therapeutic biomarkers associated with Silvestrol treatment in chronic lymphocytic leukemia (CLL).

Chapter 2 focuses on the development of histone H1 phosphorylation as a diagnostic and prognostic biomarker. The post-translational modifications of histones extracted from bladder cancer cells were analyzed by liquid chromatography mass spectrometry (LC-MS). Increased phosphorylation of the H1 histones in invasive (J82, T24 and UMUC3) compared to non-invasive (RT4) bladder cancer cell lines and cultured epithelial cells was discovered. The epithelial cells demonstrated the least histone phosphorylation compared to all others. Immunohistochemistry (IHC) was performed on one replicate of patient tissue samples to determine if H1 phosphorylation can indicate invasive cells in human tissues and preliminary results are positive. IHC results showed increased H1 phosphorylation in bladder cancer tissues compared to normal urothelium.

Chapter 3 focuses on histone H1 phosphorylation as a pharmacodynamic biomarker. Treatment with AR-42, a novel histone deacetylase (HDAC) inhibitor developed at the Ohio State University (OSU), resulted in a reduction of phosphorylation along with an increase in acetylation as determined by LC-MS. For the invasive cell lines, this reduction in phosphorylation returned the H1 histones to a relatively normal phosphorylation pattern, while reducing cell proliferation and enhancing apoptosis. Histone H1 phosphorylation status can be an informative marker for malignant bladder cancer cells and serve as a downstream end-point for drug therapy. These results further support the hypothesis that AR-42 may be effective in the treatment of human bladder cancer.

Chapter 4 evaluates H1 phosphorylation as a pharmocodynamic biomarker in treatment with the phytochemicals sulforaphane (SFN) and erucin (ECN). SFN has been reported to be a histone deacetylase inhibitor (HDACi). We used histone LC-MS profiling to ascertain the extent of H3 and H4 acetylation changes induced by SFN and ECN at treatments and time-points that mimic more physiological therapeutic dosages. While we found no evidence of an increase in acetylation on the core histones, we did find a decrease in phosphorylation of the linker histones and an increase in phosphatase activity (PP1 and PP2A). These findings have never been reported in the literature before. Our findings are consistent with the report that SFN is an HDACi, although histones may not be a robust target of the phytochemical.

In Chapter 5, a mass spectrometric workflow for the identification of therapeutic biomarkers after Silvestrol treatment in CLL was developed. It consisted of lysis of patient sample cells in RapiGest followed by trypsin digestion and LC-MS/MS. Label-free quantitation was carried out through a spectral counting technique. EdgeR was used for the statistical analysis of the data.

Committee:

Michael Freitas, PhD (Advisor); Thomas Magliery, PhD (Committee Member); Anne Co, PhD (Committee Member)

Subjects:

Chemistry

Keywords:

Mass Spectrometry; Proteomics; Biomarker; Histone H1 Phosphorylation

Brunst, Kelly J.Epigenetic Biomarkers of Diesel Exhaust Exposure and Pediatric Respiratory Health
PhD, University of Cincinnati, 2012, Medicine: Epidemiology (Environmental Health)

Background: Global, interferon-gamma (IFN-¿¿¿¿) and forkhead box transcription factor 3 (FOXP3) methylation are associated with air pollution exposures and have shown to be modified by common glutathione S-transferase (GST) variants. It has yet to be established if chronic diesel exhaust exposure during childhood is associated with global methylation levels or alterations in the promoters of IFN-γ and FOXP3 and whether the epigenetic changes are associated with the respiratory health effects, such as wheezing and asthma development, of diesel exhaust particles (DEP) exposure.

Objectives: The objectives of this dissertation were threefold; 1) determine if chronic childhood DEP exposure is associated with global methylation and gene-specific methylation of IFN-¿¿¿¿ and FOXP3, 2) examine the modifying effects of the GST Pi-1 (GSTP1) isoluecine105valine polymorphism, and 3) evaluate the relationship between global and gene-specific methylation patterns and wheezing and asthma development during childhood.

Methods: Children were randomly selected from the Cincinnati Childhood Allergy and Air Pollution birth cohort (n=92). DEP exposure from birth through age seven was estimated from a land-use regression model using a complete address history. Children with consistent DEP exposure throughout childhood and no exposure to tobacco smoke were eligible for inclusion. Global methylation was assessed as total methylated cytosine, IFN- ¿¿¿¿ promoter methylation was determined by methylation-specific polymerase chain reaction (MS-PCR), and FOXP3 promoter methylation was determined by pyrosequencing. GSTP1 genotype was dichotomized as carriers and noncarriers of the Val105 allele. The association between DNA methylation and DEP exposure was analyzed using generalized linear regression (adjusted for an interquartile range increase in DEP of 0.14 ¿¿¿¿g/m3) and logistic regression (adjusted for a 1 standard deviation increase in methylation) was used for respiratory outcomes (longitudinal wheezing phenotypes and asthma).

Results: Chronic exposure to high levels of DEP resulted in significantly lower global methylation (p<0.01); this effect was over two-times greater among children carrying the GSTP1- Val105 allele (¿¿¿¿, -0.48; 95%CI: -0.88, -0.08) compared to non carriers (¿¿¿¿, -0.21; 95%CI: -0.46, 0.03). Significant hypermethylation of the IFN-? promoter was observed as a result of increased DEP exposure (p<0.01) but was not modified by GSTP1genotype. Similar to IFN-¿¿¿¿, increases in estimated DEP exposure were associated with increases in FOXP3 methylation (P<0.05), although this effect was not modified by GSTP1 genotype. In addition, children with increased FOXP3 methylation were also at greater risk for developing persistent wheezing (OR=3.05; 95% CI, 1.54-6.05), early transient wheezing (OR=1.57; 95% CI, 1.01-2.41), and asthma (OR=2.1; 95%CI, 1.14-3.85). Neither global nor IFN-¿¿¿¿ methylation was associated with wheezing or asthma development.

Conclusions: Global methylation was significantly lower among children carrying the valine allele suggesting an indirect role of DEP exposure through oxidative stress mechanisms. Increasing DEP exposure was associated with increasing IFN-¿¿¿¿ and FOXP3 methylation but these associations were not modified by GSTP1 genotype, suggesting the changes in methylation may result directly from exposure to DEP rather than indirectly through oxidative stress. FOXP3 methylation may serve as a clinically relevant mediating predictor for wheezing and/or asthma risk in children exposed to traffic-related air pollution.

Committee:

Shuk-Mei Ho, PhD (Committee Chair); Gurjit Hershey, MD PhD (Committee Member); Yuet Kin Leung, PhD (Committee Member); Linda Levin, PhD (Committee Member); Patrick Ryan, PhD (Committee Member)

Subjects:

Epidemiology

Keywords:

Epigenetics;Asthma;Wheezing;Diesel Exhaust;Methylation;Biomarker;

Kai, JunhaiProtein Lab-on-a-Chips on Polyer Substrates for Point-of-Care Testing (POCT) of Cardiac Biomarkers
PhD, University of Cincinnati, 2006, Engineering : Electrical Engineering

Cardiac disease is the major cause of death in patients with end-stage renal disease, accounting for about 40% of all deaths. Cardiovascular diseases account for a significant number of mortalities in the United States especially in the 50 – 65 age range. The fast growing numbers of such patients generate great demand for a commercially available lab-on-a-chip which is able to carry out not only fast, reliable, and accurate measurements but also in portable or disposable format at low cost.

In this research, a disposable protein lab-on-a-chip on a Cyclic-Olefin Copolymer (COC) is designed, fabricated and calibrated for monitoring human cardiac biomarkers. The multi-analyte protein lab-on-a-chip is able to perform rapid and accurate measurement of multiple cardiac biomarkers in human serum, such as CRP, Myoglobin, c-Tnl and BNP. By integration into a portable analyzer system, point-of-care testing (POCT) of cardiac biomarker measurement can be realized.

Comparing with commercially available cardiac biomarker diagnostic systems, this work mainly focuses on an inexpensive platform for disposable biochip applied for multi-analyte point-of-care testing. Many novel methods and technologies are then developed and extensively tested such as low-cost polymer fabrication process and self-spotting antibody patterning.

The proposed protein lab-on-a-chip will be able to measure multiple types of cardiac biomarkers in human serum, such as CRP, Myoglobin, c-Tnl and BNP. In the proposed protein lab-on-a-chip, fluorescence and chemiluminescence based solid phase immunoassay techniques will be applied for sensing. In this work, multiple parameters that can affect cardiac biomarker immunoassay performances in protein lab-on-a-chip are extensively studied and used for computer-based simulation. The protein lab-on-a-chip configuration is then characterized and optimized based on the simulation and experimental results. By integration into a portable optical detection system and an automatic microfluidic control system, point-of-care testing of human cardiac biomarkers can be realized. In addition, by adopting different types of antibodies, the proposed protein lab-on-a-chip also can be easily extended to the detection of other biomarkers and proteins for clinical diagnostics.

Committee:

Dr. Chong Ahn (Advisor)

Keywords:

Lab-on-a-chip; Cardiac biomarker; multi-analyte; Protein chip; Chemiluminescence; immunoassay; BioMEMS; Polymer microfabrication

Wei, LiPart I: Oxidative Modification of Ethanolamine Phospholipids by Isolevuglandins: Detection by LC-MS/MS in Vitro and in Vivo Part II: Total Synthesis of C22-11-isolevuglandin E4
Doctor of Philosophy, Case Western Reserve University, 2010, Chemistry

Levuglandins (LGs) and isolevuglandins (isoLGs), formed by rearrangement of endoperoxide intermediates generated through the cyclooxygenase and free radical induced oxidation of arachidonates, are extraordinarily reactive, forming covalent adducts incorporating protein lysyl epsilon-amino groups. Because they accumulate, these adducts provide a dosimeter of oxidative injury.

The thesis reports the detection in vivo and quantitative analysis of LG/isoLG adducts that incorporate the amino group of phosphatidylethanolamines (PEs) into LG/isoLG-hydroxylactams. Notably, liquid chromatography-tandem mass spectrometry detection of these hydroxylactams is achieved with samples that are an order of magnitude smaller and sample processing is much simpler and less time-consuming than required for measuring protein-derived LG/isoLG-lysyl-lactams. A key feature of our protocol is treatment of biological phospholipid extracts with phospholipase A2 to generate mainly 1-palmitoyl-2-lysoPE-hydroxylactams from heterogeneous mixtures of phospholipids with a variety of acyl groups on the 2-position. LG/isoLG-PE-hydroxylactam levels in plasma from patients with age-related macular degeneration (N = 15, 5.2 ± 1.4 ng/ml) were significantly elevated (P < 0.0001) compared to healthy volunteers (N = 15, 3.4 ± 0.5 ng/ml). Two-fold higher levels (P = 0.00021) were detected in liver from chronic ethanol-fed (27% of calories for 4 weeks) mice (C57BL/6 female, N = 6, 32.4 ± 6.3 ng/g) compared to pair-fed animals (N = 4, 12.1 ± 1.5 ng/g).

C22-11-isolevuglandin E4(10-acetyl-11-formyl-14-hydroxynonadeca-4(Z),7(Z),12(E),16(Z)-tetraenoic acid) is one of 128 structurally isomeric levulinaldehyde derivatives that can be generated during the free radical-induced oxidation of docosahexaenoic acid (DHA). Its synthesis was accomplished by conjugate addition of a higher order vinyl cyanocuprate to a gamma-alkoxyenone to construct the carbon skeleton. A lower side chain synthon was prepared by nucleophilic addition of a beta-stannyl vinyllithium to a volatile and chemically sensitive beta, gamma-ethylenic aldehyde. Basic and acidic hydrolysis in a sequence of functional and protecting group manipulations delivered C22-11-isoLGE4 if precautions were taken to avoid free-radical induced oxidation of this air sensitive product. The synthesis of C22-11-isoLGE4 provides an authentic standard for characterization of biological samples and products of DHA oxidation in vitro.

Committee:

Robert G. Salomon, PhD (Advisor); Anthony J. Pearson, PhD (Committee Chair); Michael G. Zagorski, PhD (Committee Member); James D. Burgess, PhD (Committee Member); Laura E. Nagy, PhD (Committee Member)

Subjects:

Chemistry

Keywords:

levuglandin; isolevuglandin; phosphatidylethanolamine; oxidative stress; biomarker; free radical; mass spectrometry

Trexler, Ryan VincentLipid Analysis and Microbial Community Characterization of Subsurface Shale
Master of Science, The Ohio State University, 2017, Environmental Science
Black shale formations are being increasingly relied upon for energy extractions through the engineered processes of hydraulic fracturing and horizontal drilling. Although some deep terrestrial environments are known to harbor microbial life, little is know of the diversity and function of microbial communities in black shale formations. Our understanding of microbial life in black shale has been hampered by the need to drill into the subsurface with fluids and media containing surface microbial contaminants (e.g. drilling muds), the sample chemistry (i.e. high salt and organics) of the rock, and low expected densities of indigenous microbial cells. This thesis first examined the efficacy of seven extraction methods on PLFA yield, profile quality, and reproducibility from black shale. In all, three lipid extraction methods (modified Bligh and Dyer, modified Folch, and Microwave-assisted) and four variations (Citrate Buffer and Escherichia coli, Mg2+, and POPC spikes) of the modified Bligh and Dyer method were investigated and results demonstrated that adding an intact phospholipid spike (POPC) to the modified Bligh and Dyer extraction mixture provided a 6-fold increase in PLFA yield and better reproducibility among methods tested. This method was subsequently applied to sidewall cores collected from a borehole drilled into the Marcellus shale in an effort to investigate the presence of biomass across the interface of the Marcellus and Mahantango formations. Two drilling mud samples as ii well as paired samples from brine washes collected during core cleaning were also analyzed. PLFA biomass, richness, and profiles of the drilling muds were distinct from those of the brine wash and subsurface cores and were marked with significant amounts of Pseudomonas and Fungal lipid biomarkers. Drilling muds were ~7-fold higher in PLFA biomass than that of the three core samples. Core samples yielded two to six-fold higher biomass than previous PLFA studies of deep subsurface black shale formations though PLFA richness was generally similar. Here, core samples were distinguished by unique PLFAs with epoxy-, keto-, and dimethyl ester fatty acids not found in the drilling muds. This data indicates that recent viable microorganisms dwell within the Marcellus shale and Mahantango formations.

Committee:

Paula Mouser, Dr. (Advisor); Kelly Wrighton, Dr. (Committee Member); Michael Wilkins, Dr. (Committee Member)

Subjects:

Environmental Engineering; Environmental Science; Geology; Microbiology

Keywords:

Subsurface; Marcellus; Shale; PLFA Profile; PLFA Extraction; Microbial Community; Biomarker; Mahantango

Frankhouser, David EMethylome Analysis: From Computation Workflow Development to Implementation in a Breast Cancer Prevention Trial
Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences
Cancer research is rapidly advancing toward more personalized treatments and diagnostics. The major driving force of this advancement are the technological developments of sequencing which has resulted in greatly reduced costs. The democratization of sequencing assays has produced a unique set of challenges that must be addressed in order to successfully conduct clinical research. DNA methylation (DNAm) has increasingly been assayed by sequencing due to its role in human disease and cancers, including breast cancer. DNAm is an epigenetic modification and primarily functions to regulate gene expression. In high-risk breast cancer subtypes, the focus of our most recent research, DNAm is able to predict survival for subtypes with no molecular markers of risk. There are many sequencing assays for DNAm, but the two most common approaches are methylation capture (MethylCap-seq) and bisulfite conversion (BS-seq). As with any sequencing analyte, choosing the appropriate approach for a study is made more difficult by the continual improvements made to the methods and the sequencing technologies. Additionally, each DNAm assay requires a unique data analysis workflow in order to derive valid conclusions. In this work, we will describe a novel computational quantification method for MethylCap-seq and the study design and analysis of a BS-seq experiment as applied to a preventative therapy in breast cancer. Together, these results demonstrate the development and planning needed for the successful implementation of a sequencing assay in clinical research. MethylCap-seq is an approach that uses a protein that binds to DNAm to capture methylated DNA fragments. A region with a large number of captured fragments is interpreted as having high DNAm. Therefore, quantification is based on the number of fragments. Although MethylCap-seq is an inexpensive genome-wide assay, it does have a few limitations. The one we sought to address was the limited resolution of the data. Biologically, DNAm occurs at a single nucleotide; however, the resolution of MethylCap-seq is the fragment which is several hundred nucleotides. To improve the quantification of MethylCap-seq data, we developed a novel computational method. Our method, PrEMeR-CG, imputes a more accurate DNAm quantification at single nucleotide resolution. Since development, PrEMeR-CG has been used to make discoveries in multiple clinical studies. Unlike MethylCap-seq, BS-seq provides DNAm quantification at nucleotide resolution and is considered the gold-standard of DNAm sequencing assays. We utilized BS-seq to assess DNAm for an on-going clinical trial that aims to investigate the efficacy of high-dose polyunsaturated fatty acids (PUFA) treatment to prevent breast cancer in subtypes that have no preventative therapies. This work is motivated by the anti-inflammatory effect observed from PUFA treatment and by pro-cancer effects of inflammation in breast cancer. Our hypothesis is that anti-inflammatory effects of PUFA are mediated by DNAm changes in the breast tissue. In this work, we present two achievements that advance the research of PUFA treatment. First, we describe the design and the approach to investigate DNAm in the breast tissue in an on-going phase II clinical trial of PUFA treatment. One important result from this process was that we developed a novel inflammation related breast cancer specific candidate gene list for interrogating DNAm. Second, we report DNAm changes due to PUFA treatment in the PBMCs of peripheral blood. Our data showed that PUFA treatment resulted in specific DNAm changes in inflammation related pathways. Additionally, we identified potential inflammation related genes that we will follow up as treatment response biomarkers.

Committee:

Lisa Yee, MD (Advisor); Qianben Wang, PhD (Advisor); Ralf Bundschuh, PhD (Committee Member); Amanda Toland, PhD (Committee Member)

Subjects:

Bioinformatics; Biomedical Research

Keywords:

DNA methylation; breast cancer; bioinformatics; next-generation sequencing; omega-3 fatty acids; bisulfite; methylcap-seq; clinical research; biomarker; computational analysis; cancer prevention; treatment; therapy; study design

Liao, Peter Lee MingBioinformatics approaches to cancer biomarker discovery and characterization
Doctor of Philosophy, Case Western Reserve University, 2018, Systems Biology and Bioinformatics
Cancers are a heterogeneous set of diseases that are defined by uncontrolled cellular growth with the potential to invade or spread to adjacent and distant tissues. While sharing certain biological capabilities that define the development and behavior of all human malignancies, cancers are governed by complex molecular changes that are often tumor-specific. As a result, even tumors arising from the same cell-type can exhibit highly divergent prognoses and treatment responses depending upon the underlying molecular mechanisms that are dysregulated and that drive its abnormal growth and cellular processes. New data collection methods grant researchers unprecedented capability to investigate and characterize cancers on a systems level. Rather than being restricted in measurement to a specific target molecule or set of molecules, “-omics” approaches allow experiments to identify and measure thousands of molecules at a time. These “-omics” approaches can therefore characterize significant proportions of the genetic, transcript, protein, and post-translational modification landscapes that underlie and drive human malignancies. Because cancers represent such a diverse set of diseases, clinicians and researchers rely on biomarkers for a variety of uses in cancer, ranging from diagnosis to prognosis and prediction of treatment response. A good cancer biomarker is a molecular signal that is capable of distinguishing, for example, disease from normal, high-risk from low risk disease, or disease cases that may be particularly susceptible to targeted treatments. In this dissertation, I demonstrate the use of multiple bioinformatics tools for cancer biomarker discovery and characterization. Models of epigenetic age, termed epigenetic clocks, are investigated in gliomas and are shown to be associated with previously defined prognostic molecular subtypes and are independently predictive of survival. I introduce a novel method for phosphoproteomics analysis, termed pKSEA, which uses in silico kinase-substrate predictions to infer changes in kinase activity. pKSEA is described, benchmarked against previously published data, and compared to existing methods. Three examples are provided of pKSEA analysis in cancer-related data, identifying kinase activity signals that may be useful as biomarkers in identifying and targeting high risk glioblastomas, as well as identifying treatment-related phosphorylation signaling changes in response to kinase inhibition and phosphatase activation in cancer cells.

Committee:

Jill Barnholtz-Sloan, PhD (Advisor)

Subjects:

Bioinformatics; Oncology

Keywords:

cancer; biomarker; proteomics; epigenetics; phosphorylation; bioinformatics

Carter, Molly HRelationships Among Eye Gaze, Social Ability and Extracellular Signal-Regulated Kinase Pathway Activation in Children and Adolescents with Autistic Disorder
Doctor of Psychology (Psy.D.), Xavier University, 2016, Psychology
Previous eye tracking research suggested that children and adolescents with Autistic Disorder (AD) exhibit atypical gaze patterns while observing dynamic social interactions. The relationship among atypical gaze patterns, social ability, and underlying biological conditions has yet to be investigated. The present study utilized eye tracking technology to investigate the gaze patterns of 26 typically developing children and adolescents, ages 5-17, and 38 aged-matched peers with AD as they viewed a dynamic play scene. Between-group differences in the static activation level of the extracellular signal-regulated kinase (ERK) pathway in peripheral lymphocytes were also explored. Children and adolescents with AD displayed shorter fixations on faces and took longer to fixate on a face compared to participants without AD. Additionally, participants with AD had almost twice the amount of static ERK activation in peripheral lymphocytes than control participants. The atypical gaze patterns of participants with AD were not associated with social ability or ERK activation level. The findings demonstrate that atypical gaze extends to social play scenes for individuals with AD. Further investigation is needed on the implications of the ERK pathway in children and adolescents with AD and the extent to which eye tracking research directly relates to real-world social abilities.

Committee:

Janet Schultz, Ph.D. (Committee Chair); Kathleen Hart, Ph.D. (Committee Member); Craig Erickson, Ph.D. (Committee Member)

Subjects:

Behavioral Psychology; Cellular Biology; Developmental Psychology

Keywords:

Autism Spectrum Disorder; eye tracking; extracellular signal-regulated kinase pathway; social responsiveness; fixation duration on faces; latency to first face fixation; play scene; biomarker

Wang, BoNovel statistical methods for evaluation of metabolic biomarkers applied to human cancer cell lines
Doctor of Philosophy, Miami University, 2014, Chemistry
Metabonomics is a novel tool to investigate diseases and therapeutic treatments. This dissertation describes novel statistical methods for metabonomics based on nuclear magnetic resonance (NMR) spectroscopy and their application in studying the mechanism of human cancers. Chapter 1 introduces the currently used metabonomics data interpretation methods and instruments. Chapter 2 investigated how the reproducibility of metabolite resonances measured by NMR depends on signal-to-noise ratio (SNR) and normalization methods using the coefficient of variation (CV) method. An inverse correlation was detected between SNR and CV for all normalization methods, which will aid the researchers to optimize experiments. Chapter 3 demonstrates a new potential biomarker discovery method for metabonomics studies called Standard Deviation Step Down (SDSD). Unlike most of commonly used methods, SDSD gives weight to relative metabolite concentration and is progressively more sensitive for more concentrated metabolites. Chapter 4 provides a new algorithm for Principal Components Analysis (PCA) in protein sequence analysis. The method provides a way to know which amino acid position variations are most responsible for driving separation into sub-clusters. Chapter 5 describes metabonomics study of human neuroblastoma cell line induced by Neuropeptide Y (NPY) and its Y2 receptor. High conversion of glucose into lactate in abundance oxygen (Warburg effect) and lower intracellular nutrient in NPY Y2R group was observed. NPY and Y2R may influence glycolysis, glutaminolysis and possibly TCA cycle. Chapter 6 describes role of osteopontin-a in tumor progression by studying breast cancer cell metabolites. Osteopontin-a upregulates the levels of glucose in breast cancer cells, likely through STAT3 and its transcriptional targets apolipoprotein D and IGFBP5. The splice-variant-specific metabolic effects of osteopontin add a novel aspect to the pro-metastatic functions. Chapter 7 describes the microbial community and metabolites analysis of the early stage necrotizing enterocolitis (NEC) in preterm infants. A high urinary alanine:histidine ratio was found associated with microbial community difference in the first two weeks and provided good prediction of NEC. Chapter 9 summarizes the dissertation.

Committee:

Michael Kennedy (Advisor); Neil Danielson (Committee Chair); Carole Dabney-Smith (Committee Member); Gary Lorigan (Committee Member); Michael Robinson (Committee Member)

Subjects:

Biochemistry; Biostatistics; Chemistry

Keywords:

Bioanalytical methods; Bioassays; Biological samples; Chemometrics; Statistics; NMR; Metabolomics; Metabonomics; Principal components analysis; Protein sequence analysis; Cancer biology; Metabolic regulation; Biomarker validation

Jia, GuangMR imaging biomarkers for benign prostatic hyperplasia pharmacotherapy
Doctor of Philosophy, The Ohio State University, 2006, Biophysics

Benign prostatic hyperplasia (BPH) is a highly prevalent disease in older men and occurs in more than 50% of men aged 60 to 70 years. BPH results in prostate enlargement with bladder outflow obstruction. Treatment with a 5α-reductase inhibitor such as finasteride induces apoptosis in epithelial cells and leads to the reduction of prostate volume. However, pharmacologic treatment is not uniformly effective in shrinking the prostate and in relieving symptoms, so the ability to predict how each patient will benefit best from varying pharmacotherapy is a question of great medical and economic importance.

Finasteride is also used as a prophylaxis of BPH-associated hematuria and to reduce blood loss at surgical resection of the prostate. The important questions to be addressed include what is the optimum dose and how long should the patients be treated. An effective non-invasive tool may be helpful to solve these questions by monitoring the changes in prostatic blood flow.

Twenty-four male beagles with benign prostatic hyperplasia were enrolled in a drug trial and imaged at five time points by magnetic resonance imaging (MRI). The capabilities of different MRI-based methodologies for measuring prostate volume were evaluated from anatomical MR images. The possibility of using pharmacokinetic parameters as a predictor of MRI prostate volume changes were evaluated and the use of DCE-MRI as a biologic marker of in-vivo changes in microcirculation in prostatic suburethral region was assessed.

The segmented MRI prostate volume significantly correlated with post necropsy volume. The changes in prostate volume at the end of the treatment exhibited a significant linear correlation to the initial parenchymal Maximum Enhancement Ratio (MER) (p < 0.02) in the finasteride group. After completion of the therapeutic regiment, Tmax on prostatic suburethral area was significantly longer in the finasteride group compared to controls (p < 0.01), and the pharmacokinetic parameters amplitude A and exchange rate constant kep decreased 39% and 34% respectively in the finasteride group at the end of the treatment.

In conclusion, MRI of prostate can supply important in-vivo biomarkers, such as organ volume and pharmacokinetic parameters, in the development of BPH pharmacotherapies such as 5α-reductase inhibitors.

Committee:

Michael Knopp (Advisor)

Subjects:

Health Sciences, Radiology

Keywords:

Prostate; Benign Prostatic Hyperplasia; Magnetic Resonance Imaging; Dynamic Contrast-Enhanced MRI; Biomarker; Pharmacotherapy

Turnier, Jessica LUrine S100 Proteins as Potential Biomarkers of Lupus Nephritis Activity
MS, University of Cincinnati, 2017, Medicine: Clinical and Translational Research
Objective: Improved, non-invasive biomarkers are needed to accurately detect lupus nephritis (LN) activity. The purpose of this study was to evaluate five S100 proteins (S100A4, S100A6, S100A8/9 and S100A12) in both serum and urine as potential biomarkers of global and renal-specific disease activity in childhood-onset systemic lupus erythematosus (cSLE). Methods: In this multicenter study, S100 proteins were measured in the serum and urine of four cSLE cohorts and healthy controls using commercial ELISAs. Patients were divided into cohorts based on biospecimen availability: (1) longitudinal serum, (2) longitudinal urine, (3) cross-sectional serum, (4) cross-sectional urine. Global and renal disease activities were defined using the SLE disease activity index 2000 (SLEDAI-2K) and SLEDAI-2K renal domain score (SLEDAI-R). Results: All urine S100 proteins were elevated in active LN when compared to active extrarenal disease and healthy controls. All urinary S100 protein levels decreased with LN improvement, with S100A4 demonstrating the most significant decrease. Urine S100A4 levels were also higher with proliferative as compared to membranous LN. S100A4 staining in the kidney localized to mononuclear cells, podocytes and distal tubular epithelial cells. Irrespective of the S100 protein tested, serum levels did not change with cSLE improvement. Conclusion: Higher urine S100 levels are associated with increased LN activity in cSLE whereas serum S100 levels do not correlate with disease activity. Urine S100A4 shows the most promise as an LN activity biomarker given its pronounced decrease with LN improvement, isolated elevation in urine and positive staining in resident renal cells.

Committee:

Erin Haynes, Dr.P.H. (Committee Chair); Hermine Brunner, M.D. (Committee Member); Alexei Grom (Committee Member); Sherry Thornton, Ph.D. (Committee Member)

Subjects:

Surgery

Keywords:

SLE;lupus nephritis;biomarker;S100 protein;S100A4

Mainville, Gisele NadiaThe Prognostic Significance of Insulin-like Growth Factor II mRNA-Binding Protein 3 (IMP3) Expression in Oral Epithelial Dysplasia: a Retrospective Case-Control Study
Master of Science, The Ohio State University, 2013, Dentistry
Oral epithelial dysplasia (OED) is a potentially precancerous lesion with an estimated rate of malignant transformation ranging from <1% to 17.5%. Although risk of progression to oral squamous cell carcinoma (OSCC) is greater in higher-grade lesions, we are currently unable to predict the transformation potential of individual dysplastic lesions. Given the poor overall prognosis of OSCC, identification and elimination of pre-invasive lesions would presumably reduce disease-related morbidity and mortality. To achieve this, a better predictor of cancer risk is needed. Prognostic biomarkers capable of targeting at-risk OED lesions could help direct lesion-specific management. Insulin-like growth factor II mRNA-binding protein-3 (IMP3) is a recently identified oncofetal protein that is overexpressed in numerous malignancies, but rarely identified in benign adult tissue. In addition, throughout many organ systems, histologically high-grade precursor lesions overexpress IMP3. Most OSCC tumors produce IMP3, yet little is known on IMP3 expression in OED. Recently, IMP3 expression has been shown to indicate imminent progression of cervical intraepithelial neoplasia to overt cancer. The possibility of a similar predictive expression pattern in OED lesions warranted investigation. The objective of this retrospective case-control pilot study was to characterize the immunohistochemical (IHC) expression profile of IMP3 in OED lesions using two different methods: routine light microscopy and digital image analysis with Tissue Studio 3.5 software. We hypothesized that 1) IMP3 would be expressed in OED lesions shortly before malignant transformation, and absent in non-progressive OED lesions; and 2) IMP3 expression would increase over time in OED lesions that progressed to cancer. IHC staining was performed on 29 OED biopsies from 5 patients who developed OSCC (Progressive group), and on 32 OED biopsies from 5 patients who did not develop OSCC (Non-progressive group). The minimum follow-up period was 56 months. IMP3 was expressed in both groups’ biopsies. All final biopsies in the Progressive group showed IMP3+ OSCC or carcinoma in situ, and were immediately preceded by an IMP3+ OED biopsy in 4 out of 5 patients. In the Non-progressive group, 2 patients demonstrated consistently negative-staining OED biopsies, while the rest exhibited IMP3+ biopsies during follow-up. One patient from in the Non-progressive group may have been mis-assigned, and was therefore removed from statistical analyses. The presence of IMP3 expression, although skewed toward the predicted direction, did not discriminate between both groups (Fisher’s Exact test, 2-tailed p=0.1667). As detected with Tissue Studio 3.5, IMP3 expression in dysplastic epithelium did not significantly increase over time in either group (Pearson’s r, Non-progressive group: r=0.36, p=0.08; Progressive group: r=-0.07, p=0.73), and therefore, did not predict imminent transformation to OSCC. Although IMP3 expression paralleled malignant transformation (p=0.0009), it did not increase with higher grades of dysplasia (p=0.23). In conclusion, IMP3 was not found to be differentially expressed between OED lesions with and without eventual malignant progression. However, many limitations affected our results. Further studies are warranted to better elucidate the utility of IMP3, either alone, or in conjunction with other markers, as a prognostic biomarker in OED lesions.

Committee:

Susan R. Mallery, D.D.S., Ph.D. (Advisor); Carl M. Allen, D.D.S., M.S.D. (Committee Member); M. Scott Herness, B.S., Ph.D. (Committee Member); Kristin K. McNamara, D.D.S., M.S. (Committee Member)

Subjects:

Dentistry; Pathology

Keywords:

Insulin-like Growth Factor II mRNA-Binding Protein 3; IGF2BP3; IMP3; KOC; oral epithelial dysplasia; malignant transformation; prognostic biomarker; Tissue Studio 3.5

Daniels, Alexander R.Extreme exposure biomarker levels: do physicians want to be informed?
MPH, University of Cincinnati, 2015, Medicine: Epidemiology
BACKGROUND: Improving technology has allowed researchers to detect ever smaller amounts of environmental toxins in blood, serum and urine. Occasionally, participants are found to have concentrations well above national averages. There are few systems of support in place for participants to whom these results have been returned, and no guidelines for researchers or community physicians concerning how to address participants’ concerns. This study aims to assess whether or not community physicians are ready and willing to assist their patients who have participated in biomonitoring studies. METHODS: The study group recruited 100 internists, and 100 pediatricians selected via multiple online directories to ensure the greatest coverage of the Greater Cincinnati area. A survey consisting of two scenarios, with 8 or 9 multiple choice questions each, was mailed to these 200 community physicians. The scenarios presented were an 11 year old female with extremely high urinary phthalates, and a 55 year old male with extremely high urinary cadmium. STATISTICAL ANALYSIS: Descriptive statistics were calculated for each scenario separately for strata of physician specialty, gender, and age range. RESULTS: The majority of physicians surveyed indicated that they would like to receive the information about their patient's exposure levels (93.4%). Nearly all indicated that they did not have the knowledge base to offer their patient any guidance or treatment, and that they would require additional information. Many of the comments left on the surveys asked for the researchers to provide informational material to the physicians along with their patient's results, so that they might be able to better serve the patient. DISCUSSION: Based on the large percentage of physicians who indicated a desire to receive this kind of information, we believe this topic warrants further study in order to address the lack of guidance for researchers and community physicians.

Committee:

Susan Pinney, Ph.D. (Committee Chair); Frank Biro, M.D. (Committee Member); Jun Ying, Ph.D. (Committee Member)

Subjects:

Environmental Health

Keywords:

Report findings;extreme exposure biomarker;physician communication;environmental exposure

NI, JIAQIANPlasma Biomarkers for Age-Related Macular Degeneration
Master of Science in Chemistry, Cleveland State University, 2009, College of Science
The goal of this thesis research was to develop methods to predict susceptibility to age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elderly in developed countries, and early identification of susceptibility is important for preventing this progressive disease. New and original evidence was found supporting the prognostic potential of plasma advanced glycation endproducts (AGEs) as possible biomarkers for AMD, specifically the oxidative protein modifications Nε-(carboxymethyl)lysine (CML) and pentosidine. Quantitative analyses of plasma from AMD (n = 60) and normal control (n = 30) donors showed that mean CML and pentosidine levels were elevated in AMD plasma by ~ 60% and ~ 80%, respectively, while furosine was largely unchanged. Another type of oxidative modification, carboxyethylpyrrole (CEP) adducts, were found to be ~ 2-fold higher in AMD (n = 56) than in controls (n = 30), consistent with previous findings. C-statistics were determined from receiver operating characteristic curves that indicate for these plasma samples, CML discriminated between AMD and control donors with ~ 80% accuracy, CEP adducts with ~ 81% and pentosidine with ~ 92% accuracy. In combination with CEP, CML provided ~ 89% accuracy and pentosidine provided ~ 95% discrimination accuracy. Overall, these results provided evidence that CML and pentosidine are both elevated in AMD plasma and may prove useful for predicting AMD susceptibility and for monitoring AMD therapeutics, especially in combination with CEP biomarkers.

Committee:

John Crabb, PhD (Committee Chair); David Anderson, PhD (Committee Member); Baochuan Guo, PhD (Committee Member)

Subjects:

Analytical Chemistry; Biochemistry; Bioinformatics; Biology; Biomedical Research; Biostatistics; Chemistry; Pathology

Keywords:

Advanced Glycation End-products; Age-related Macular Degeneration; Biomarker; Plasma; LC-MS

Romick-Rosendale, Lindsey ElizabethUSE OF NMR-BASED METABONOMICS TO STUDY ANIMAL MODELS AND HUMAN DISEASE
Doctor of Philosophy, Miami University, 2011, Chemistry
This dissertation describes the effects that both antibiotics and opportunistic pathogens had on the host and gut metabolome, as well as the physical effects that these experimental factors had on the distal ileum of the study mice. The dissertation also presents urinary metabolic profiles in order to identify statistically significant changes in metabolites that could be used as a biomarker for early disease diagnosis and treatment. In chapter 1 a background of both human and animal model metabonomics studies is presented, including an overview of NMR and the multivariate statistical analysis method, PCA, which was utilized in all work in this dissertation. Chapter 2 illustrates the effects that a broad spectrum antibiotic, Baytril, has on the urinary and fecal metabolic profiles of Balb/c mice. It was found that eliminating bacterial species from the gut caused significant changes in metabolic profiles of study mice. Many of the metabolic changes were a direct result of bacterial cell death and a lack of bacterial processes that naturally occur in the host intestinal tract. Chapter 3 takes the study design in Chapter 2 a few steps further and following antibiotic treatment an opportunistic pathogen, Clostridium butyricum was introduced to the mice by an intragastric gavage procedure. C. butyricum has been linked to necrotizing enterocolitis (NEC) in past studies and has been isolated from the blood and fecal cultures of neonates diagnosed with NEC. It was determined that administering a broad spectrum antibiotic to the mice altered both the urine and fecal metabolic profiles and also had negative effects on the structural integrity of the distal ileum of the same mice. Rather than further amplify the intestinal injury in the mice, the Clostridial species brought the metabolic profile of the mice back to that which was similar to the control mice, and the intestinal villi that were damaged by the antibiotics was able to repair as bacterial colonization became more prominent. Chapter 4 describes the metabolic changes in the urine samples of 20 preterm infants over the first 14 days of life with the goal of identifying a marker for acute kidney injury. Three infants of the extremely low birth weight (ELBW) group were found to have elevated levels of one metabolite, carnitine, and this increase was also correlated to spikes in neutrophil gelatinase-associated lipocalin (NGAL), the current biomarker for acute kidney injury in adults. Chapter 5 is a study comparing the urinary metabolic profiles of patients suffering from class IV and class V lupus nephritis (LN) and focal segmental glomerulosclerosis (FSGS). Two metabolites, citrate and taurine, were found to be changing significantly between class IV and class V patients, and hippurate was identified as changing when comparing class V LN and FSGS patients. The metabolic changes in these three metabolites were all linked to a more severe renal tubular dysfunction in the class V LN patients. The results contained in this dissertation demonstrated the ability of NMR to be used as a tool to identify markers of injury and disease in both mouse models and human diseases.

Committee:

Michael Kennedy, PhD (Advisor); Ann Hagerman, PhD (Committee Chair); Michael Crowder, PhD (Committee Member); Gary Lorigan, PhD (Committee Member); Annette Bollmann, PhD (Committee Member)

Subjects:

Biochemistry; Biomedical Research; Chemistry; Medicine

Keywords:

metabonomics; nuclear magnetic resonance spectroscopy; biomarker; necrotizing enterocolitis; acute kidney injury; lupus nephritis

Norris, LauraUtilization of Biomarkers to Validate an Omega-3 Fatty Acid Food Frequency Questionnaire for Overweight and Obese Pregnant Women
MS, University of Cincinnati, 2010, Allied Health Sciences: Nutrition

Objectives. To determine if an omega-3 food frequency questionnaire (FFQ) will provide a valid estimate of omega-3 fatty acid intake in overweight and obese pregnant women when compared to biomarker values.

Design. Cross-sectional validation study

Subjects. Twenty-seven overweight and obese pregnant women (pre-pregnant BMI > 25) between 18 and 40 years old were recruited from the greater Cincinnati area.

Methods. An omega-3 FFQ was administered, and a fasting venous blood sample was collected to assess erythrocyte fatty acid levels in 27 pregnant women in the last trimester of pregnancy. Average intake for total omega-3 fatty acids, alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were calculated and compared to erythrocyte concentrations of the same fatty acids. The mean intake of DHA was also calculated and compared to the recommended intake levels for pregnant women. Results. Dietary DHA intake was significantly correlated with both total omega-3 (r = +0.491, p < 0.01) and DHA (r = +0.438, p < 0.05) in the red blood cell. There was no significant correlation between dietary intake of total omega-3 fatty acids, ALA, or EPA and erythrocyte concentrations of the same. Mean DHA intake levels for this sample were 75 mg/day, significantly lower than the recommended level of 200 mg/day.

Conclusion. The omega-3 FFQ was a valid tool for assessing DHA status in overweight and obese pregnant women.

Acknowedgements. Supported in part by USPHS Grant #UL1 RR026314 from the National Center for Research Resources, NIH and NIH, R21 HL093532-0231.

Committee:

Debra Ann Krummel, PhD (Committee Chair); Seung-Yeon Lee, PhD (Committee Member)

Subjects:

Nutrition

Keywords:

omega-3; DHA; pregnant; food frequency questionnaire; biomarker

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