Title: Designing a Survey Instrument to Evaluate K-12 Educators Perception of Well-being Authors: Trystan Innes, Amanda M.S. Frasier PhD, and Chad R. Frasier PhD Introduction: For several years, the United States has faced an increasingly growing teacher shortage as measured by increased vacancies for certified positions 1–3. This shortage is caused by both a decreased enrollment in teacher preparation programs (reduction in those entering the profession) and greater numbers of staff turnover (increase in those leaving the profession) and schools in the South are disproportionately impacted 4. The use of non-invasive measures of heart rate has recently gained an increase in attention, especially with the prevalence of watch-based measurements. These emerging technologies have increased the availability of these measures outside of clinical settings, but the utility of these measures has not been validated. A growing measure companies claim to use is heart rate variability (HRV), which closely approximates the output of the autonomic nervous system. Several studies have linked changes in HRV to mood 15–17, stress18–22, cardiovascular health23–25, and exercise performance and recovery 26–28. However, the utility of the wrist base measurements may be overstated, and expert consensus suggests the use of these devices may become disruptive to healthcare until more is known about how the data relates to a person's health and well-being (29. Overall, there is a gap in research on teacher health and well-being that includes the use of objective measures 34. Methods: A review of current literature for keywords such as occupational stress, teacher, well-being, health, and heart-rate variability. Surveys that addressed well-being were isolated from these studies. Surveys were evaluated on scoring system, question format, experimental group population and relevance to well-being. The findings were incorporated into a modified Teacher Stress Inventory (TSI) survey. The finalized survey was uploaded into Qualtrics for dissemination. The survey will be administered to K-12 educators at schools in northeast Tennessee. These schools will be comprised of both rural and city schools. Participants will also be from schools with variations in socioeconomic levels and population density. Surveys will be scored in accordance with the guidelines established in the Teacher Stress Inventory. In addition, questions from the Effort-Reward Imbalance (ERI) survey were included. Where there was a potential overlap in questions between the TSI and ERI, answers were coded for scaling according to ERI in addition to the TSI. Results: Once the survey has been completed, participants who fit the inclusion criteria will be selected and fitted with an Equivital monitor to track their movement, breathing rate, and ECG over a 24-hour period. In addition, during the fitting of the device, participants will answer the WHO-5 questionnaire which will be used to longitudinally track well-being during the study. ECG data will be used to analyze heart rate variability (HRV) in addition to other ECG parameters. Survey scores and Equivital measures will then be investigated for potential correlation. Conclusion: The survey constructed will serve the foundation in the future research project for evaluating the relationship between HRV and teacher perceptions of well-being.

Indiana University-Purdue University Indianapolis (IUPUI) / Alternative splicing is a regulatory mechanism that generates multiple mRNA transcripts from a single gene, allowing significant expansion in proteome diversity. Disruption of splicing mechanisms has a large impact on the transcriptome and is a significant driver of complex diseases by producing condition-specific transcripts. Recent studies have reported that mis-spliced RNA transcripts can be another major source of neoantigens directly associated with immune responses. Particularly, aberrant peptides derived from unspliced introns can be presented by the major histocompatibility complex (MHC) class I molecules on the cell surface and elicit immunogenicity. In this dissertation, we first developed an integrated computational pipeline for identifying IR-induced neoantigens (IR-neoAg) from RNA sequencing (RNA-Seq) data. Our workflow also included a random forest classifier for prioritizing the neoepitopes with the highest likelihood to induce a T cell response. Second, we analyzed IR neoantigen using RNA-Seq data for multiple myeloma patients from the MMRF study. Our results suggested that the IR-neoAg load could serve as a prognosis biomarker, and immunosuppression in the myeloma microenvironment might offset the increasing neoantigen load effect. Thirdly, we demonstrated that high IR-neoAg predicts better overall survival in TCGA pancreatic cancer patients. Moreover, our results indicated the IR-neoAg load might be useful in identifying pancreatic cancer patients who might benefit from immune checkpoint blockade (ICB) therapy. Finally, we explored the association of IR-induced neo-peptides with neurodegeneration disease pathology and susceptibility. In conclusion, we presented a state-of-art computational solution for identifying IR-neoAgs, which might aid neoantigen-based vaccine development and the prediction of patient immunotherapy responses. Our studies provide remarkable insights into the roles of alternative splicing in complex diseases by directly mediating immune responses. / 2023-08-16

No / The advance of high throughput biotechnology enables the generation of large amount of biomedical data. The microarray is increasingly a popular approach for the detection of genome-wide gene expression. Microarray data have thus increased significantly in public accessible database repositories, which provide valuable big data for scientific research. To deal with the challenge of microarray big data collected in different research labs using different experimental set-ups and on different bio-samples, this paper presents a primary study to evaluate the impact of two important factors (the origin of bio-samples and the quality of microarray data) on the integrated analytics of multiple microarray data. The aim is to enable the extraction of reliable and robust gene biomarkers from microarray big data. Our work showed that in order to enhance biomarker discovery from microarray big data (i) it is necessary to treat the microarray data differently in terms of their quality, (ii) it is recommended to stratifying (i.e., sub-group) the data according to the origin of bio-samples in the analytics.

Kidney transplantation is a lifesaving treatment for end stage kidney disease that offers considerable benefits to recipients in terms of survival and quality of life. The growing demand for transplants to treat conditions stemming from a rising prevalence of end stage renal disease, diabetes and cardiovascular diseases has to be met increasingly with donors who are older with a high incidence of comorbid conditions. Organs obtained from these higher risk donors are more likely to have either suboptimal short and long-term transplant outcomes or even fail to function altogether. Transplant clinicians, who have to balance the risk of patients dying while waiting for a transplant against the uncertainty of outcomes, often decline organs as transplants. These clinical challenges entail difficult decisions, and more refined tools to assess and quantify the risks of marginal donor organs are lacking. Diagnostic markers of donor kidney quality that can predict transplantation outcomes are highly desirable in order to discriminate the suboptimal from those allografts that will recover and have good long-term function. My doctoral research using donor samples collected within the Quality of Organ Donation (QUOD) programme has shown for first time that it is possible, on the basis of a tissue proteomic profile, to discriminate donor kidneys at the time of retrieval that will have suboptimal allograft function from those kidneys that could recover and have good function at three and 12 months post transplantation. Despite AKIN classification and Remuzzi scoring showing no evidence of acute kidney injury or chronic kidney disease in the analysed biopsies, quantitative mass spectrometry and degredomics experiments with a subsequent validation analysis on an independent cohort of biopsy samples confirmed the increased levels of inflammation and pro-fibrotic proteins in the allografts with suboptimal function, while increased levels of cytoprotective proteins were detected in the kidneys that recovered with a good function one year after transplantation. Furthermore, the kidneys with suboptimal function demonstrated enhanced degradation of cytoskeletal proteins that are vital in sustaining the glomerular basement membrane cytoskeleton. In addition, I conducted a pilot study using proteomic analysis of serum and urine of donors whose kidneys either developed delayed graft function or functioned immediately. This study confirmed that is feasible to identify alterations in the blood and urine proteome that are biologically meaningful. In preparation of the discovery and development of diagnostic biomarkers of long-term outcome after kidney transplantation using QUOD plasma samples, I assessed the pre-analytical variability associated with the processing of whole blood during QUOD sample collection in order to identify a baseline proteomic and peptidomic profile against which candidate protein markers relevant for clinical correlates can be selected. Based on the findings reported in this thesis, future work should aim to identify and develop better diagnostic tools that can more reliably predict donor organ quality. In addition, novel intervention strategies can be explored that either attenuate pro-fibrotic and proteolytic activities or enhance antioxidant and cytoprotective mechanisms in deceased donor kidneys prior to transplantation.

The action of overtraining may lead to the different states of overreaching or the overtraining syndrome (OTS). Chronic maladaptation in endocrine and immune mechanisms, and performance decrements occur with the incidence of these states. Circulating cortisol and testosterone have been proposed as endocrine markers of overreaching/OTS. Salivary measurements of these hormones have been used as a non-invasive surrogate for circulating levels. Chapter 4 (study 1) on this Thesis examined the influence of consuming water 10 min, 5 min and 1 min before providing a saliva sample in diluting saliva and consequently providing invalid salivary cortisol and testosterone concentration levels. No trial effect was found. However, exercise-induced salivary cortisol and testosterone significantly elevated in response to the 10 min and 5 min trials only, with lower absolute-changes observed in the 1 min trial. No differences were found in the resting samples. It was suggested that consuming water up to 5 min before providing a saliva sample does not appear to influence the hormone concentrations at rest and during exercise. However, the recommended guidelines for saliva collection have been followed in the subsequent studies. Chapter 5 (study 2) examined the reproducibility of salivary cortisol and testosterone responses to a 30-min cycle named as the 55/80. This test has been proposed as a suitable indicator of hormonal alterations associated with overreaching/OTS. Reproducibility of salivary cortisol and testosterone to the 55/80 was confirmed by determining intra-individual coefficients of variation (CVi). However, the 55/80 is a cycle test and therefore, may not be appropriate for runners. Chapter 6 (study 3) focused on designing a 30-min, running bout (i.e. the RPEtreadmill) to reproduce the effects of the 55/80. The RPEtreadmill is a self-paced test and therefore, will not require aVO2maxtest to determine exercise intensities. An acute elevation of plasma and salivary testosterone, but not cortisol was observed in response to the RPEtreadmill. These responses have been shown to be reproducible. The data from Chapter 6 suggest that the RPEtreadmill may be a suitable tool to indicate hormonal alterations associated with overreaching/OTS. This led to the design of study 4 (Chapter 7). Plasma and salivary cortisol and testosterone responses were examined before and after a 12-day intensified-training period. Immunity markers (specifically salivary immunoglobulin A (SIgA), leucocyte subset proliferation and phagocytic activity) were examined before and after training. Plasma and salivary cortisol were unaffected by acute exercise and training. However, testosterone elevated to the RPEtreadmill Pre-Training, and these responses were reduced Post-Training. Total leucocytes and mucosal immunity were unaffected by exercise and training. However, increased upper respiratory tract infection symptoms were found Post-Training. Baseline phagocytic function was 47% lower Post-Training. This Thesis suggests that testosterone may be a more reliable exercise-stress marker. Moreover, the RPEtreadmill may be a suitable tool to highlight alterations in testosterone when in an overreached state in an attempt to avoid the incidence of OTS, and that this tool may be practically applied in the field of exercise science. Additionally, this Thesis shows that a 12-day intensified-training period induced a marked decrease in phagocytic responses, and therefore using the RPEtreadmill to highlight overreaching may be important to also prevent further impairments in immunity status.

Around 1% of the population worldwide suffer from epilepsy, a condition which is characterized by recurring seizures. The development of reliable biomarkers for both prediction and targeted treatment of seizures is critical, as they can pave the way towards personalized therapy in epilepsy. In addition, sensitive biomarkers can be utilized for the detection of epilepsy in its early stages and allow for early treatment intervention. Various types of biomarkers have been studied in relation to epilepsy, with blood markers emerging as major candidates. Blood biomarkers offer the benefit of being cost and time efficient, in addition to being less invasive to sample in contrast to cerebrospinal fluid markers. Importantly, they can enhance patient diagnosis and prognosis when supplemented with other diagnostic methods, such as EEG. In this pilot study, five blood biomarkers of brain injury are studied in epilepsy, post-stroke epilepsy and single seizure patients. The aim is to analyze whether S100B, NSE, GFAP, NfL and tau are promising indicators of epilepsy after a first seizure in adults. The results present S100B as the most promising biomarker, with potential to predict early epilepsy.

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Alzheimer's Disease (AD) is a neurodegenerative disorder affecting over 35 million people. Early diagnosis and intervention are crucial for improving outcomes. Digital Cognitive Biomarkers (DCBs) offer a promising approach for early detection and disease management, quantifying cognitive processes of encoding and retrieval through a hierarchical Bayesian cognitive processing model using wordlist memory tests. We hypothesize that DCBs will correlate with classic AD cerebrospinal fluid (CSF) biomarkers (Aβ42, T-tau, p-tau) in patients with varying cognitive decline levels compared to healthy elderly controls. Using Alzheimer's Disease Neuroimaging Initiative (ADNI) data and paired Pearson correlation coefficient analysis, our results support the hypothesis, indicating that DCBs correlate with CSF biomarkers and demonstrating their potential as a noninvasive diagnostic tool for AD. Furthermore, DCBs exhibited improved diagnostic accuracy compared to classic AD CSF biomarkers, as indicated by the area under the Receiver Operating Characteristic curve analysis. DCBs hold promise for monitoring disease progression, response to therapeutics, and identifying patients at earlier disease stages. Future research should validate these findings in diverse populations and conduct longitudinal studies to assess DCBs' potential in tracking disease progression and treatment response. Integrating DCBs with other diagnostic approaches, such as neuroimaging, could enhance overall AD diagnosis accuracy and provide a comprehensive understanding of an individual's cognitive health. In conclusion, DCBs may offer a valuable, noninvasive tool for early diagnosis and management of Alzheimer's Disease, supporting the initial hypothesis.

One group of chemotherapeutics that are used successfully to treat breast cancer, alone or in combination with other agents, are the taxanes; paclitaxel and docetaxel. They act by interfering with the spindle microtubule dynamics of the cell causing cell cycle arrest. However, the complexities underlying the mechanism of action are yet to be fully elucidated. Arguably, one of the most significant problems with taxanes is chemoresistance. Unfortunately, some patients are intrinsically resistant to taxanes and others acquire resistance to taxanes as treatment advances. This problem is exacerbated by a lack of understanding of the mechanisms underlying taxane resistance. Isogenic breast cancer cell lines that were taxane resistant were generated to use as an experimental model. Paclitaxel resistant (PACR) MDA-MB-231, paclitaxel resistant ZR75-1 and docetaxel resistant (DOCR) ZR75-1 cell lines were successfully generated by incrementally increasing taxane dose in respective native cell lines in vitro. An extensive characterisation of each of the resistant cell lines was conducted, focussing primarily on the 25nM resistant cells which were determined to be the most clinically relevant dose of taxane. A suboptimal dose of 5nM, a “superoptimal” dose of 50nM and the native, taxane sensitive cells was included. Dose response cell count experiments were performed that confirmed taxane resistant cells had been generated. It was shown that MDA-MB-231 native cells were more sensitive to paclitaxel than the ZR75-1 native cells, suggesting that ZR75-1 cells may already have low level inherent resistance. The MDA-MB-231 25nM PACR cells were tested to determine whether they retained PACR when maintained in media containing no paclitaxel. MDA-MB-231 25nM PACR cells were maintained in a taxane free environment for six months and then rechallenged with taxane. When rechallenged, the PACR cells previously maintained in the absence of paclitaxel mirrored the pattern of growth of corresponding PACR cells that had been maintained in the presence of paclitaxel. This proved that in the absence of paclitaxel, PACR cells did not revert to parent phenotype. This meant that experiments could be designed to grow cell lines as xenografts in mice, (in the absence of paclitaxel) & bring in vitro experiments into an in vivo setting. Effects of taxane treatment on both native and resistant cells were analysed using flow cytometry. Paclitaxel treatment exerted G2/M block in native MDA-MB-231 cells but when PACR cells were treated with the same dose of paclitaxel no G2/M block was observed, suggesting that PACR cells had developed a mechanism for escaping G2/M block. ZR75-1 native lines were also investigated and we established that treatment with paclitaxel also exerted a G2/M block in these lines. In future studies this process will be repeated to investigate the effect of taxane treatment on the ZR75-1 PACR and DOCR lines. CD 1 nude mice were injected with cells from all five cell lines to grow xenografts, unfortunately MDA-MB-231 PACR cells failed to grow so they could not be used for further xenograft experiments. PACR, DOCR and Native ZR75-1 cells did successfully grow as xenografts in mice and confirmed that all 3 groups showed very similar growth patterns. A cross resistance experiment was conducted and it was determined that the DOCR xenografts maintained a taxane resistant phenotype to docetaxel, and not paclitaxel and the PACR xenografts may be perpetuate the paclitaxel resistant phenotype in xenografts and that there may be cross resistance to docetaxel in the paclitaxel resistant xenografts. This is the first time that taxane resistant cell lines grown in this way have been established as xenografts in mice. These cross resistance experiments represent novel findings and merit further investigation. Extensive genomic and transcriptomic analyses were carried out on the cell lines to help identify potential taxane resistance markers. aCGH experiments were carried out to compliment the illumina experiments. The first set of experiments used DNA from pooled whole female blood as ref sample and DNA from each of the native and taxane resistant cell lines as test samples. The second set of experiments used DNA from native cells as a ref sample and DNA from their respective taxane resistant cells as a test, which allowed areas of loss or gain to be tracked in the genome as resistance increased. In the MDA-MB-231 cell lines the following areas of loss extended with increasing resistance: 1p36.13-q44, 6p25.3-q12, 8p, 10p, 19q, X Chr and the following areas of gain 2p25.3-23.3, 3p24.3-q13.3, 4p16.1-q12, 5q14.3-q31.1, 8q21.13-24.3, 11q15.1-q25, centromeric 12, and centromeric 14. In the ZR75-1 PACR and DOCR cell lines the areas of loss extended with increasing resistance in the following regions: 7q, 12p and 16q. For gene expression analysis RNA was extracted from the MDA-MB-231 cell lines, labelled and hybridised them to illumina human ref 8 vs. 2 chips. Data showed a progressive increase in mRNA dysregulation as paclitaxel resistance increased. Eleven genes were dysregulated across all resistance levels in the PACR MDA-MB-231 cells when compared to the relative cell lines; RGS16, CLDN1, IL7R, P&PP1R14C, COBL, TRPV4, TSPAN8, CD33, NLRP2, P13, and PAGE5. The experiment was repeated using MDA-MB-231 PACR, ZR75-1 PACR and DOCR cells and resulting data was analysed to determine genes commonly dysregulated across resistance levels, between MDA-MB-231 PACR and ZR75-1 PACR and between ZR75-1 PACR and DOCR cell lines. An extensive literature search was conducted and established four genes of interest in the context of our genomic and transcriptomic experiments including AURKA, Mdr-1, Stathmin and YY1. The novel biomarkers identified in the illumina experiments were validated with complimentary qPCR gene expression experiments looking at expression levels of the eleven commonly dysregulated genes identified and a panel of 19 other genes with significantly increased or decreased expression as resistance increased including AURKA, Mdr-1, Stathmin and YY1. Western blots were performed with lysates from the cell lines using a standard panel of predictive breast cancer markers and AURKA, Mdr-1, Stathmin and YY1. Combining the data from the genomic study, the gene expression profile, qPCR and Western blotting it was established that Mdr-1 had increased expression in the taxane resistant ZR75-1 lines and YY1 had increased expression in the MDA-MB-231 PACR line. Material from the LAPATAX trial was used to observe any transcriptomic changes occurring in tumours following treatment with docetaxel and to compare them to changes identified in our in vitro and xenograft models, this allowed the final step to be taken into a translational environment. LAPATAX (EORTC 10054) is a phase I-II study of Lapatanib and Docetaxel as neoadjuvant treatment for HER-2 +ve locally advanced/inflammatory or large operable breast cancer. Tumour material from eighteen core biopsies pre and post treatment was obtained, the mRNA was extracted, labelled and hybridised to the illumina array. This allowed the changes in gene expression pre and post docetaxel treatment to be tracked. The gene expression data from the LAPATAX trial was combined with gene expression data from our cell line panel and identified two novel putative markers of taxane resistance DUSP1 and FOS. Although sample size is small this has provided extremely valuable evidence directly from the clinic. These two novel putative biomarkers are extremely intriguing and certainly merit further investigation, ideally using additional taxane treated breast tumour tissue. Ultimately, an isogenic in vitro model of taxane resistance was developed in two different cell lines and with two different taxanes within one cell line. The cell lines were characterised and the effect of the taxanes on the cell cycle was determined in the native and taxane resistant lines. Selected cell lines were grown as xenografts in mice and performed successful cross resistance studies upon them. A large transcriptomic and genomic analysis was conducted and has identified a panel of potential taxane resistance markers and areas of loss and gain in the genome perpetuated by increasing taxane resistance. This analysis was validated using qPCR and Western blotting. This allowed a panel of novel taxane resistance markers to be identified. In future studies it is hoped that these targets will be knocked down with shRNA to observe if the taxane resistant cell lines revert to the parental phenotype. In vitro studies will be conducted to find agents that may be used to reduce expression of these markers and restore sensitivity to taxanes and consequently restore the efficacy of these drugs in a clinical setting. As far as the author is aware this is the first time that isogenic taxane resistant cell lines have been generated and investigated in this way.