Protein Ligand Interactions Probed by NMR: A Dissertation

Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena. In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d. Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research. This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease

THE PREVALENCE OF PRACTICING SAFETY PROFESSIONALS’ KNOWLEDGE, INVOLVEMENT, AND COMPETENCY ASSOCIATED WITH WORKPLACE WELLNESS PROGRAMS: GAUGING THE SAFETY PROFESSION’S ENGAGEMENT WITH THE TOTAL WORKER HEALTH INITIATIVE

The morbidity and mortality burden of occupational injuries and illnesses in the United States became such a public health concern that in 1970 the Occupational Safety and Health Act was signed into law. This law requires employers to provide workplaces “free from recognized hazards” and spurred the development of specific regulations along with the creation of academic and professional training programs to educate individuals about the safety sciences and careers in safety fields. Today there are an estimated 26,000 professionally trained and board certified safety professionals supporting workplace programs across the country, alongside numerous non-certified, but degreed, practicing safety professionals. While the collective efforts of these professionals has served to significantly reduce the rate of workplace injuries and illnesses, an emerging body of scientific knowledge indicates that the health status of any worker is affected by both occupational and non-occupational exposures. The failure to consider both impacts an employee’s “total worker health”. Led by the U.S. National Institute for Occupational Safety and Health (NIOSH) Total Worker Health® (TWH) initiative, some employers have created workplace wellness initiatives and programs. While these efforts are quite laudable, it is unclear to what extent, if any, safety professionals are involved in developing or operating TWH-related programs. Involvement with a wellness program carries with it certain sensitivities and risks. To gain a better sense of the level of safety professional involvement with workplace TWH-related programs, this research project was implemented to: Ascertain the prevalence of the safety professionals’ knowledge of wellness programs in general and specifically the TWH initiative; Establish what level of involvement, if any, safety professionals have with their institutions’ workplace wellness programs; Identify training or credentialing safety professionals have, if any, in wellness topics; and Examine the associations between outcomes (knowledge, involvement and competency level) and selected covariates/determinants (age, gender, smoking status, ethnicity, self-reported health, years of work experience, years of experience in safety, years of experience in wellness, field of work and number of employees at place of employment). We administered a web-based survey emailed to 5150 ASSE members during the fall of 2017. We received completed surveys from 654 responders (12.7%). Results showed that the majority of safety professionals (73%) are not familiar with the TWH program, but 78% reporting their company having a workplace wellness program. Safety professionals do implement and train in some of the topics covered in TWH. The main correlates of TWH knowledge were being female (aOR 1.49, 95%CI:1.02-2.16), African-American (aOR 3.33, 95%CI: 1.53-7.23) and having years of experience in wellness. Increasing years of experience in wellness was also a positive determinant for involvement with their institution’s workplace wellness program and having prior training in TWH topics, with adjusted odds ratios ranging from 3.5 to 35.5. Poor self-rated health was inversely associated with knowledge of TWH (aOR 0.44, 95%CI: 0.22-0.89). Company size was inversely associated with the likelihood that a safety professional is familiar with TWH. Only 284 (43.4%) of safety professionals reported receiving training in wellness topics. With these low rates of knowledge and training in TWH, increased awareness is needed. Safety professionals’ involvement with TWH is moderately high for traditional safety topics, but low in other areas. Training opportunities in TWH are minimal and credentialing is non existent. These findings, in turn, suggest that there is an opportunity to develop educational materials and training programs specifically designed for safety professionals as well as potentially developing a credentialing program for TWH

In Adult Smokers Unwilling or Unable to Quit, Does Changing From Tobacco Cigarettes to Electronic Cigarettes Decrease the Incidence of Negative Health Effects Associated With Smoking Tobacco? A Clin-IQ

Data from a randomized controlled trial and systematic review support the claim that switching from tobacco cigarettes to electronic cigarettes (e-cigarettes) can reduce the short-term negative health effects of smoking. In adult smokers unwilling or unable to quit, exhaled carbon monoxide levels, total number of cigarettes smoked, and exposure to nitrosamine chemicals were reduced within a 12-month period. While the e-cigarette industry remains largely unregulated thus far, these studies provide encouraging hope in the uphill battle toward helping patients make informed and healthy choices

Alu element in the RNA binding motif protein, X-linked 2 (RBMX2) gene found to be linked to bipolar disorder

Objective We have used long-read single molecule, real-time (SMRT) sequencing to fully characterize a similar to 12Mb genomic region on chromosome Xq24-q27, significantly linked to bipolar disorder (BD) in an extended family from a genetic sub-isolate. This family segregates BD in at least four generations with 24 affected individuals. Methods We selected 16 family members for targeted sequencing. The selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed hybrid capture probes enriching for 5-9Kb fragments spanning the entire 12Mb region that were then sequenced to screen for candidate structural variants (SVs) that could explain the increased risk for BD in this extended family. Results Altogether, 201 variants were detected in the critically linked region. Although most of these represented common variants, three variants emerged that showed near-perfect segregation among all BD type I affected individuals. Two of the SVs were identified in or near genes belonging to the RNA Binding Motif Protein, X-Linked (RBMX) gene family-a 330bp Alu (subfamily AluYa5) deletion in intron 3 of the RBMX2 gene and an intergenic 27bp tandem repeat deletion between the RBMX and G protein-coupled receptor 101 (GPR101) genes. The third SV was a 50bp tandem repeat insertion in intron 1 of the Coagulation Factor IX (F9) gene. Conclusions Among the three genetically linked SVs, additional evidence supported the Alu element deletion in RBMX2 as the leading candidate for contributing directly to the disease development of BD type I in this extended family.Peer reviewe

Recovery of macular pigment spectrum in vivo using hyperspectral image analysis

We investigated the feasibility of a novel method for hyperspectral mapping of macular pigment (MP) in vivo. Six healthy subjects were recruited for noninvasive imaging using a snapshot hyperspectral system. The three-dimensional full spatial-spectral data cube was analyzed using non-negative matrix factorization (NMF), wherein the data was decomposed to give spectral signatures and spatial distribution, in search for the MP absorbance spectrum. The NMF was initialized with the in vitro MP spectrum and rank 4 spectral signature decomposition was used to recover the MP spectrum and optical density in vivo. The recovered MP spectra showed two peaks in the blue spectrum, characteristic of MP, giving a detailed in vivo demonstration of these absorbance peaks. The peak MP optical densities ranged from 0.08 to 0.22 (mean 0.15+/−0.05) and became spatially negligible at diameters 1100 to 1760 μm (4 to 6 deg) in the normal subjects. This objective method was able to exploit prior knowledge (the in vitro MP spectrum) in order to extract an accurate in vivo spectral analysis and full MP spatial profile, while separating the MP spectra from other ocular absorbers. Snapshot hyperspectral imaging in combination with advanced mathematical analysis provides a simple cost-effective approach for MP mapping in vivo

The outcomes of Perthes’ (TOP) study: development of a core outcomes set for clinical trials in Perthes’ disease

Aims To identify a suite of the key physical, emotional, and social outcomes to be employed in clinical practice and research concerning Perthes' disease in children. Methods The study follows the guidelines of the COMET-Initiative (Core Outcome Measures in Effectiveness Trials). A systematic review of the literature was performed to identify a list of outcomes reported in previous studies, which was supplemented by a qualitative study exploring the experiences of families affected by Perthes’ disease. Collectively, these outcomes formed the basis of a Delphi survey (two rounds), where 18 patients with Perthes’ disease, 46 parents, and 36 orthopaedic surgeons rated each outcome for importance. The International Perthes Study Group (IPSG) (Dallas, Texas, USA (October 2018)) discussed outcomes that failed to reach any consensus (either ‘in’ or ‘out’) before a final consensus meeting with representatives of surgeons, patients, and parents. Results In total, 23 different outcome domains were identified from the systematic review, and a further ten from qualitative interviews. After round one of the Delphi survey, participants suggested five further outcome domains. A total of 38 outcomes were scored in round two of the Delphi. Among these, 16 outcomes were scored over the prespecified 70% threshold for importance (divided into six main categories: adverse events; life impact; resource use; pathophysiological manifestations; death; and technical considerations). Following the final consensus meeting, 14 outcomes were included in the final Core Outcome Set (COS). Conclusion Core Outcome Sets (COSs) are important to improve standardization of outcomes in clinical research and to aid communication between patients, clinicians, and funding bodies. The results of this study should be a catalyst to develop high-quality clinical research in order to determine the optimal treatments for children with Perthes’ disease

Applying the Risk of Bias Tool in a Systematic Review of Combination Long-Acting Beta-Agonists and Inhaled Corticosteroids for Persistent Asthma

Background: The Risk of Bias (RoB) tool is used to assess internal validity of randomized controlled trials (RCTs). Our objectives were to: 1) evaluate inter-rater agreement of the RoB tool; 2) determine the time to access supplemental study information; 3) compare the RoB tool with the Jadad scale and Schulz allocation concealment (AC); and 4) examine the relationship between RoB and effect estimates. Methods: We conducted a systematic review of long-acting beta agonists (LABA) combined with inhaled corticosteroids (ICS) for adults with persistent asthma. Two reviewers independently assessed 107 trials using RoB, Jadad, and AC. One reviewer searched for study protocols. We assessed inter-rater agreement using weighted Kappa (k) and the correlation between tools using Kendall’s Tau (t). Mean differences in effect sizes for RCTs with different RoB were calculated using inverse variance method and random effects model. Results: Trials had good Jadad scores (median 4, IQR 3-4); however, 85 % had unclear AC and 87 % high RoB. The factor that most influenced RoB was the potential inappropriate influence of study sponsors (95 % industry funded). Agreement on RoB domains was fair (k = 0.40) to almost perfect (k = 0.86), and moderate for overall RoB (k = 0.41). Median time to complete RoB assessments was 21 minutes (IQR 14-27) and 12 minutes (IQR 9-16) to search for protocols. Protocols were identified for 5/42 studies (12%); in 3 cases the assessment of selective outcome reporting changed. There was low correlation between overall RoB vs. Jadad (t =0.04

Mathematical model of blood and interstitial flow and lymph production in the liver.

We present a mathematical model of blood and interstitial flow in the liver. The liver is treated as a lattice of hexagonal \u2018classic\u2019 lobules, which are assumed to be long enough that end effects may be neglected and a two-dimensional problem considered. Since sinusoids and lymphatic vessels are numerous and small compared to the lobule, we use a homogenized approach, describing the sinusoidal and interstitial spaces as porous media. We model plasma filtration from sinusoids to the interstitium, lymph uptake by lymphatic ducts, and lymph outflow from the liver surface. Our results show that the effect of the liver surface only penetrates a depth of a few lobules\u2019 thickness into the tissue. Thus, we separately consider a single lobule lying sufficiently far from all external boundaries that we may regard it as being in an infinite lattice, and also a model of the region near the liver surface. The model predicts that slightly more lymph is produced by interstitial fluid flowing through the liver surface than that taken up by the lymphatic vessels in the liver and that the on-peritonealized region of the surface of the liver results in the total lymph production (uptake by lymphatics plus fluid crossing surface) being about 5 % more than if the entire surface were covered by the Glisson\u2013peritoneal membrane. Estimates of lymph outflow through the surface of the liver are in good agreement with experimental data. We also study the effect of non-physiological values of the controlling parameters, particularly focusing on the conditions of portal hypertension and ascites. To our knowledge, this is the first attempt to model lymph production in the liver. The model provides clinically relevant information about lymph outflow pathways and predicts the systemic response to pathological variations