The objective of this study is to investigate heart rate variability (HRV) over a period of 24 hours with special focus on patterns during the sleep period. Comparisons were made between sleep and the full 24-hour data. The Polar M430 watch was used to record heart rate (HR) data for about 24 hours, and the subject kept a journal of the activities they performed throughout the day. The data was visualized as heart rate in beats per minute (BPM) as a function of time. To check the accuracy of the Polar watch, a 7-minute recording of HR from the watch was performed simultaneously with a gold standard (IX-TA 220 and IX-ECG12, iWorx, Dover, NH). The data was downloaded into Excel spreadsheet, then analyzed. Analysis included calculations such as: standard deviation, range, mean, and the Fast Fourier Transform (FFT). HR data analysis was performed for the 24hr and sleep period. Since daily activities (which can affect HR) were difficult to control in the study subjects, the sleep period was chosen for more detailed analysis. A trend of lower HR and HRV was seen during sleep. Although the watch had modest accuracy, it was able to show some HR patterns during sleep. FFT analysis of the sleep data suggested existence slow HR cycles(which corresponds one cycle every > 5 minutes).

The central dogma of biology states that genetic information describes the flow ofinformation from DNA to RNA and then finally resting in proteins. The fundamental aspect that underlies all aspects of life is the expression and modification of proteins. One can even argue that there is no life without proteins. As such, many human diseases are either directly or indirectly related to dysfunction of proteins and can potentially be solved through protein therapeutics. Consequently, scientists have begun to harness the diversity of proteins to treat the diseases which plague man in the form of protein therapeutics such as clotting factors, cytokines, and growth factors. Unfortunately, the short circulation half-life of proteins is a major limiting factor which must be overcome before their widespread adoption as a platform for therapeutic development. Contributing factors to this short circulation half-life include renal elimination, proteasomal degradation, and metabolism in the liver. Namely, renal elimination is the main challenge for protein therapeutics and warrants clinicians to resort to higher doses and more frequent administrations to maintain necessary concentrations in the body. Unfortunately, side effects of this approach are dose limiting toxicities and reduced therapeutic outcomes as drug concentrations fluctuate drastically. As a result, addressing the challenge of renal elimination for protein therapeutics would allow for the development of novel treatments which were previously not viable. The human glomerulus readily filters out any particle smaller than approximately 30 kDa in weight. As a result, strategies adopted all share a common theme of endowing the protein with a greater effective size without compromising their natural activity against the intended target. The current approaches include conjugation to a polymer (i.e., PEGylation), covalent or non-covalent binding to a larger protein, or conjugating to the neonatal Fc receptor. Major limitations of these approaches include compromised activity caused by steric hinderance rooted inconjugation to moieties of larger size. This issue applies to all of the aforementioned reported approaches wherein activity becomes reduced, thus necessitating higher dosages. Furthermore, other limitations also exist such as humoral immune responses against polymers through anti-PEG antibodies, occurrence of organ damage, and solubility issues. A novel approach was recently developed by the Alhamadsheh lab which demonstratedthe ability of a small molecule linker termed “Transthyretin Ligand for Half-life Extension” (TLHE) to extend the circulation half-life of Gonadotropin releasing hormone. Most essentially, this was accomplished without compromising the potency or introducing a major sterically bulky group to the original peptide. Furthermore, additional concerns such as solubility issues was demonstrated to not be an issue either. In this work, human Interleukin-2 (IL-2) was chosen as a proof of concept to demonstrate application of the TLHE technology in a protein to address the aforementioned half-life challenge. Previously, a mixture of IL-2 and TLHE-IL-2 was demonstrated to maintain comparable activity to control IL-2 in both in vitro and ex vivo efficacy assays. Furthermore, a pharmacokinetic evaluation in rodents demonstrated significant half-life extension of TLHE-IL-2. The objective of this work was to shift the ratio of the IL-2/TLHE-IL-2 mixture to majority TLHE-IL-2 and or enhance the yield of the mixture for further in vivo efficacy evaluation. What would happen to a viral infection if suddenly there were a billion fake receptors forevery real target receptor. A version of this question is what led to the development of a novel HIV therapy at Duke University around 1996. Fast forward more than 30 years, man still lacks the proper tools to combat viral infections. One can argue that the Achilles’ heel of viral infections is their need to bind a specific receptor. This protein-protein interaction between viral proteins and human receptors is arguably the fundamental point behind all viral infections. Recently, the COVID-19 pandemic again challenged man to develop new weapons at a revolutionary pace in order to save lives. During this time, the Pentelute lab at Massachusetts Institute of Technology reported a humanized version of the peptide sequence thought to represent the binding face of the human ACE2 receptor41. The 23 amino acid sequence was derived from the α1 helix of ACE2 peptidase domain and referred to as, spike-binding peptide 1 (SBP1). It was this sequence which was postulated to be responsible for binding the receptor binding domain of the notorious COVID-19 spike protein. However, a major limitation of peptide is their short in vivo half-life (through serum proteases and renal filtration). Therefore, the main aim of our proposed research was to employ the TLHE approach to extend the in vivo half-life of the SBP1 peptide. This would allow the creation a COVID-19 entry inhibitor that could help combat the COVID-19 pandemic.

Gamma-hydroxybutyric acid (GHB) is an endogenous shorty chain fatty acid that is used clinically as Xyrem to treat narcolepsy. GHB is best known for its illicit use and abuse due to its sedative/hypnotic and euphoric effects. It is used in body building, for recreational use and sexual assault. Nonlinear toxicokinetics of GHB has been described in humans and rats with decreased total clearance at higher doses due to capacity-limited metabolism resulting in a higher plasma exposure. Renal clearance increases with increasing dose and becomes the major route of GHB elimination in overdose cases due to saturation of GHB metabolism. Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) have been identified as major transporters in the renal reabsorption of GHB moving it from filtrate back to systemic circulation. Our laboratory has previously investigated sex differences in GHB toxicokinetics at 600 mg/kg in rats and identified sex differences in MCT expression in the liver and kidney. These data suggest that individual sex hormones may be involved in altering MCT and SMCT expression in drug disposition tissues and as a result alter GHB toxicokinetics. The present study had three objectives:1) GHB toxicokinetics were evaluated over the estrous cycle, and in the presence and absence of sex hormones following a dose of 1000 mg/kg iv in rats; 2) The role of individual female sex hormones on altering GHB toxicokinetics was investigated at 1000 mg/kg and 1500 mg/kg iv following sex and cross-sex hormone treatment with 17β-estradiol and progesterone, alone or in combination; and 3) renal MCT1, MCT4, SMCT1 and CD147 mRNA and membrane protein expression were quantified in response to female sex and cross-sex hormone treatment. We have demonstrated that GHB toxicokinetics and renal clearance vary between sexes, over the estrus cycle in females and in the absence of female and male sex hormones. In hormone-treated animals, GHB toxicokinetics were altered following sex and cross-sex hormone treatment with significantly increased total clearance and decreased GHB plasma exposure at 1000 mg/kg. Significant differences in renal and metabolic clearance were observed following 1000 mg/kg and 1500 mg/kg GHB suggesting altered regulation of the underlying clearance pathways. Additionally, we have investigated the renal mRNA and membrane-bound protein expression of MCT1, MCT4, CD147 and SMCT1 which were significantly altered in response to female sex hormones in both OVX (ovariectomized female rats, ovary removal surgery performed) and CST (castrated male rats, testicles removal surgery performed) rats. The mechanisms underlying MCT/SMT regulation by female sex hormones appear to vary based on the specific transporter. The alteration of renal monocarboxylate transporters in response to female sex hormones may contribute to the observed differences in GHB toxicokinetics, which may benefit the potential antidotes of GHB as a combined therapeutic strategy in clinic. In future, inhibition studies should be performed with the coadministration of MCTs/SMCTs inhibitor with GHB to further confirm the contribution of monocarboxylate transporters to GHB toxicokinetics following sex and cross-sex hormone treatment. The influence of female sex hormones on GHB-related metabolic enzymes and monocarboxylate transporters in liver should be evaluated to further explore the mechanism of underlying the observed alterations in metabolic clearance. Sex hormone receptor expression should be evaluated by western bolt and correlated with transporter expression, combined with analysis of sex and cross-sex hormone replacement with coadministration of sex hormone receptor antagonists, to further elucidate the mechanisms underlying MCTs/SMCTs regulation in response to female sex hormones. Additionally, GHB TK studies should also be conducted, combined with sex and cross-sex hormone replacement with coadministration of sex hormone receptor antagonists, to further confirm the effect of sex hormone receptor antagonist in GHB toxicokinetics. The MCT/SMCT expression is a key determinant of their substrates’ drug disposition; sex differences and altered regulation in response to sex and cross-sex hormone treatment may contribute to differences in GHB toxicokinetics and toxicity.

The aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, has been acknowledged as a critical regulator of xenobiotic-induced toxicity and carcinogenesis. In the absence of ligand, the AHR is cytoplasmic in a complex with Hsp90, p23, XAP2, and Src. The AHR complex translocates to the nucleus upon ligand binding. After releasing its chaperones, it forms a heterodimer with ARNT, which subsequently binds to a dioxin-responsive element (DRE) for target genes transcription. Multiple aspects of cells are altered by the substantial expression of AHR target genes. Even without the AHR ligand, the cytoplasmic AHR plays a critical role in tumor progression by affecting various cellular functions. Thus, understanding the mechanisms of AHR degradation is crucial, which provides novel ways to control the AHR target genes transcription and cellular functions. In addition to the 26S proteasomal degradation triggered by ligand or geldanamycin treatment, we discovered a novel AHR degradation pathway mediated by autophagy-lysosome in A549 cells. Specifically, the chaperone-mediated autophagy (CMA) facilitates the degradation of basal AHR in the lysosome. It can be activated by 6-AN, resulting in downregulated AHR protein levels and functions, including the ligand-dependent target genes transcription and cell migration/invasion process in A549 cells.p23 as a part of the AHR cytoplasmic complex has been continuously studied in our lab over the past decade. The most prominent role of p23 is protecting AHR from degradation in both immortalized cancer cell lines (mouse hepatoma Hepa1c1c7, human hepatoma Hep3B, human cervical HeLa) and untransformed human lung bronchial/tracheal epithelial (HBTE) cell lines. It encouraged us to investigate the mechanisms further. In A549 cells, downregulation of p23 content reduced AHR protein levels, partially due to an elevated AHR protein degradation. This degradation was not reversed by proteasome inhibitor MG132 but partially restored by lysosome inhibitor CQ. We cannot rule out the possibility that selective macroautophagy was involved in the basal AHR degradation in A549 cells since the PLA results showed a positive interaction between AHR and LC3B. So far, Hela cells could be the best expression system for HaloTag-AHR overexpression. Thus, we can use the HaloTag technology as a powerful tool to study the AHR degradation mechanism via protein labeling and LC-MS/MS analysis.

Spinal cord injury results in loss of motor function and sensory perception. A myriad of obstacles prevent axonal regeneration and ultimately functional recovery in those afflicted with spinal cord injury. Combinatorial strategies addressing many of these obstacles simultaneously have shown promising results. Laboratories investigating contusional spinal cord injuries must overcome the formation of a fluid filled cyst, a physical gap that axons must traverse, at the injury epicenter. To fill the cyst, our lab has generated a 3-D electrospun matrix that is capable of directing neurite outgrowth, delivering neurotrophic support, and reducing the activity of neuroinhibitory compounds. These electrospun matrices were surgically implanted into female Long Evans Hooded rats aged approximately 60 days using a complete transection model of SCI. Following injury, rats with implants showed greater functional recovery than controls. In Chapter 1, we introduce spinal cord injury, the epidemiology, pathology and potential for regeneration, followed by our novel electrospun implant. Chapter 2 details the materials and methods. In Chapter 3, we relate the functional recovery seen to a histological analysis. The histological analysis consists of three parts: the implant integration into the host, the axons above, in and below the implant, and the functional vascular supply found within the implant. In Chapter 4, we designed a modified implant and discuss the use of this implant in vivo. With our modified implant we were able to demonstrate cellular influx and the generation of a vascular network within the implant, but poor axonal regeneration. Finally in chapter 5, I discuss potential future modifications to our electrospun matrix as well as suggestions to consider for improved functional outcome.

Endothelial dysfunction, or the inability of an artery to dilate sufficiently when subjected to excessive shear stress, serves both as a predictor of future cardiovascular events as well as an early indication of atherosclerosis. Several chronic disease states, including obesity, have been shown to alter endothelial function, which may be mediated through circulating pro- and anti-inflammatory adipokines. Still, the mechanisms by which obesity-related low-grade inflammation alters endothelial function are not fully elucidated. Acute and chronic endurance exercise training has previously been shown to be effective in improving endothelial function; however, chronic resistance exercise training is not universally regarded as beneficial to vascular functioning. Far fewer studies have examined the effect of acute resistance exercise on vascular function and adipokine release. To further understand the effects of resistance exercise training on vascular function, a meta-analysis was completed to examine the effects of resistance training on brachial artery flow mediated dilation (FMD), a common measure of endothelial function. The results of the meta-analysis indicate that resistance training has a small positive effect on FMD. Additionally, the effects of an acute bout of lower body resistance exercise on forearm blood flow (FBF) and two inflammatory cytokines were evaluated in obese (>30% body fat) and non-obese (≤30% body fat) subjects. It was hypothesized that the resistance exercise bout would increase FBF, that those changes would be greater in obese versus non-obese subjects, and that the changes in circulating cytokines (adiponectin and tumor necrosis factor-α) would be related to changes in FBF. The results indicate that FBF measures in obese and non-obese subjects react in a divergent pattern immediately following resistance exercise but return to baseline within 24 hours. These changes were not related to changes in adiponectin or TNF-α although changes in adiponectin were related to changes in TNF-α. In conclusion, resistance exercise training programs may have a small positive effect on vascular function which may reduce overall cardiovascular disease risk. Additionally, obese and non-obese subjects display differing patterns of vascular responses to an acute bout of resistance exercise, supporting the view that obesity, and its associated low-grade inflammatory response, may negatively alter vascular homeostasis.

The human Class Ib gene, HLA-E inhibits both Natural Killer Cells and a subset of CD8+ cytotoxic T lymphocytes by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for the killing of target cells. Since HLA-E has negative effects on immune killing of target cells, we have sought to identify locus specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrate that tumor cells that express GATA factors as determined by western blot and quantitative PCR, mediate a 2.4 to 4.0 fold enhanced response to IFN-γ stimulation. Functional constructs containing mutations of the core nucleotides in the GATA binding site had a 4.8 fold decreased response to IFN-γ in A2780 cells and a 8.5 to 14.0 fold decreased response to IFN-γ in SKOV3 cells. Knockdown of GATA-6 using siRNA resulted in a 40% decrease in HLA-E induction in Seg1 cells and a 30% decrease in HLA-E induction in HCT116 cells. Tetracycline regulated shRNA knockdown of GATA-6 expression in the SKOV3 cell line revealed a 3 fold decrease in the IFN-γ response of HLA-E reporter driven constructs. Additionally we observed a decreased IFN-γ response in SKOV3 cells transfected with siRNA specific for CBP and IRF-9. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression and that IRF-9 may be a target for the differential manipulation of classical MHC and HLA-E.

ANESTHESIA RECORDKEEPING: ACCURACY OF RECALL WITH COMPUTERIZED AND MANUAL ENTRY RECORDKEEPING By Thomas Corey Davis, PhD A dissertation submitted in partial fulfillment of the requirements for the degree of PhD in Health Related Sciences at Virginia Commonwealth University. Virginia Commonwealth University, 2011 Major Director: Dr. Chuck Biddle Director of Research, Department of Nurse Anesthesia And Dr. Jeffery A. Green Assistant Chief of Anesthesiology, Department of Anesthesia Introduction: Anesthesia information management systems are rapidly gaining widespread acceptance. Aggressively promoted as an improvement to manual-entry recordkeeping systems in the areas of accuracy, quality improvement, billing and vigilance, these systems record all patient vital signs and parameters, providing a legible hard copy and permanent electronic record. At risk is a potential loss of “connectedness” to the patient with the use of computerized recordkeeping, perhaps jeopardizing vigilance. Methods: This research analyzed differences in the accuracy of Certified Registered Nurse Anesthetists' (CRNAs) recall of specific patient variables during the course of an actual anesthetic case. CRNAs using computerized recordkeeping systems were compared to CRNAs using manual entry recordkeeping. Accuracy of recalled values of 10 patient variables was measured - highest and lowest heart rate, systolic blood pressure, inspiratory pressure, and end-tidal carbon dioxide levels, lowest oxygen saturation and total fluid volume. In addition, a filmed educational vignette was presented to evaluate any effect on accuracy of recall following this presentation. Four tertiary care facilities participated in this research. A Solomon four-group research design was selected to control for the effect of pretesting on results of the filmed educational treatment. Results: 214 subjects participated in this study; 106 in the computerized recordkeeping group, and 108 in the manual entry recordkeeping group. Demographic covariates were analyzed to ensure homogeneity between groups and facilities. No significant statistical differences were identified between the accuracy of recall among the groups. There was no statistically significant effect of the educational film vignette on accuracy of recall. Conclusions: There was no difference in the accuracy of practitioners’ recall of patient variables when using computerized or manual entry recordkeeping systems, suggesting little impact on vigilance. The educational film presented did not have an effect on accuracy of recall following the discussion of benefits and limitations of methods of recordkeeping.

CD23, the natural negative regulator of IgE, has been shown to be involved in asthma progression through its regulation of IgE. To investigate if its sheddase, ADAM10, is also involved in asthma progression, three mouse models were utilized; an IgE/mast cell dependent model, an IgE dependent, mast cell independent model and a mast cell and IgE independent model. Experimental asthma was then induced in mice which were selectively deficient for ADAM10 in B cells (ADAM10-/-) and compared to WT controls. The ADAM-/- mice had decreased signs of asthma, including eosinophilia, AHR and IgE synthesis in the IgE dependent model compared to LM controls, while with the IgE independent model there was no significant difference. Thus, CD23Tg and ADAM10-/- B cell mice have reduced IgE dependent lung inflammation in mouse models compared to WT controls. As a follow up, ADAM10 was inhibited in WT mice by intranasal administration of an ADAM10 inhibitor, compared to carrier (DMSO) treated mice. As with ADAM10-/- mice, inhibition of ADAM10 was only able to control IgE dependent models. These results thus show that ADAM10 is a possible target in controlling IgE dependent allergic disease, possibly as blocking ADAM10 would cause an increase in CD23 membrane expression. To better understand how ADAM10 cleaves CD23 we first sought to confirm previous studies that CD23 is internalized, with the hypothesis that shedding takes place intracellularly, rather than at the cell surface as previously assumed. Indeed, ADAM10 is more highly expressed intracellularly than at the cell surface. At 37 ºC, crosslinking CD23, especially with the anti-stalk mAb 19G5, resulted in extensive CD23 internalization. In addition, the expected increase in soluble CD23 (sCD23) production when 19G5 was added was blocked by the addition of NH4Cl. NH4Cl is known to block the progression of the endosomal pathway. These findings thus confirmed our hypothesis that cleavage of CD23 requires internalization and progression through the endosomal pathway before it is released into the extracellular space. We further demonstrated that ADAM10 is not only involved in cleaving CD23, but also in sorting CD23 into exosomes, as B cells lacking ADAM10 do not incorporate CD23 into exosomes. In addition, we found that exosomes secreted from the cell contain full length CD23, thus showing that they could bind IgE/antigen complex and be involved in the known CD23 dependent enhancement of antigen presentation by the injection of IgE/antigen complexes compared to antigen alone. These results also show that the change in ADAM10 expression specifically in a B cell could be involved in enhancement of IgE dependent inflammation. To determine what signals change ADAM10 expression, ADAM10 promoter studies were initiated. We found that both IL-21 and anti-CD40 increased ADAM10 promoter activity, while IL-4 and IL-13 had no effect. Overall our data show that increasing ADAM10 activity and expression leads to increased inflammation and IgE and is a possible target in controlling IgE dependent diseases.

PURPOSE: To create a study designed to assess patient satisfaction and preference for oral versus intravenous sedation in conjunction with periodontal surgical procedures. METHODS: Twenty-six patients who required at least two periodontal surgery procedures and requested sedation for treatment, participated in our study at VCU Department of Periodontics. This was a randomized, cross-over design with groups which received an intravenous sedative regimen with or without oral sedation premedication for one surgery and oral sedation medication alone for the other surgery. The primary outcome measurement was the type of sedation preferred by the subject. RESULTS: 14/26 (53.8%) subjects indicated a preference for intravenous sedation, compared with 7/26 (26.9%) subjects who preferred oral sedation alone. 1/26 (3.8%) subject reported that they would prefer no sedation after experiencing both oral and oral/intravenous combination sedation methods. 4/26 (15.3%) of the subjects who completed the study reported “No Difference” with regards to their preference for either method of sedation. CONCLUSION: More subjects preferred intravenous sedation and would consent to the sedation again for any future needed surgery. This study supports the need to offer intravenous sedation with periodontal surgery