<p> <b>BACKGROUND:</b> The 2009 Health Information Technology for Economic and Clinical Health (HITECH) Act created meaningful use (MU) incentive program to promote the nationwide adoption of certified electronic health record (EHR) systems. Computerized physician order entry (CPOE) system is a part of the EHR system and a cornerstone of the MU incentive program, which helps to reduce prescribing errors and enhance care coordination for treatment between providers. </p><p> <b>OBJECTIVES:</b> The main objective of this study was to investigate trends in the adoption of CPOE system for medication orders and determine factors associated with meeting the meaningful use criteria for health information technology. </p><p> <b>METHODS:</b> A cross-sectional analysis was conducted using 10 years of data from the 2006&ndash;2015 National Ambulatory Medical Care Survey (NAMCS), 10 years of 2006&ndash;2015 data from the National Hospital Ambulatory Medical Care Survey (NHAMCS)&mdash;emergency department (ED) component, the 2016 American Hospital Association (AHA) Annual Survey Database, and the 2016 AHA Annual Survey Information Technology (IT) Supplement. The outcomes of the study included the adoption of CPOE for medication orders, drug-drug interaction alerts (DDI), guideline reminders, electronic prescribing (eRx), health information exchange (HIE), and compliance with the MU criteria. Descriptive statistics were calculated for all study variables. Bivariate analysis using the chi-square test was used to determine if there is a significant relationship between the adoption of CPOE for medication orders and timing (pre-post meaningful use). Chi-square test for trend was used to determine the significance of the change in the adoption of several EHR functionalities between 2006 and 2015. Logistic regression analyses were performed to identify factors that influence the adoption of several EHR functionalities. All analyses were performed using SAS 9.3 at an alpha of 0.05. </p><p> <b>RESULTS:</b> In NAMCS 2006&ndash;2015, the weighted surveyed physicians&rsquo; responses were weighted to represent 325,070 ambulatory based physicians throughout the U.S. The majority (66%) of respondents worked in group practices, and 34% worked as solo practitioners. The overall AHA annual survey sample had 6,239 hospitals. Of these, a total of 3,656 hospitals responded to the AHA IT supplement survey, representing a response rate of 59%. Primary care physicians&rsquo; adoption of CPOE systems for medication orders was significantly higher than specialists (p &lt; 0.0001). The adoption of CPOE for medication orders was higher in the Post-MU incentive payments period (2012&ndash;2015) compared to pre-MU incentive payments period (2006&ndash;2011) in both the ambulatory care and ED settings (p &lt; 0.0001). From 2006 through 2015 there was a statistically significant increase in the percent of ambulatory care practices adopting CPOE medication ordering system with clinical decision support (CDS) tools and eRx in the ambulatory care setting (p-trend &lt; 0.001). In the same period, group practices compared to solo practices were significantly more likely to adopt these EHR functionalities (p &lt; 0.0001). From 2013 to 2015, physician offices that generated > 50% of their revenue from Medicaid in the ambulatory care setting were less likely to adopt EHR systems that meet the MU criteria compared those generate &le; 50% (p &lt; 0.01). </p><p> <b>CONCLUSION:</b> Findings indicate that physician specialty, practice size, and percentage of revenue from Medicaid are significantly associated with the adoption of selected EHR functionalities. The CPOE for medication orders adoption rates significantly increased post-MU incentive payments. No significant association was found between for-profit hospitals and sending electronic notification to the patient&rsquo;s primary care physician upon ED visit. These results may be important to design interventions to improve EHR adoption.</p><p>

<p> The overarching goal of my thesis work was to utilize the combination of mathematical and experimental models towards an effort to resolve chemotherapy-induced peripheral neuropathy (CIPN), one of the most common adverse effects of cancer chemotherapy. In chapter two, we have developed quantitative-structure toxicity relationship (QSTR) models using machine learning algorithms that enable the prediction of peripheral neuropathy incidence solely from a chemicals molecular structure. The QSTR models enable the prediction of clinical neurotoxicity, which could be potentially useful in early drug discovery to screen out compounds that are highly neurotoxic and identify safer drug candidates to move forward into further development. The QSTR model was used to suggest modifications to the molecular structure of bortezomib that may reduce the number of patients who develop peripheral neuropathy from bortezomib therapy. In the third chapter, we conducted a network-based comparative systems pharmacology analysis of proteasome inhibitions. The concept behind this work was to use <i>in silico</i> pharmacological interaction networks to elucidate the neurotoxic differences between bortezomib and carfilzomib. Our theoretical results suggested the importance of the unfolded protein response in bortezomib neurotoxicity and that the mechanisms of neurotoxicity by proteasome inhibitors closely relate to the pathogenesis of Guillian-Barr&eacute; syndrome caused by the Epstein-Barr virus. In chapter four we have written a review article to introduce the concept of Boolean network modeling in systems pharmacology. Due to the lack of knowledge about parameter values that govern the cellular dynamic processes involved in peripheral nerve damage, the development of a quantitative systems pharmacology model would not be feasible. Therefore, in chapter five, we developed a Boolean network-based systems pharmacology model of intracellular signaling and gene regulation in peripheral neurons. The model was used to simulate the neurotoxic effects of bortezomib and to identify potential treatment strategies for proteasome-inhibitor induced peripheral neuropathy. A novel combinatorial treatment strategy was identified that consists of a TNF? inhibitor, NMDA receptor antagonist, and reactive oxygen species inhibitor. Our subsequent goals were aimed towards translating this finding with the endeavor to hopefully one-day impact human health. Initially we had proposed to use three separate agents for each of these targets, however the clinical administration of three agents to prevent the neurotoxicity of one is likely unfeasible. We then came across a synthetic cannabinoid derivative, dexanabinol, that promiscuously inhibits all three of these targets and was previously developed for its intended use to treat traumatic brain injury. We believe that this drug candidate was worth investigating due to the overlapping pharmacological activity with suggested targets from network analyses, previously established favorable safety profile in humans, notable <i>in vitro/vivo</i> neuroprotective properties, and rising popularity for the therapeutic potential of cannabinoids to treat CIPN. In chapter six we assessed the efficacy of dexanabinol for preventing the neurotoxic effects of bortezomib in various experimental models. Due to the limited translatability of 2D cell culture techniques, we investigated the pharmacodynamics of dexanabinol using a microphysiological model of the peripheral nerve. Bortezomib caused a reduction in electrophysiological endpoints, which were partially restored by dexanabinol. In chapter 7 we evaluated the possible interaction of dexanabinol on the anti-cancer effects of bortezomib. We observed no significant differences in tumor volume between bortezomib alone and in combination with dexanabinol in a multiple myeloma mouse model. Lastly, we are currently investigating the efficacy of dexanabinol in well-established rat model of bortezomib-induced peripheral neuropathy. We believe that positive results would warrant a clinical trial. In conclusion, the statistical and mechanistic models of peripheral neuropathy that were developed could be used to reduce the overall burden of CIPN through the design of safer chemotherapeutics and discovery of novel neuroprotective treatment strategies.</p><p>

Bakgrund: Det finns många orsaker till ofrivillig barnlöshet men tack vare nya tekniker har det blivit möjligt att få barn för många. Först och främst är det in vitro fertilisering (IVF)- / Intracytoplasmatisk spermieinjektion (ICSI)-behandlingar genom kontrollerad ovariestimulering (COS) av gonadotropiner som har ökat möjligheten att få flera ägg för att öka chansen att få en lyckad graviditet. Under de senaste årtiondena har en markant utveckling gjorts inom gonadotropins område från urin extraherade gonadotropiner till rekombinanta gonadotropiner. De två välstuderade och mest använda gonadotropinerna för COS vid IVF/ICSI är högrenat humant menopausalt gonadotropin (HP-hMG) och rekombinant humant follikelstimulerande hormon (r-hFSH, follitropin alfa). Syfte: Syftet med föreliggande arbete var att jämföra effektivitet och farmakologiska skillnader på Menopur ® (HP-hMG) och GONAL-f ® (r-hFSH) för COS vid IVF/ICSI-behandling. Metod: Denna litteraturstudie byggde på vetenskapliga artiklar, som söktes fram via PubMed, Webb of Science. Fem kliniska studier, vilka främst fokuserade på effektiviteten och farmakologiska skillnader av två gonadotropinpreparat (r-hFSH och HP-hMG) för COS vid IVF/ICSI, granskades närmare. Resultat: Granskningen visade att resultatet med avseende på pågående graviditetsgrad är jämförbart mellan HP-hMG och follitropin alfa oavsett fast eller flexibel dos både med GnRH-agonist- och antagonistprotokoll. Resultat med avseende på farmakologiska skillnader för COS visade att antalet erhöllna oocyter och progesteronnivån var signifikant högre hos r-hFSH-gruppen. Däremot var serum E2-nivån signifikant högre hos HP-hMG-gruppen medan serum LH-nivån var liknande i båda grupperna. Slutsats: Utifrån denna litteraturstudie demonstrerades icke-inferioritet med avseende på den pågående graviditeten för HP-hMG jämfört med r-hFSH. Men det fanns tydligt farmakologiska skillnader för COS mellan dessa två gonadotropinpreparat exempelvis serum E2-nivå, progesteronnivå och antal erhöllna oocyter. Detta skulle kunna bero på att LH-aktivitet spelade en viktig roll både för att optimera kvalitet och utvecklingspotential hos oocyterna. r-hFSH är rekombinant och därmed kan tillverkas oberoende av tillgång på humant material. Detta bör på längre sikt innebära att tillverkningskostnaderna minskar och att preparatet blir billigare samt mera tillgängligt för patienterna. Då effekten mellan preparaten i denna studie inte tycks skilja sig åt i någon högre utsträckning kanske framtida investeringar skulle sikta på att försöka utveckla effektivare r-hFSH-preparat. Dessutom bör större kunskap om betydelsen av de olika farmakologiska profilerna av dessa två gonadotropiner med avseende på reproduktiva resultat utredas genom ytterligare undersökningar i större effektstudier. Ur ett kliniskt perspektiv bör mer forskningsfokus läggas på att undersöka behandlingsprotokoll som förbättrar oocytkvaliteten och kliniska utfall istället för att maximera antalet erhållna oocyter.

<b>Background:</b> Sustained elevations in catecholaminergic signaling, mediated primarily through β-adrenergic receptor (β-AR) stimulation, are a hallmark neurohormonal alteration in heart failure (HF) that contribute to pathophysiologic cardiac remodeling. An important pathophysiological change during sustained β-AR stimulation is functional inhibition of the slow delayed rectifier potassium current, IKs, which has been demonstrated to prolong action potential duration (APD) and increase ventricular arrhythmogenesis in HF. Though functional inhibition of IKs has been consistently reproduced in cellular, animal, and limited human studies of HF, the mechanisms that mediate IKs inhibition during HF remain poorly understood.<div><br></div><div>In addition, HF results in aberrant calcium handling that is known to contribute to the disease. HF has been demonstrated to increase the expression and function of calcium/calmodulin-dependent protein kinase II (CaMKII), a key regulator of calcium homeostasis and excitation-contraction coupling in cardiomyocytes. Enhanced CaMKII signaling has been consistently demonstrated to contribute to increased arrhythmogenesis in a number of cardiac diseases, including HF. CaMKII is a known pathological regulator of many cardiac ion channels resulting in APD prolongation and the development of arrhythmias.<br></div><div><br></div><div><b>Objective:</b> This investigation aims to assesses the potential for CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and to characterize the potential functional implications on IKs. Furthermore, this investigation seeks to elucidate the mechanism underlying CaMKII-mediated IKs inhibition during sustained β-AR stimulation.<br></div><div><br></div><div><b>Methods: </b>Phosphorylation of KCNQ1 was assessed using a tandem liquid chromatography- mass spectrometry/ mass spectrometry (LCMS/MS) approach during sustained β-AR stimulation via treatment with 100 nM isoproterenol (ISO) for 4-24 hours and during co-expression with KCNE1. Whole-cell, voltage-clamp patch clamp electrophysiology experiments were performed in HEK 293 cells transiently co-expressing wild-type (WT) or mutant KCNQ1 (mutations conferring mimics of dephosphorylation and phosphorylation were introduced at phosphorylation sites identified by LCMS/MS) and KCNE1 (auxiliary subunit) during ISO treatment, treatment with CaMKII or protein kinase A (PKA) inhibitors, or during lentiviral δCaMKII overexpression. A robotic peptide synthesizer was used to create fifteen residue peptide fragments on a nitrocellulose membrane corresponding to KCNQ1 intracellular domains and the KCNQ1 residues identified via LCMS/MS; membranes were incubated with activated CaMKII or PKA in the presence of radiolabeled ATP to identify potential sites of phosphorylation. Bimolecular fluorescence complementation (BiFC) experiments were performed in HEK 293 cells to assess the impact of CaMKII-mediated KCNQ1 phosphorylation on the interaction of KCNQ1 and KCNE1 subunits. Protein immunoblot experiments were performed to (1) assess CaMKII activation during ISO treatment and (2) to assess plasma membrane expression of KCNQ1 and KCNE1 subunits with mimics of differential KCNQ1 phosphorylation following a membrane protein biotinylation procedure.<br></div><div><br></div><div><b>Results:</b> In Aim 1, we investigated the regulation of the KCNQ1 carboxyl terminus during sustained β-AR stimulation and assessed the associated functional implications on IKs. An LCMS/MS approach identified five novel KCNQ1 carboxyl terminal sites that demonstrated basal phosphorylation, with T482 and S484 having enhanced phosphorylation during treatment with 100 nM ISO for 24 hours (p<0.01 at both sites). Using patch clamp electrophysiology, we demonstrated that treatment with 100 nM ISO for 12-24 hours reduced IKs current density (p=0.01) and produced a depolarizing shift in the voltage dependence of activation (p<0.01) relative to vehicle. Mimics of phosphorylation (mutations to aspartic acid; Triple-D KCNQ1) at S457, T482, and S484 in combination, meanwhile, reduced IKs activation current density relative to dephosphorylation (mutations to alanine; Triple-A KCNQ1) mimics (p=0.02) but did not affect the voltage dependence of activation (p=0.66). Functional assessment of these sites individually revealed that phosphorylation mimics at S457 (p=0.02) and S484 (p=0.04), but not at T482 (p=0.53), reduced IKs current density relative to mimics of dephosphorylation. Similarly, the voltage dependence of activation was right-shifted with phosphorylation mimics at S457 (p=0.03) and S484 (p=0.02), but not at T482 (p=0.99), relative to mimics of dephosphorylation.<br></div><div><br></div><div>The focus of Aim 2 was to assess the potential for CaMKII signaling to regulate increased KCNQ1 phosphorylation and reduced IKs function during sustained β-AR stimulation. Peptide fragments corresponding to the KCNQ1 carboxyl terminal sites demonstrating basal phosphorylation via LCMS/MS analysis were synthesized on a nitrocellulose membrane and exposed to activated δCaMKII. Only peptide fragments corresponding to S484 demonstrated CaMKII phosphorylation. Patch clamp experiments demonstrated that CaMKII inhibition via the chemical inhibitor KN-93 (p=0.02) and the peptide inhibitor CN21 (p<0.01) reversed ISO-treatment associated inhibition of IKs activation current density relative to appropriate controls (KN-92 and CN21-Alanine, respectively). Inhibition with KN-93 and CN21 (p<0.01 for both) also reversed ISO-treatment associated right shifts in the voltage dependence of activation relative to appropriate controls. The ability of ISO treatment to activate CaMKII in HEK 293 cells was confirmed via protein immunoblot wherein T287 phosphorylation (CaMKII residue conferring constitutive activity) was increased during ISO treatment (p<0.05). Lentiviral overexpression of δCaMKII inhibited IKs activation current density with WT IKs (p=0.01) but not with Triple-A IKs (p=0.20) relative to lentiviral control. Inhibition of IKs activation current density during δCaMKII overexpression was attenuated with S484A IKs (p=0.04) but not with S457A (p=0.99) relative to WT IKs during δCaMKII overexpression. The voltage dependence of activation was also right-shifted during δCaMKII overexpression relative to lentiviral control (p=0.03). PKA inhibition with the peptide inhibitor PKI did not reverse ISO-treatment associated inhibition of IKs activation current density (p=0.51), and PKA did not phosphorylate peptide fragments corresponding to any of residues identified via LCMS/MS.<br></div><div><br></div><div>Aim 3 investigated the mechanism through which CaMKII-mediated phosphorylation at KCNQ1 S484 inhibits IKs function. To assess whether interaction with KCNE1 affects KCNQ1 phosphorylation, we performed LCMS/MS experiments during expression of KCNQ1 alone and during co-expression with KCNE1. Phosphorylation at S484 was reduced during co-expression with KCNE1 relative to expression of KCNQ1 alone (p<0.01). In addition, mimics of phosphorylation at S484 (S484D) did not affect activation current density (p=0.96) or the voltage dependence of activation (p=0.51) relative to dephosphorylation mimics (S484A). Based on these results, we hypothesized that S484 phosphorylation affected the interaction between KCNQ1 and KCNE1 subunits; accordingly, we assessed the KCNQ1-KCNE1 interaction using BiFC experiments in HEK 293 cells. In accordance with our hypothesis, Venus fluorescent intensity (corresponding to KCNQ1-KCNE1 interaction) was reduced during ISO treatment relative to vehicle (p<0.05) and with S484D KCNQ1 relative to S484A (p<0.01). The role of CaMKII in mediating this disruption of KCNQ1-KCNE1 interaction was demonstrated BiFC experiments that showed co-treatment with ISO and KN-93 attenuated reduced Venus intensity during co-treatment with ISO and KN-92 (p<0.01). These results were corroborated by BiFC experiments with Long QT Syndrome Phenotype 1 (LQT1) mutations that demonstrated that an LQT1 mutation predicted to disrupt CaMKII phosphorylation at S484 (R481I) attenuated reduced Venus intensity during ISO treatment relative to an LQT1 mutations predicted to not affect CaMKII regulation of S484 (S484T; p<0.01). The ability of S484 phosphorylation to affect KCNQ1 and/or KCNE1 trafficking was assessed via protein immunoblot experiments to detect KCNQ1 and KCNE1 following a biotinylation procedure to isolate plasma membrane-bound proteins. Biotinylation experiments demonstrated that KCNQ1 and KCNE1 plasma membrane expression were reduced by ~15% and ~33%, respectively, with S484D KCNQ1 relative to S484A (p<0.05 for both).<br></div><div><b><br></b></div><div><b>Conclusion: </b>CaMKII phosphorylates KCNQ1 S484 during sustained β-AR stimulation to inhibit IKs function. S484 phosphorylation inhibits IKs function by disrupting the interaction between KCNQ1 and KCNE1 subunits and by reducing the plasma membrane expression of KCNQ1 and KCNE1. Pathological regulation of KCNQ1 by CaMKII (and subsequent inhibition of IKs) during sustained β-AR stimulation may contribute to increased arrhythmogenesis during physiologic states of chronically increased catecholaminergic tone, such as during HF.<br></div>

Poor prognosis and resistance to therapy in malignant gliomas is mainly due to highly dispersive nature of glioma cells. Aggressive infiltration in the brain parenchyma poses a serious challenge to complete tumor resection and effective treatment. Moreover, absence of valid biomarkers confounds definitive diagnosis and therapy response prediction. Identification of novel markers will require improved understanding of glioma genetics, particularly as it relates to key regulatory signals that control glioma cell migration. Since, these tumors are genetically heterogeneous, genomic explorations could improve understanding of molecular mechanisms underlying glioma migration and aid in discovery of molecular markers of clinical impact. Thus the central aim of our study is to identify novel genetic markers in patients with glioma for better prognosis, diagnosis and prediction of response to treatment. In this study we screened for genome wide copy number alterations and loss of heterozygosity in a representative glioma population of sixty patients. Alterations were detected at multiple chromosomal loci with putative tumor suppressor genes and oncogenes. In relation to molecular determinants of glioma cell migration, alterations at the Protein Tyrosine Phosphatase Receptor type Kappa (PTPRK) locus were the most frequent in our glioma population. PTPRK alterations are relevant to glioma biology as PTPRK is a cell adhesion molecule that is highly expressed in brain. However, function of PTPRK has not been described previously in gliomas. This led to our central hypothesis that PTPRK is a novel biomarker that suppresses diffusive capacity of glioma cells and thereby may improve glioma therapeutic outcome. Overexpression and knockdown experiments were performed to study PTPRK function using malignant glioma cell lines. We explored molecular mechanisms underlying PTPRK function and its effect on response to therapy. Moreover, we discovered novel PTPRK mutations by sequencing full length PTPRK transcripts in numerous glioma biopsies. Effect of these mutations on PTPRK function and response to anti-glioma therapeutics were subsequently analyzed using in vitro cell based assays. Our results suggest that PTPRK is an independent prognostic marker and a glioma tumor suppressor that is altered at both transcriptional and post-translational levels. Proteolytic processing of transmembrane PTPRK protein generates a series of fragments which are the only detectable PTPRK forms in glioblastoma. Short hairpin RNA (shRNA) mediated downregulation of PTPRK reduces glioblastoma cell migration suggesting that proteolysis of PTPRK contribute to migration of glioblastoma cells. Additionally, overexpression of wild-type PTPRK suppresses growth and migration of malignant glioma cells which correlates with inhibition of EGFR and beta-catenin signaling, and improves effect of conventional therapies for glioma. However, PTPRK mutations abrogate tumor suppressive effects of wild-type PTPRK and alter sensitivity of glioma cells to chemotherapy. These results indicate that the cell surface PTPRK protein is a tumor suppressor of prognostic and predictive value which is frequently inactivated in malignant gliomas.

Over recent decades, intranasal drug delivery has received considerable attention. The intranasal route is a non-invasive route that provides rapid drug absorption with a quick onset of action and enhanced bioavailability by avoiding hepatic first-pass metabolism and GIT degradation. The unique anatomical connections between the nasal cavity and the CNS provided by the olfactory and trigeminal nerves also makes the nasal route an interesting administration site for the purpose of CNS targeting. Epithelial transporters in the nasal mucosa modulate the uptake and efflux of substrates and their subsequent systemic absorption and/or CNS targeting. Ribavirin, a nucleoside-analog antiviral drug compound, inhibits the replication of various strains of viral encephalitis, a progressive and fatal CNS disorder, in cell culture. However, it has been reported that ribavirin was ineffective in the treatment of encephalitis following intramuscular, subcutaneous, or intraperitoneal injection. These findings suggest that ribavirin is unable to cross the blood brain barrier. The nasal route, which shows promise in targeting drugs to the brain via the olfactory region might be a potential administration route for ribavirin in the treatment of encephalitis. Ribavirin may also be a potential substrate for uptake via nucleoside transporters in the nasal mucosa. The objective of this study was to measure the permeation of ribavirin across the nasal mucosa in vitro and to evaluate the role of nucleoside transporter-mediated uptake and P-gp-mediated efflux in the total permeability of ribavirin across the nasal respiratory and olfactory tissues. The permeation of ribavirin across the nasal mucosa was investigated using bovine nasal tissues. Both respiratory and olfactory nasal tissues were exposed to ribavirin-containing solutions (concentration range 50µM - 20mM), and a non-linear uptake of ribavirin was observed in the olfactory mucosa. These results suggest the contribution of saturable transporters, specifically nucleoside transporters, in the nasal uptake of ribavirin. A linear increase in permeation with increasing ribavirin concentration was observed across the respiratory nasal mucosa which suggests a lower activity of the nucleoside transporters in these tissues. To further evaluate the role of nucleoside transporters on ribavirin intranasal uptake, NBMPR (nitrobenzyl mercaptopurine ribonucleoside) was used as an equilibrative nucleoside transporter (ENT) inhibitor. When bovine nasal tissues were exposed to solutions containing ribavirin and NBMPR, ribavirin flux was decreased by 45% suggesting that ENTs play an important role in the nasal uptake of ribavirin. The effect of P-gp-mediated-efflux on ribavirin uptake was also studied. Two inhibitors were used to evaluate the role of general energy-dependent transporters (sodium azide) and the specific role of P-glycoprotein (verapamil). Sodium azide (known to inhibit ATP synthesis and deplete cellular ATP) only resulted in a slight increase in ribavirin flux across the nasal respiratory and olfactory tissues. However, the flux of ribavirin across the respiratory nasal tissues increased significantly when 50 uM of verapamil (specific P-gp inhibitor) was included. These findings may be attributed to the effect of sodium azide to inhibit cellular ATP synthesis, and thus it might affect other transporters such as concentrative nucleoside transporters (CNTs) that utilize ATP indirectly to transport ribavirin across the cell membrane. Knowledge of the contributions of nucleoside transporters and P-gp in the nasal uptake of ribavirin is valuable in the evaluation of the potential absorption and subsequent distribution of drugs across the nasal mucosa. Reducing P-gp-mediated efflux and enhancing nucleoside transporter activity may enable the nasal administration of ribavirin to treat encephalitis.

No description available.

G protein-coupled receptors (GPCRs) play a central role in numerous biological processes, from olfaction to vision to neurotransmission and more, leading to their classification as important drug targets. Signaling through GPCRs is carried out by a number of intracellular effector proteins, such as the α subunits of heterotrimeric G proteins and Adenylyl Cyclases (AC). In this work, inhibition of protein-protein interactions to reduce GPCR-mediated cAMP formation is explored to identify compounds with potential therapeutic value. Regulators of G Protein Signaling (RGS) inhibit Gαi/o signaling, resulting in persistent AC-mediated cAMP formation. Fragment-based screening against RGS17, which has been implicated in a number of cancers, identified seven hits that bind RGS17, though only one leads to inhibition of RGS17 function in vitro. Importantly, these compounds represent a starting point for future structure-based drug discovery targeting RGS17 for the development of new chemotherapeutics. As different RGS proteins serve different functions throughout the body, achieving isoform selectivity is important for reducing potentially deleterious side effects. Analysis of the selectivity of previously described RGS4 inhibitors revealed that several are more potent inhibitors of RGS14. This work also identified the RGS proteins for which inhibitors are likely to be found (RGS14, RGS4, RGS1) and those which are likely to be more difficult drug targets (RGS6 and RGS7). Finally, inhibition of AC8 as a means to decrease intracellular levels of cAMP is explored. AC1 and AC8 are robustly stimulated by CaM, so inhibition of the AC/CaM interaction was explored as a novel mechanism of AC inhibition. Biochemical and cell-based assays were developed, and a pilot screen of 1,000 FDA-approved compounds identified six capable of inhibiting the AC8/CaM interaction. This ultimately resulted in reduced AC8 activity and cAMP accumulation, validating this interaction as a druggable target. The compounds identified were not selective for AC8 over AC1, but they have pre-clinical utility in evaluating the biology of CaM-stimulated AC activity.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by two pathological hallmarks, selective loss of dopaminergic neurons and intraneuronal protein aggregation. The presence of an endogenous neurotoxin has been implicated in the pathogenesis of the disease, to explain the observed neurodegeneration. Dopamine (DA) has been indicated to be an endogenous neurotoxin as DA readily undergoes auto-oxidation to an o-quinone capable of protein modification. However, DA is metabolized by monoamine oxidase to form the intermediate 3,4-dihydroxyphenylacetaldehyde (DOPAL) and several studies have demonstrated DOPAL to be orders of magnitude more toxic than DA. An accumulation of DOPAL may cause dopaminergic cell death via the formation of free radicals, inhibition of the mitochondrial transition pore or protein modification. The hypothesis of this work is that DOPAL, a potential endogenous neurotoxin relevant to PD, is capable of protein modification and protein cross-linking through reactivity with amine and thiol nucleophiles. Results demonstrate that elevated DOPAL concentrations in striatal synaptosomes will yield considerable protein modification. In addition, DOPAL was demonstrated to be highly reactive towards amine nucleophiles in comparison to thiol nucleophiles. However, DOPAL was demonstrated to mediate protein cross-linking through reactivity with protein thiols subsequent to modification of amines, indicating DOPAL to be a bifunctional electrophile. Furthermore, a novel isolation procedure was developed, and through a proteomics-based approach, twelve proteins were identified to be relevant to PD and susceptible to DOPAL modification. This research demonstrates increased concentrations of DOPAL lead to significant cellular consequences (i.e. protein modification) and implicate DOPAL as a potential neurotoxin relevant to the pathogenesis of PD.

The objective of this research was to estimate prophylactic G-CSF effectiveness among patients on the extensive margin, whose treatment decisions are most likely to be affected by policy changes intended to alter the G-CSF treatment rate. Using the national Surveillance, Epidemiology and End Results (SEER)-Medicare Linked Database, we studied patients 66 years or older diagnosed with Non-Hodgkin's Lymphoma (NHL) and on anthracycline in one of the 13 SEER registry areas from 1994-2002. Prophylactic G-CSF use was designated if a patient had a G-CSF claim within the first five days of the first chemotherapy cycle. The dependent variable of neutropenia hospitalization (NH) was identified within 6 months of diagnosis and was further specified as NH incidence within 21, 42, 63 and 126 days after anthracycline initiation in sensitivity analyses. Multivariate regression estimates were used to examine whether treated patients actually benefited from G-CSF. Instrumental variable estimates using local area prophylactic G-CSF treatment rates as instruments were used to estimate whether increases in the G-CSF utilization rate could lead to further reductions in the rate of neutropenia hospitalization. We found only 9.85% of study patients had prophylactic G-CSF. After adjustment for patient demographic and clinical risk factors, multiple regressions indicated prophylactic G-CSF significantly reduced NH events within six months of diagnosis date for the patients who received G-CSF (OR=0.595, 95% CI=0.384-0.922). This estimate of G-CSF's effect may be biased low from the true values of Average Treatment effect on the Treated (ATT) because patients may be selected into treatment based on unobservable risk factors. Chow F-statistics showed our instrumental variable of area prophylactic G-CSF treatment rate described a statistically significant portion in the variation of G-CSF use (F=60.46, P Among elderly NHL patients on anthracycline-based chemotherapy, our multiple regression estimates suggest that patients treated with prophylactic G-CSF reduced their neutropenia risk within six months of diagnosis date. The effect of prophylactic G-CSF on neutropenia hospitalization among marginal patients whose choices varied with local area G-CSF treatment rate was negative. Substantial G-CSF treatment benefits within the first cycle of chemotherapy were available for patients on the extensive margin. Higher treatment rates may be guaranteed to improve patient short-term benefits from G-CSF.