This research is focused on improving health outcomes for older people by improving the continuum of care at the critical hospital-community interface. The elderly are at particular risk of medication misadventure following hospital discharge. The smooth transition of patients across of the hospital-community interface requires the availability of quality information in a timely fashion to patients and the community-based health professionals. The principal objective of this thesis was to improve medication-related communication through the provision of a community-linked Medication Liaison Service involving hospital and community-based practitioners in the identification of at risk patients, delivery of services to those patients whilst in the hospital and immediately post-discharge in their own homes, and appropriate follow-up services.

<p>Breast cancer is the most frequently diagnosed malignancy and the second most lethal cancer in women. Metastasis in breast cancer is invariably responsible for patient death and is comprised of many steps, of which proliferation in vital organs is responsible for morbidity and mortality due to vital organ failure. Patients with the metastatic disease are limited to chemotherapy, which non-specifically targets proliferating cells. Despite it being initially effective, chemotherapy is associated with high toxicity and many patients develop resistance. Thus, there is an urgent need to characterize the biology of metastatic breast cancer to develop targeted therapies for the late-stage disease.</p> <p>EGFR is a member of the ErbB family of receptor tyrosine kinases, which have particular relevance in breast tumorigenesis. Clinical studies show that high expression levels of EGFR in the primary mammary tumors correlate with poor prognosis and decreased survival of breast cancer patients due to metastasis. Patient data is supported by experimental and pre-clinical studies, which describe various signaling pathways that mediate the oncogenic effects of EGFR, such as the MAPK, STAT3, and PI3K pathways. Despite these well-documented roles of EGFR, clinical trials evaluating EGFR inhibitors (EGFRi) in metastatic breast cancer have been unanimously unsuccessful in improving patient prognosis, and the mechanisms that contribute to this intrinsic resistance are unknown.</p> <p>To characterize the signaling events that govern EGFR behavior in metastatic breast cancer resistant to EGFRi, we utilized multiple pre-clinical breast cancer progression series and patient-derived cells that display the intrinsic resistance phenomenon. In these models, EGFR functions as a pro-apoptotic molecule whose ligand-mediated activation results in growth inhibition and/or apoptosis of metastatic breast cancer cells. Here we show that in the later stages of metastasis, increased nuclear translocation of EGFR leads to increased physical access to STAT1 and STAT3 molecules residing in the nucleus. Indeed, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1/3 phosphorylation. Additionally, specific inhibition of nuclear EGFR function using the EGFR kinase inhibitor gefitinib linked to a nuclear localization signal (NLS-gefitinib) prevents EGF-induced STAT1/3 phosphorylation. Altogether, these findings implicate nuclear localization of EGFR in downstream STAT1/3 signaling in metastatic breast cancer.</p> <p>Subsequently, we examined the involvement of nuclearly-activated STAT1/3 signaling in the apoptotic function of EGFR. NLS-gefitinib treatment or genetic/pharmacologic inhibition of STAT1/3 efficiently blocks EGF-induced apoptosis in metastatic breast cancer cells resistant to EGFRi. These findings were utilized therapeutically by activating EGFR with EGF treatment while simultaneously blocking the downstream proliferative MAPK:ERK1/2 pathway using the MEK1/2 inhibitor trametinib. EGF + trametinib combination preserved STAT1 signaling while effectively blocking the MAPK pathway, thus potentiating EGF-mediated apoptosis in metastatic breast cancer cells. Importantly, combined administration of trametinib and EGF resulted in STAT1-mediated apoptosis of primary mammary tumor cells, which respond to EGF in a proliferative fashion. These data provide a novel approach of targeting metastatic breast cancer by biasing EGFR signaling towards nuclear activation of STAT1/3 signaling resulting in apoptosis.</p> Our studies herein also examined the role of STAT3 in primary mammary tumor cells overexpressing EGFR. Depletion of STAT3 expression normalized the transformed phenotype of these cells <i>in vitro</i> and resulted in smaller mammary tumors <i>in vivo</i>. These results implicate STAT3 in EGFR-driven breast tumorigenesis localized to the mammary tissues. Further, systemic dissemination of breast cancer is associated with activation of the JAK1/2:STAT3 signaling axis. Despite the involvement of STAT3 in EGFR-mediated oncogenesis in the primary tumor setting, targeting JAK1/2:STAT signaling with the JAK1/2 inhibitor ruxolitinib proved ineffective in inhibiting the growth and invasion of metastatic cells derived from these primary tumors. These results are in agreement with the role of STAT1/3 in driving the pro-apoptotic function of EGFR in metastatic breast cancer cells. Altogether, these investigations provide a plausible explanation for the inability of JAK1/2 inhibitors to effectively target metastatic breast cancer in clinical and experimental investigations. Further, these findings indicate that the development of therapeutics or molecular tools that efficiently activate STAT1/3 signaling in metastatic breast cancer may represent an important concept for eradicating tumors resistant to targeted therapies.

<p>  </p> <p>Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood. </p> <p>To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that <em>INS2</em> transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells</p> <p>We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.</p> <p>Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.</p>

<div> <div> <div> <p>Medication non-adherence and non-compliance left unaddressed can compound into severe medical problems for patients. Identifying patients that are likely to become non-adherent can help reduce these problems. Despite these benefits, monitoring adherence at scale is cost-prohibitive. Social forums offer an easily accessible, affordable, and timely alternative to the traditional methods based on claims data. This study investigates the potential of medication adherence prediction based on social forum data for diabetes and fibromyalgia therapies by using transfer learning from the Medical Expenditure Panel Survey (MEPS). </p><p><br></p> <p>Predictive adherence models are developed by using both survey and social forums data and different random forest (RF) techniques. The first of these implementations uses binned inputs from k-means clustering. The second technique is based on ternary trees instead of the widely used binary decision trees. These techniques are able to handle missing data, a prevalent characteristic of social forums data. </p><p><br></p> <p>The results of this study show that transfer learning between survey models and social forum models is possible. Using MEPS survey data and the techniques listed above to derive RF models, less than 5% difference in accuracy was observed between the MEPS test dataset and the social forum test dataset. Along with these RF techniques, another RF implementation with imputed means for the missing values was developed and shown to predict adherence for social forum patients with an accuracy >70%. </p> </div> </div> <div> <div> <p><br></p> </div> </div> </div> <div> <div> <div> <p>This thesis shows that a model trained with verified survey data can be used to complement traditional medical adherence models by predicting adherence from unverified, self-reported data in a dynamic and timely manner. Furthermore, this model provides a method for discovering objective insights from subjective social reports. Additional investigation is needed to improve the prediction accuracy of the proposed model and to assess biases that may be inherent to self-reported adherence measures in social health networks. </p> </div> </div> </div>

<p>  G Protein Coupled Receptor (GPCR) signaling plays a key role in intercellular communication and regulates many physiological processes relevant to disease. Approximately 30-40% of all FDA approved drugs target GPCR pathways, but limitations and off-target side effects remain obstacles. Regulator of G protein Signaling (RGS) proteins negatively modulate GPCR signaling by accelerating deactivation of the Gα subunit and thus represent a novel alternative to current approaches. While research on RGS proteins and how they are regulated has expanded rapidly, there are still gaps in knowledge for some members of the RGS family. One example is RGS2, which is selective for Gαq signaling. Lowered RGS2 levels are implicated in numerous diseases, and while the E3 ligase responsible for facilitating degradation of RGS2 has been identified more work needs to be done to viably drug it to enhance RGS2 protein levels. In this thesis, I explore how FBXO44, an E3 ligase substrate recognition component responsible for RGS2 degradation, interacts with RGS2 to explore approaches to inhibit degradation.</p> <p><br></p> <p>While the FBXO44-RGS2 interaction has been demonstrated previously, the degron sequence of RGS2 remained unknown. We hypothesized that FBXO44 binds RGS2 at its Nterminus and investigated this using N-terminally truncated RGS2 constructs. Our results indicated that FBXO44 binds between residues 5 and 16 of RGS2, as removal of these stabilized RGS2 against proteasomal degradation. Based on these results we designed a peptide microarray to identify important residues and properties for FBXO44 in vitro and found that Cys13 is essential for FBXO44 binding.</p> <p><br></p> <p>We also developed and optimized a high-throughput split luciferase screen to identify potential inhibitors of the FBXO44-RGS2 interaction. After forming a cell-line stably expressing tagged FBXO44 and RGS2 and optimizing assay condition, we achieved a robust assay for screening as determined by Z’-factor. <br>  </p>

<p dir="ltr"><b>Background</b></p><p dir="ltr">Heart failure (HF) is a chronic clinical syndrome characterized by symptoms including breathlessness, fatigue, swelling of the ankles, and signs such as edema pulmonary crackles etc. During HF, pathogenic mechanisms including hemodynamic overload, ventricular remodeling, aberrant calcium handling, excessive neurohormonal stimulation contribute to the worsening and progression of the condition. Ventricular arrhythmias are the common cause of sudden cardiac death (SCD) in HF patients.</p><p dir="ltr">Hyperactivation of the sympathetic nervous system (SNS), a characteristic of HF, causes an increase in circulating catecholamines which becomes detrimental to-adrenergic receptors (-AR) leading to signaling dysfunction, and decrease in contractility and the ionotropic reserve. Expression of calcium/calmodulin-dependent protein kinase II (CaMKII), a downstream effector of-AR and a key regulator of calcium homeostasis, has been shown to be enhanced in HF. CaMKII-mediated mechanisms have been demonstrated to contribute to cardiac remodeling, arrhythmias by pathological regulation of ion channels, and contractile dysfunction.</p><p dir="ltr">The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the voltage-gated potassium channel that conduct the rapid component of the delayed rectifier potassium current, <i>I</i>_Kr. The gating kinetics of <i>I</i>_Krmakes it a crucial determinant of the duration of the plateau phase of atrial and ventricular action potential (AP). Reduced <i>I</i>_Kr density due to loss-of-function mutations or pharmacological blockage of hERG channels precipitate arrhythmias. Downregulation of <i>I</i>_Kr density and protein have been reported in HF. Recent studies suggest that microRNAs (miRNAs) are involved in pathological downregulation of hERG.</p><p dir="ltr">miRNA are small non-coding RNAs of approximately 22 nucleotides in length that function as gene expression regulatory elements by repression translation. Aberrant miRNA expression has associated with cancer, cardiovascular, autoimmune, and inflammatory disorders.</p><p dir="ltr"><b>Objective</b></p><p dir="ltr">The overarching objective of this study is to investigate the mechanisms of CaMKII-mediated regulation of hERG function, including assessment of an interplay with miR-362-3p during sustained β-AR stimulation. In Specific Aim 1, the effect of CaMKII activation through sustained β-AR stimulation on hERG function and miR-362-3p expression will be assessed. The mechanism of miR-362-3p upregulation will be evaluated in Specific Aim 2, and in Specific Aim 3, the interactome of miR-362-3p and binding sites will be characterized and predicted, respectively.</p><p dir="ltr"><b>Methods</b></p><p dir="ltr">Whole-cell, voltage clamp electrophysiology experiments were performed in HEK 293 cells stably expressing hERG (hERG-HEK) and both hERG and wild-type CaMKIIδ<br>(hERG/CaMKII-HEK) following treatment with isoproterenol for 48 hours, and after transfection with miR-362-3p. The effect of CaMKII activation on miR-362-3p was assessed using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated 48 hours after isoproterenol treatment and the TaqMan assay was used to reverse transcribe and analyze miR-362-3p expression. Cells were transfected with cJun siRNA and precursor miR-362-3p to assess the role of cJun miR-362-3p upregulation during sustained β-AR stimulation with isoproterenol. The interactome of miR-362-3p was assessed in both cell lines using enhanced crosslinking immunoprecipitation (eCLIP) assay. miR-362-3p binding sites were predicted using RNAStructure Duplexfold after identification of miR-362-3p chimeric molecules from eCLIP experiment. Interaction analysis was performed using GeneMania in Cytoscape to identify genes that were potentially downregulated by miR-362-3p and been reported to interact with hERG.</p><p dir="ltr"><b>Results</b></p><p dir="ltr">In Specific Aim 1, the effect of sustained β-AR stimulation on hERG currents and endogenous miR-362-3p was assessed in hERG-HEK and hERG/CaMKII-HEK cells. Using whole-cell voltage clamp electrophysiology, we demonstrated that 48 hours treatment with 100 nM isoproterenol reduced hERG currents in hERG/CaMKII-HEK cells (p = 0.032) but had no effect on the voltage dependence of activation (p = 0.61) relative to control vehicle. Isoproterenol treatment for 48 hours, however, had no effect on hERG currents (p = 0.58) and the voltage dependence of activation (p = 0.99) in hERG-HEK cells. The effect of sustained isoproterenol treatment on miR-362-3p was also assessed using RT-qPCR. In hERG/CaMKII cells, 48 hours isoproterenol treatment increased miR-362-3p expression (2.3 folds; p = 0.038) relative to control vehicle. hERG/CaMKII-HEK cells were also treated with 500 nM KN-93 or its inactive analogue, KN-92, in an attempt to reverse CaMKII effect on miR-362-3p expression. Treatment with KN-93 decreased miR-362-3p expression (0.5-fold; p = 0.002) relative KN-92 treatment. Isoproterenol treatment had no effect on miR-362-3p expression in hERG-HEK cells (p = 0.38).</p><p dir="ltr">The regulatory mechanism of miR-362-3p expression was evaluated in Specific Aim 2. The role of an activator protein-1 (AP-1)-like sequence located at 98 base pairs upstream of miR-362-3p transcription start site was probed using siRNA inhibition of cJun, a central protein of the AP-1 complex, and deletion of the site sequence. The effect of exogenous miR-362-3p on hERG currents were first assessed. Precursor miR-362-3p decreased hERG currents (p = 0.003) compared to control plasmid. The effect of CaMKII overexpression was also assessed on exogenous miR-363-3p expression. Isoproterenol treatment in hERG/CaMKII-HEK cells transfected with precursor miR-362-3p increased mature miR-362-3p expression (0.029) compared to control vehicle treatment. Inhibition of cJun inhibition with cJun-specific siRNA decreased mature miR-362-3p expression (0.5-fold; p = 0.027) compared to scramble siRNA in hERG-HEK cells. In hERG-HEK cells transfected with mutated precursor miR-362-3p (AP-1-like site deleted), cJun inhibition with siRNA had no effect on miR-362-3p expression (p = 0.40).</p><p dir="ltr">The focus of Specific Aim 3 was to characterize the interactome of miR-362-3p as well as predict the miRNA response element (MRE) of its target mRNAs using enhanced crosslinking immunoprecipitation. A network analysis was also performed to identify miR-362-3p targets that have been reported to interact with hERG. Approximately 23% of miR-362-3p mRNA targets from the eCLIP assay have also been catalogued in miRNA database, TargetScanHuman, as miR-362-3p targets. miR-362-3p chimeric molecules with 853 unique targets, of which 75 were identified to interact with hERG through the network analysis. Four unique chimeric molecules between miR-362-3p and hERG mRNA were identified, but the interactions were non-canonical (located in the coding sequence of hERG and outside the seed region of miR-362-3p). Thirty five of the 75 miR-362-3p targets that were identified to interact had a chimeric read ≥ 3, a cutoff number indicating non-random chimeric formation. Using RNAStructure DuplexFold, miR-362-3p was predicted to form canonical binding with 12 of 35 mRNA targets. HSPA4, a heat shock protein involved in the maturation and trafficking of hERG, was identified in a canonical interaction (8-mer) with miR-362-3p.</p><p dir="ltr"><b>Conclusion</b>:</p><p dir="ltr">Sustained β-AR stimulation increases miR-362-3p expression and decreases hERG currents in CaMKII overexpressing cells. cJun mediates miR-362-3p upregulation by interacting with an AP-1-like sequence upstream of miR-362-3p transcription start site. Pathological regulation of <i>I</i>_Kr by CaMKII mediated by miR-362-3p during sustained-AR may contribute to increased risk of arrhythmias in states of increase catecholaminergic activity, such as HF.</p>

<p>G-protein coupled receptors (GPCRs) are membrane proteins that constitute ~30% of the FDA-approved drug targets. Opioid receptors are a subtype of GPCRs with four different receptor types: delta, kappa, mu, and nociception opioid receptors. Opioids such as morphine have been used for thousands of years and are deemed the most effective method for treating pain. However, opioids can have detrimental effects if used illicitly or over an extended period of time. Intriguingly, most of the clinically used opioids act on the mu opioid receptor (µOR). Hence, efforts in recent decades have focused on other opioid receptors to treat pain and other disorders. The delta opioid receptor (δOR) is one of four opioid receptors expressed in the central and peripheral nervous system. The δOR has attracted much attention as a potential target for a multitude of diseases and disorders including substance and alcohol use disorders, ischemia, migraine, and neurodegenerative diseases. However, to date, no δOR agonists, or drugs that act directly at the δOR, have been successful as clinical candidates. Nonetheless, the therapeutic potential of the δOR necessitates the targeting its pharmacologically. In this dissertation, I highlight peptide-based modulation as well as the identification of novel agonists at the δOR. I report research findings in the context of biased agonism at δOR, which is a hypothesized cellular signaling mechanism with potential therapeutic benefits. The focus on this work is the molecular determinants of biased agonism, which were investigated using a combination of cellular and computational approaches.  </p>

<p dir="ltr">My research projects focuses on the dual targeting of small molecules to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to reduce the spread of AD and related dementias. Not very many drug discovery programs focus on the specific isoforms of the tau protein. We established two series of compounds: aminoindole compounds connected by a carboxamide and triazine compounds connected by a triazine linker. Using biophysical methods we evaluated the effectiveness of both series of compounds in decreasing the amount of misfolded α-syn and tau protein in order to explore their anti-aggregation potential.</p><p><br></p>

<p>This dissertation describes the effects of a <i>de novo</i> mutation of CaMKII found in children with learning disabilities and describes its effect on catalytic activity. We develop a malachite green assay for the measurement of CaMKII activation and use it for high-throughput chemical screening to identify CaMKII inhibitors and enhancers. We also propose a new mechanism of regulation of CaMKII activity by ADP.</p><p><br></p>

<p dir="ltr">The recent global pandemic, COVID-19, has revealed to all the importance of understanding the complex relationship between viruses and hosts. Before COVID-19, I started my study of viral protein-host lipid interactions in the hemorrhagic fevers Ebola and Marburg viruses. These viruses contain a matrix protein that interacts with the plasma membrane to facilitate the formation of both authentic viruses and virus-like particles. My goal was to understand the limitations of their specific host lipid interactions. However, when the COVID-19 pandemic began, so to be our swift response in the development of a biosafety level 2 compatible model. This model can be used for studying severe acute respiratory distress syndrome 2 (SARS-CoV-2) assembly, egress, and entry. This model enabled exponentially greater access to more facilities to study the intricacies of SARS-CoV-2 assembly. With more access to studying the virus in a safe model, our goal is to push the understanding of viral assembly faster. I then began to take apart the individual pieces of the model and started to look at understanding the roles that they play independently. The membrane protein is the most abundant structural protein and I studied the specific lipid interactions of the soluble fraction of the protein. Physicians observed nucleocapsid protein mutations in the clinic with the increasing number of SARS-CoV-2 variants that are on the rise. The microscopy data collected can give us more insight into perhaps how the nucleocapsid protein induces the formation of filopodia structures at the plasma membrane. The envelope protein proved to be a challenge, but I determined a specific envelope and ceramide interaction in cells. The envelope protein was also causing the formation of microvesicles for an undefined function. I was able to determine the subcellular localization of the protein to the mitochondria. The localization to the mitochondria appears to induce depolarization of the mitochondria membrane action potential and induces the increase in mitochondria dysfunction signal, cytochrome c. Although the mitochondria were dysfunctional, there was no increase in apoptosis signal in the presence of the protein alone.</p>