Pharmacy

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Advancing Understanding of the Bacteriophage Lambda T4RII Exclusion (REX) Phenotype
(University of Waterloo, 2025-10-20) Alattas, Hibah
The Bacteriophage Lambda (λ) T4rII exclusion (Rex) system is a finely tuned antiviral defense mechanism in Escherichia coli (E. coli) lysogens that prevents the propagation of T4rII mutant bacteriophage. This exclusion phenotype is defined as the inability of T4rII mutant bacteriophage to propagate within λ-lysogenized E. coli cells. This phenomenon is encoded by two genes of λ (rexA and rexB), whose expression is primarily regulated and driven by the λ PM promoter under the regulation of the λ cI repressor gene. The T4rII infection of Rex+ lysogen triggers the onset of the Rex phenotype. It is characterized by a harsh cascade of cellular responses, including rapid membrane depolarization and the induction of a stress environment that mimics the physiology of the stationary phase. In addition, the disruption of the RexA:RexB balance, particularly the overexpression of rexA relative to rexB, can lead to the same manifestations without infection, indicating that the stoichiometry of RexA:RexB is relevant. Rex “activation” results in significant cell death and has been proposed to serve as an altruistic cell death system to protect the species. In contrast, despite some cell killing, infected lysogens do, to an extent, recover from Rex activation. As such, the phenotype may additionally serve as a mutualistic mechanism that protects both the λ prophage and its stability, as well as its host, by limiting superinfection. For a long time, researchers have hypothesized that E. coli host proteins are involved in this pleiotropic phenotype and identifying them has been a key goal in elucidating the Rex mechanism. Notably, Rex activation depends not only on gene expression and the stoichiometry of RexA to RexB but also on an optimal monovalent cationic environment, underscoring the importance of membrane ion channels and transport systems. Identifying all host mutations that attenuate or abrogate Rex will help in understanding the role of individual components, directly or indirectly, in triggering and inducing the cellular (physiological) manifestations of the Rex phenotype. While many E. coli membrane proteins control the passage of solutes and ions through the membranes, mutations in these proteins may disrupt the ionic environment, thereby resulting in the attenuation or abrogation of Rex. This finding was observed and confirmed by the deletion of several outer membrane proteins, including ompW, ompX, ompA, and ompF (Alattas et al., 2020). Beyond these membrane-associated proteins, the ability of additional genetic and regulatory components to modulate and influence Rex and Rex escape to varying degrees was demonstrated, including tolA, mglC, and rpoS (S). Several of the identified genes play a critical role in cellular osmotic balance and are governed by the stationary phase sigma factor (S) or envelope stress sigma factor (E) as part of the stationary phase and/or envelope distress regulons, under the control of many non-coding regulatory small RNAs. The impact of mutations in these regulatory proteins on Rex activity was examined following superinfection by T4 and T4rII, and the role of the S regulatory proteins, including RssB and IraD, in the Rex and Rex escape mechanisms was reported for the first time. However, the mechanism by which this occurs remains unclear. The impact of fluorescence fusions on colony morphology and texture was investigated, showing variations in colony size among fusion sites and Rex types. These experiments suggested that the fusion site and the expression status of the partner significantly affect Rex activity, likely by interfering with proper protein folding, complex formation, or the stoichiometric balance of RexA to RexB. N-terminal fusions severely attenuated Rex activity, while dual N-terminal tagging almost entirely abolished it. In contrast, C-terminal fusions only partially affected Rex activity, with dual C-terminal fusions showing minimal impact on exclusion capability. Together, these findings highlight the Rex system as a multifactorial, tightly regulated antiviral mechanism that integrates gene stoichiometry, membrane composition, sigma factor regulation and its RNA-mediated control. This work adopts a genetic approach to enhance and refine the existing model of Rex-mediated T4rII exclusion, leading to a new model of Rex that offers new insights into its underlying molecular determinants and paves the way for further exploration of bacteriophage-host dynamics, superinfection exclusion strategies, and programmed cell death in prokaryotes. Keyword: Rex exclusion, RexA, RexB, T4rII, T4, Rex phenotype, λ lambda, sigma factors, E. coli, bacteriophage, phage
Theranostic Gold Nanoparticles for Enhanced Prostate Cancer Radiotherapy
(University of Waterloo, 2025-09-23) Aborig, Mohamed
Theranostic gold nanoparticles (GNPs) were engineered to enhance external-beam radiotherapy for prostate cancer while enabling quantitative imaging readouts. I synthesized biocompatible, polyphenol-functionalized GNPs using epigallocatechin gallate (EGCG-GNPs) and curcumin (Curc-GNPs), optimized for colloidal stability, cell-receptor affinity, and antioxidative properties. Comprehensive physicochemical characterization (DLS/ζ-potential, TEM, UV–Vis) and analytical assays (HPLC for drug loading; ICP-MS for Au quantification) established reproducible formulations. In vitro studies in PC-3 cells demonstrated efficient cellular uptake and radiosensitization, evidenced by reduced clonogenic survival compared with radiation alone. In vivo, murine and canine models were used to evaluate biodistribution, acute/sub-acute toxicity, and imaging. Computed-tomography (CT) phantom and tissue studies confirmed a linear relationship between Hounsfield units and gold concentration, enabling noninvasive estimation of intraprostatic nanoparticle burden. A physiologically based pharmacokinetic (PBPK) model captured organ kinetics and supported translation of exposure–response. Finally, I piloted an image-guided intra-arterial delivery paradigm adapted from prostatic arterial embolization: nanoparticle infusion into prostate lobes followed by embolization to promote local retention, reduce systemic exposure, and potentiate radiation dose deposition. This minimally invasive procedure was tested in 3 lab beagles and 1 clinical canine case with naturally occurring prostate cancer. Collectively, these data establish a dual-functional GNP platform that couples CT-visible quantification with meaningful radiosensitization, laying the preclinical and procedural groundwork for image-guided nanoparticle-augmented radiotherapy in prostate cancer.
Design and Evaluation of Targeted mRNA Delivery Systems to the Retina
(University of Waterloo, 2025-09-18) Hakim, Antoine
Glaucoma is a neurodegenerative eye disease traditionally linked to elevated intraocular pressure (IOP). However, recent studies have revealed a complex pathogenic process involving the interplay of multiple factors, leading to the death of retinal ganglion cells (RGCs) and ultimately resulting in vision loss. Current treatments primarily aim to normalize IOP levels. Nonetheless, they are limited by issues and have often failed to prevent eventual vision loss. Neuroprotective gene therapy presents a promising approach to safeguarding RGCs and promoting their survival against potential damage from elevated IOP. This can be achieved by increasing neurotrophic factors (NTFs) and reducing the activation of apoptotic pathways. In this project, the main objective was to develop an effective non-viral lipid nanoparticle (LNP) system for mRNA for intravitreal (IVT) delivery in the eye using a second-generation parent 18-7NH-18 gemini surfactant (GS) and three derived cell-adhesive-peptide (CAP p1, p3 and p5) conjugated GSs: 18-7Np1-18, 18-7Np3-18, and 18-7Np5-18, along with other lipids. The goal was to enable effective mRNA delivery to the retina as an initial step towards developing a non-viral neuroprotective gene therapy for glaucoma. Gem++ION hybrid lipid nanoparticle (LNP) system was developed and prepared using Ultrasonication Method 1-3h (USM1-3). The parent Gem++ION Supra formulation and two peptide-conjugated Gem++ION (pGem++ION) Supra formulations were evaluated in vitro in two retinal cell lines and in vivo in CD-1 mouse model. The results demonstrated >90% transfection efficiency (TE) in vitro for all three formulations in both cell lines. In the in vivo study, the two pGem++ION Supra formulations, after 48 hours of intravitreal administration, showed GFP expression in various retinal layers, consistent with the Cy5-labelled LNP biodistribution results by confocal microscopy. Additionally, the three Gem++ION Supra LNP formulations exhibited GFP expression in the anterior eye segment, including the ciliary body, iris, and cornea. Overall, in this project, a novel hybrid dicationic+ionizable lipid LNP system with CAP targeting ligands, termed Gem++ION Supra, and pGem++ION Supra formulations, with high efficiency to deliver and transfect retinal cells, particularly the RGCs, both in vitro and in vivo, were developed, paving the way for further development in neuroprotective gene therapy for glaucoma.
The Effects of Sequence Variations in the Structural Dynamics and Ligand Interactions of Main Proteases in Betacoronaviruses
(University of Waterloo, 2025-09-15) Ramgoolam, Shubham Devesh
The main protease (Mpro) of betacoronaviruses is an essential enzyme for viral replication and a premier target for antiviral drug discovery. The high conservation of its active site across the genus makes it an ideal candidate for developing pan-coronavirus therapeutics to combat future outbreaks. In response to the Coronavirus disease 2019 (COVID-19) pandemic, numerous large-scale screening campaigns were initiated to identify Mpro inhibitors. However, these efforts were frequently hindered by the fundamental pose classification problem, which is the inability to distinguish between correct and incorrect binding poses. Compounding this issue, the research response generated an unprecedented volume of Mpro structural data, yet existing bioinformatics platforms lack the integrated tools for its systematic, comparative analysis. These combined challenges hinder the rational design of next-generation inhibitors. In this work, we address these challenges by developing a novel, integrated computational toolkit. We first present CoviProDigy, a web-based platform designed for the comprehensive and comparative analysis of Mpro-ligand interactions, featuring specialized tools for subpocket occupancy analysis and elucidating common and unique interactions across different ligands. This functionality is designed to support scaffold hopping and medicinal chemistry optimization based on the molecular-level insights gained from known structures. To overcome the major issue of inaccurate pose selection in virtual screening, we then developed and validated a fine-tuned machine learning model that demonstrates enhanced accuracy in classifying ligand binding poses for the Betacoronavirus Mpro family. Together, these contributions provide a robust, end-to-end framework that accelerates the discovery of potent inhibitors, presenting a valuable resource for pandemic preparedness and the ongoing search for broad-spectrum antiviral agents.
Antisense Oligonucleotide-Loaded Liposomes for Gene Silencing in Bacterial Biofilms
(University of Waterloo, 2025-09-03) Orellana, Kaidy
The key pathogens found in chronic wound infections include Pseudomonas aeruginosa [P. aeruginosa] and Staphylococcus aureus [S. aureus], which can form biofilms on compromised skin barriers. Once bacteria attach and proliferate in the affected areas, they begin to secrete various virulence factors and other extracellular molecules, such as exopolysaccharides, for their survival and persistence at these sites of infection. These factors are secreted through a complex cell-to-cell communication system termed ‘quorum sensing,’ through which bacterial phenotypes are modulated. These phenotypic changes occur in response to cell density as induced by small diffusible elements termed autoinducer factors capable of regulating gene expression involved in biofilm production. The present work aimed to disrupt the production of an autoinducer (AI) factor involved in interspecies communication between S. aureus and P. aeruginosa through the fabrication of cationic liposomes encapsulated with antisense oligonucleotides. The liposomes themselves were synthesized via a microfluidic system to ensure uniform particle size distribution conducive to bacterial biofilm uptake. Furthermore, the various liposome samples exhibited favourable encapsulation efficiency of nucleic acids for effective delivery into Gram-positive and Gram-negative co-cultured biofilms. While antisense oligonucleotide (ASO)-loaded liposomes demonstrated a decrease in the production of an AI factor, there was no significant decrease in the biofilm production; nonetheless, this technology represents a steppingstone in the development of liposome-based strategies for delivery of nucleic acids to bacterial biofilms with the potential to expand to other pathogenic bacterial biofilms.
Novel 1,4-Diazepane Derivatives as Amyloid Beta (Aβ) Aggregation Inhibitors
(University of Waterloo, 2025-07-16) Karuturi, Rahul Chowdary
Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disease that represents one of the most pressing medical and social challenges of our time. Characterized by cognitive decline and memory loss, AD is primarily driven by the accumulation of amyloid-β (Aβ) plaques and tau tangles in the brain. The Aβ aggregation is indeed the primary pathological event, playing a key role in the initiation and progression of the disease. Despite extensive research, effective disease-modifying treatments remain elusive, and current therapies offer only symptomatic relief. While monoclonal antibody therapies targeting Aβ have emerged as a potential treatment option, their clinical effectiveness is limited. Small molecules, however, represent a more versatile and economical approach, with the potential for targeted action within the brain. In this regard, this thesis focuses on the design, synthesis, and biological evaluation of small molecules that specifically target Aβ aggregation, aiming to identify effective therapeutic candidates for AD. To this end, the present work explores the use of nitrogen-containing heterocyclics as small-molecule inhibitors of Aβ aggregation. Specifically, small molecules containing a flexible 1,4-diazepane scaffold were investigated for their ability to target the two major isoforms of amyloid-β, Aβ42 and Aβ40. A library of 38 derivatives based on the 1,4-diazepane scaffold was synthesized and evaluated for their ability to inhibit Aβ aggregation. The anti-aggregation activity of these compounds was determined through a combination of fluorescence-based aggregation kinetic assays, transmission electron microscopy (TEM), and computational modelling studies. Additionally, cytotoxicity assessments were performed using mouse hippocampal HT22 neuronal cells, alongside antioxidant assays and blood-brain barrier permeability evaluations. An overview of each chapter is outlined below: Chapter one provides an in-depth background on AD, addressing its prevalence, clinical diagnosis, and associated brain alterations. It reviews major pathological hypotheses, mainly cholinergic deficits, amyloid-β toxicity, tau pathology, and oxidative stress, and their roles in disease development. The chapter also reviews current therapeutic strategies, including small molecules, and their limitations. Chapter two provides a rationale for investigating the proposed 1,4-diazepane derivatives in the context of AD drug design by discussing the medicinal chemistry principles and structural features of bioactive natural compounds with amyloid-β inhibition properties. From this rationale, an AD-related hypothesis was formulated, guiding the selection and modification of chemical templates. A design strategy was developed, supported by computational studies, to propose a library of 1,4-diazepane derivatives for evaluation. Chapter three describes the design, synthesis, and evaluation of the first series of (1,4-diazepan-1-yl)(phenyl)methanone derivatives (4a–n). A library of 14 derivatives was synthesized, featuring varying functional groups at the para-position of the phenyl ring. The design incorporated 3,4-positions of the phenyl ring with known Aβ inhibition pharmacophores and antioxidant moieties, including a masked catechol group to study its effect on Aβ42 and Aβ40 inhibition. The compounds were synthesized by coupling acid chlorides or carboxylic acids with secondary amines. This study identified compounds with moderate to good inhibition of Aβ42 aggregation (32–52%) and enhanced inhibition towards Aβ40 (53–77%). This chapter also reports derivatives showing dual inhibitory effects on both Aβ42 and Aβ40. Chapter four describes the design, synthesis, and evaluation of a series of symmetric (1,4-diazepane-1,4-diyl)bis(phenylmethanone) derivatives (6a–p). This series contains 16 derivatives and includes derivatives incorporating functional groups at the para-position of the bisphenyl rings. This design strategy also incorporated 3,4-positions of the bis-phenyl rings with known Aβ inhibition pharmacophores and antioxidant moieties, including a masked catechol group, to investigate their impact on the inhibition of Aβ42 and Aβ40 aggregation. Additionally, this design also featured bicyclic aromatic rings, such as naphthyl derivatives. This study identified compounds exhibiting moderated inhibition of Aβ42 aggregation (31– 50%) and better inhibition of Aβ40 (60–63%), along with dual-targeting activity. Chapter five describes the design, synthesis, and evaluation of a series of (4-substituted-1,4-diazepan-1-yl)(phenyl)methanone derivatives (9a–h). This series comprised eight derivatives, each incorporating alkyl substituents of varying chain lengths at the N4 position of the 1,4-diazepane ring, along with a para-substituent on the phenyl ring. The findings revealed that the N-alkylated 1,4-diazepane derivatives exhibited reduced inhibitory activity toward Aβ42 (34%), in contrast to the derivatives described in Chapters 3 and 4. However, these compounds demonstrated improved inhibition of Aβ40 aggregation (55–67%) compared to those from Chapter 4, with one derivative identified as a dual-targeting agent. Chapter six focused on the biological evaluation of the most active compounds identified through the biophysical studies conducted in Chapters 3, 4, and 5. The lead 1,4-diazepane derivatives exhibited significant neuroprotective potential and effectively rescued the cells from Aβ42-induced cytotoxicity in HT22 cells (47.9–57.4%) and were not toxic to cells. Furthermore, compounds containing pharmacophores with antioxidant properties demonstrated reactive oxygen species (ROS) scavenging activity (13.2–90.7%). The findings also indicated that these derivatives possessed the ability to cross the blood-brain barrier (BBB). Chapter seven provides a comprehensive summary of the discovery of 1,4-diazepane derivatives as novel templates to design amyloid aggregation inhibitors and discusses the key findings of this study, including the physicochemical properties of the lead derivatives, the significance of this work, and future research directions to consider.
Hepatitis D virus among immigrants in Canada: an estimation of the prevalence and a cost-effectiveness analysis of screening and treatment
(University of Waterloo, 2025-05-08) Zhao, Jiahao
Background: Hepatitis D virus (HDV) is an important health concern in Canada. HDV is a viral infection can cause a rapid progression to inflammatory response in the liver, resulting in liver cirrhosis, hepatocellular carcinoma (HCC) and liver-related death. HDV is a derivative viral infection of hepatitis B virus (HBV). HDV infections can be acute or chronic. Chronic infections can lead to long term illness, liver cirrhosis or liver death. HDV infections can happen simultaneously, HBV and HDV infections happen at the same time, known as coinfection. HDV infections can also occur chronologically, patients already infected with Chronic hepatitis B (CHB) will then be infected with HDV, known as superinfection. Currently, no known cure for HDV infection, only available treatment is pegylated interferon alfa. Various new therapies are being developed due to limited effectiveness of pegylated interferon alfa. Bulevirtide (BLV) is a treatment that has shown promising efficacy. Objective: The objectives of this thesis are to: (1) estimate the overall prevalence of HDV among immigrants in Canada. (2) assess the cost-effectiveness of HDV screening and treating patients with bulevirtide, pegylated interferon alfa-2a and combined therapy compared to no screening for HDV. Methodology: A literature review was conducted to gather all relevant data on HDV. The prevalence estimation for HDV among immigrants in Canada was calculated based on a weighted average analysis by estimating the HBV population in Canada and then estimating HDV population using the HBV population estimation. A state transition model representing the natural progression of CHB and HDV was developed in TreeAge Pro to assess the cost-effectiveness of three screening and treating strategies. Analyses were performed from a public payer perspective with a lifetime time horizon and a 1.5% annual discount rate. One way sensitivity analysis and probabilistic analysis have been conducted to assess uncertainty. Results: Estimated HDV prevalence among HBV infected immigrants in Canada in 2021 was to be 7.7%. This is equivalent to an overall HDV prevalence rate of 0.24%, around 20,102 immigrants. HDV screening and treating with pegylated interferon alfa-2a monotherapy is cost-effective. Our analysis resulted in mean cost of $288,062 and a 22.96 QALY for screening and treating with pegylated interferon alfa-2a monotherapy versus $283,716 and 22.76 QALY for no HDV screening and treatment. With an ICER of $21,808/QALY. However, HDV screening and treating with BLV monotherapy and HDV screening and treating with combined therapy (BLV plus pegylated interferon alfa-2a) were both not cost-effective compared to no HDV screening and treatment. Our analysis resulted in ICER values of $329,015/QALY and $199,841/QALY for screening and treating with BLV and screening and treating with combined therapy, respectively. From a clinical perspective, all screen and treat HDV strategies showed a reduction in cases of liver related outcomes in decompensated cirrhosis, hepatocellular carcinoma and liver death. Conclusion: The estimated prevalence of HDV showed that a reasonable number of immigrants living in Canada with HDV remain undiagnosed. The results of cost-effectiveness study showed that screening for HDV and treating can increase clinical benefits. The results of the thesis can provide policy makers with actionable recommendation in re-assessing the current HDV screening recommendation in Canada among our immigrant population.
Harnessing Exosomes from Human Dermal Fibroblasts and Pirfenidone-exosomes as Innovative Strategies for Scarless Tissue Repair in Wound Healing
(University of Waterloo, 2025-03-11) Wang, Jin; Emmanuel, Ho
The wound healing process often leads to scar formation that can negatively affect patients both physically and psychologically. The management and treatment of scars also place a considerable financial burden on healthcare systems. Significant efforts are being made to improve wound healing outcomes by accelerating closure while simultaneously minimizing scar formation. To facilitate scarless wound healing, developing an anti-scarring treatment that modulates dermal fibroblast activity is a promising strategy, with pirfenidone (PFD) showing potential due to its anti-fibrotic properties by targeting intracellular pathways that regulate collagen disposition. PFD, particularly when delivered via dermal fibroblast-derived exosomes, may further enhance therapeutic effectiveness and promote scarless healing. To achieve this goal, we began by isolating high-purity exosomes from in vitro cultured human dermal fibroblasts. Two common isolation methods—PEG precipitation and affinity-based techniques—were compared to identify the most efficient approach for obtaining high-purity and relatively homogenous exosomes. A range of characterization techniques, including transmission electron microscopy (TEM), atomic force microscopy (AFM), antibody arrays, and enzyme-linked immunosorbent assays (ELISA), confirmed the successful isolation of high-purity exosomes. The affinity-based method demonstrated superior performance, yielding well-dispersed and highly pure exosomes. Due to the difficulties in achieving efficient drug encapsulation in exosomes, the following chapter specifically focused on the encapsulation and formulation optimization of the antifibrotic compound PFD and explored the use of exosomes as a drug delivery platform. We optimized an active drug loading method using sonication to enhance encapsulation efficiency (EE%) and loading efficiency (LE%), ensuring that careful control of the sonication process maintained exosome integrity. The optimal formulation of PFD-exosomes achieved an EE% of 11.14% ± 1.27% and an LE of 10.01% ± 1.03%, with a particle recovery rate of exosomes at 64.21% ± 8.49%. Then, we investigated how to harness exosomes from dermal fibroblasts and PFD-exosomes as innovative strategies for achieving scarless tissue repair in wound healing. Our findings showed that exosomes enhanced fibroblast migration and proliferation through an autocrine mechanism, highlighting their potential as a stand-alone cell-free therapy for wound healing. Additionally, this study was ground-breaking in demonstrating that exosomes can improve the efficacy of PFD as a drug carrier, amplifying its anti-fibrotic effects in both in vitro and in vivo models. The in vivo results indicated that PFD-exosomes accelerated wound healing while organizing the extracellular matrix (ECM) by reducing excessive collagen deposition. Overall, PFD-exosomes present an innovative strategy for pre-scarring interventions, offering benefits of enhanced wound healing outcomes while minimizing scarring.
Health Care Costs Associated with Minor Ailments and Cost Minimization Analysis of Pharmacists Prescribing for Minor Ailments in Ontario, Canada
(University of Waterloo, 2025-01-29) Koo, Vanessa; Wong, William
Background: Pharmacists in Ontario, Canada are now able to prescribe for certain minor ailments. Minor ailments are defined as health conditions that can be managed with minimal treatment and/or self-care strategies. Economic evaluations with costs and burden associated with minor ailment conditions using Ontario health administrative data are needed to accurately evaluate the economic impact of the pharmacist prescribing for minor ailment (PPMA) program. Objectives: The objectives of this research thesis are to: 1) assess the baseline characteristics, mean health care costs, and predictors of health care costs of minor ailment cases using a retrospective analysis of Ontario health administrative data for patients presenting with minor ailment conditions and 2) perform a cost-minimization analysis to determine the economic impact of a remunerated program for PPMA compared with usual care. Methodology: First, using Ontario health administrative data from ICES, baseline characteristics, mean health care costs, and predictors of health care costs were determined for patients diagnosed with the 16 studied minor ailments. Second, a decision-analytic model was implemented to perform the cost-minimization analysis for the minor ailments. Two prescribing strategies were considered in this analysis: PPMA and usual care. In the PPMA strategy, patients have the option of either seeking care from a community pharmacist or a physician. In the usual care model, all patients seek care from physicians. Probabilities and costs used in the model were derived from mostly Ontario health administrative data, literature, or expert opinion when there was insufficient literature. This analysis used a public payer perspective and outcomes were expressed in costs in 2019 Canadian dollars. Results: Analysis of Ontario health administrative data from ICES identified that the minor ailments with the highest number of unique patients billed were musculoskeletal sprains and strains (8,099,393; 24%), gastroesophageal reflux disease (5,822,495; 17%), dermatitis (5,649,829; 17%), urinary tract infection (3,356,887; 10%), and insect bites and urticaria (2,699,684; 8%). Health care costs varied by minor ailment and cost category, with older age, lower income quintiles, urban residency, and comorbidities as predictors of higher total health care costs. Cost-minimization analyses from a public payer perspective provided evidence that implementing a PPMA program for the studied minor ailments could yield cost savings for the Ontario government compared to the usual care model, with savings ranging from $19.05 to $77.38 per patient. One-way sensitivity analyses showed that results were most sensitive to the likelihood of patients receiving care from a pharmacist rather than a physician. In probabilistic sensitivity analyses, the PPMA model proved cost-saving in 100% of the simulations for all 16 minor ailments studied. Conclusion: The results of the thesis research identified the baseline characteristics, health care cost burden, and predictors of total health care costs for patients presenting with minor ailments using Ontario health administrative data. In addition, the cost-minimization analyses conducted from a public payer perspective provided evidence that implementing a PPMA program provided cost-savings for the Ontario government when compared to the usual care model for the studied minor ailments. The results of this research can continue to help shape implementation strategies of a PPMA program in Ontario, Canada.
Development of Clinical Pharmacy Key Performance Indicators for Ambulatory Oncology Using a Modified Delphi Approach
(University of Waterloo, 2025-01-16) Sothilingam, Rozhannaa; Tom, McFarlane
Introduction: Ambulatory Oncology Clinical Pharmacy Key Performance Indicators (AOcpKPIs) are quantitative measures that reflect the quality of care provided by pharmacists to ambulatory oncology patients. In this research project, AOcpKPIs were selected to advance pharmacy practice to improve patient outcomes. Research completed prior to this work involved a cross-sectional survey as part of a Canadian environmental scan. This survey revealed that 98% of respondents supported the development of AOcpKPIs for ambulatory oncology pharmacy practice. The primary objective of this project was to identify targeted processes of care aimed at improving ambulatory oncology patient outcomes represented by critical activity topics (pharmacist interventions), which were used to develop quantifiable AOcpKPIs. Methods: A literature review on evidence-informed critical activity topics revealed that certain care processes delivered by ambulatory oncology pharmacists had a positive impact on patient outcomes. A steering group comprised of expert ambulatory oncology pharmacists created evidence summary tables by critically appraising and assessing articles to identify “Ambulatory Oncology Critical Activity Topics”. A list of 15 candidate AOcpKPIs was generated by the steering group to serve as metrics to represent 9 evidence-linked critical activity topics. A sample of 18 pharmacists was purposively selected by the steering group to participate in a Delphi panel with the goal of reaching consensus on AOcpKPIs to adopt. This research project employed a modified Delphi process (a structured method to reach consensus among experts through multiple rounds of surveys and discussion in a meeting) where panelists ranked the a priori selected candidate AOcpKPIs on a 9-point Likert scale in the initial round and then reviewed the rankings and comments of the first round to re-rank the 15 AOcpPKIs in round 2. A final consensus was reached in the third round following a live meeting discussing marginal candidate AOcpPKIs from the second round. Results: After the first round of the Delphi process, 9 candidate AOcpKPIs reached preliminary consensus. This number increased to 12 after the second round. During a live meeting, discussions about the wording and meaning of the candidate AOcpKPIs led to the addition of 2 new candidates, totaling to 17 for the third round. Following the third round, 14 AOcpKPIs achieved consensus. These cpKPIs highlight essential pharmacist contributions, such as reviewing clinical orders, providing patient education on medications, assessing medication adherence, pharmaceutical care plan (oncology), pharmaceutical care plan (non-oncology), follow-up, bundled patient care interventions, medication history assessment and addressing drug therapy issues. Significance: This research project identified 14 quantifiable AOcpKPIs, based on ambulatory oncology patient outcomes, that represent critical activity topics. Through a systematic, evidence-informed consensus-building process, the final suit of consensus AOcpKPIs that improve patient outcomes were formed. Future efforts will focus on implementing these metrics in ambulatory oncology pharmacy practice to measure and ensure the quality of patient care.
Design, Synthesis, and Biological Evaluation of Novel Phenoselenazine Derivatives as Amyloid Aggregation Inhibitors
(University of Waterloo, 2024-09-19) Abdallah, Ahmed; Rao, Praveen Nekkar
One of the leading challenges of modern medicine is Alzheimer’s disease (AD), a chronic and debilitating neurodegenerative disorder that poses a global health threat with profound implications for individuals and societies. The inception of AD in 1907 can be attributed to the pioneering research conducted by a German psychiatrist, Dr. Alois Alzheimer’s, who first identified two prevalent pathological features, plaques, and tangles, in the brain of his patient. These distinct plaques are made up of an amyloid protein called beta-amyloid (Aβ), as the chief component in AD`s plaques and a principal culprit throughout the progression of AD. The recent noteworthy accomplishments in monoclonal antibodies (mAbs) have marked a pivotal milestone, ushering in a new era where treatments targeting the amyloid cascade in Alzheimer's disease have emerged as a plausible avenue. Consequently, the amyloid cascade hypothesis is known as the dominant factor to develop diagnostics and therapies for AD. Despite the scientific breakthroughs made in the last few decades, there remains a notable lack of effective treatments for impeding disease progression. Therefore, researchers are now more desperate than ever to develop amyloid-cascade targeted small molecules, aiming to pave the way toward successful outcomes in AD treatment, as small molecules have a number of advantages over biological therapies. In this regard, this thesis research presented herein aimed to design and develop novel small molecules that have the potential to reduce/prevent the disease progression through targeting the aggregation cascade of the two common forms of Aβ, known as Aβ42 and Aβ40. Besides, our ring scaffold was able to target another key factor in AD pathology, the reactive oxygen species (ROS), and has the potential to mitigate their toxicity. A library of 47 compounds based on a novel fused tricyclic ring template was designed and developed by incorporating a selenium atom as a part of its heterocyclic ring, to obtain the phenoselenazine (PSZ) derivatives. The synthesized compound libraries were evaluated as potential inhibitors of Aβ42 aggregation by carrying out fluorescence aggregation kinetics experiments, transmission electron microscopy studies, neuroprotection experiments in mouse hippocampal HT22 neuronal cells exposed to Aβ42, evaluation of their antioxidant properties, blood-brain barrier permeability experiments and computational modeling studies.
Construction and Characterization of a Targeting M13-Based Phagemid Carrying an Anti-Angiogenic DNA-Encoded Virus-Like Particle
(University of Waterloo, 2024-09-18) Li, Jiayang; Slavcev, Roderick; Aucoin, Marc
Over the past several years, molecular targeted therapy has emerged as a promising strategy for cancer treatment. Unlike broad-spectrum cytotoxic drugs used in conventional chemotherapy, targeted therapy aims to address specific molecular alterations unique to cancer cells. To develop effective targeted therapies, numerous delivery platforms have been investigated to optimize safety, specificity, and efficiency. The work presented here investigates the construction and characterization of a miniphagemid-mediated cancer therapy delivering anti-angiogenetic DNA-encoded virus-like particles (VLPs). VLPs have shown a robust ability to stimulate potent immune responses and overcome the immunosuppressive state of the tumour microenvironment (TME). Additionally, the filamentous bacteriophage (phage) M13 has been explored as a safe and efficient vehicle for delivering therapeutic genes and drugs. Phage-based vectors (phagemids) can be engineered to transfer exogenous genetic material to mammalian cells safely, as they possess no natural tropism. The present study aims to combine the advantages of both VLPs and phagemids to construct a hybrid biological platform for the specific delivery of DNA encoding VLP-displaying anti-tumour peptides, specifically VGB4, to tumour cells via M13 – a filamentous phage capable of phagemid production as well as phage display. The VGB4 peptide has demonstrated potent ability to inhibit angiogenesis in the tumour vasculature by blocking the downstream signalling pathways of vascular endothelial growth factor receptor (VEGFR). The human papillomavirus (HPV) type 16 L1 capsid gene with an inserted VGB4 peptide sequence was cloned into a miniaturized phagemid (miniphagemid) engineered by our lab. This genetically engineered miniphagemid was produced in Escherichia coli using a novel non-packaging M13 helper plasmid. The helper plasmid not only complements phagemid packaging but also enables the display of a cell-specific targeting ligand, the epidermal growth factor (EGF), which promotes receptor-mediated endocytosis for specific phage uptake by tumour cells overexpressing epidermal growth factor receptors (EGFRs). This thesis project investigated the formation of VGB4-displaying HPV VLPs within HEK 293T and HeLa cells. Our results demonstrated that the EGF-displaying miniphagemid improves gene delivery to cells compared to non-displaying miniphagemids. Furthermore, the VGB4-displaying HPV VLPs do not form in cells treated with miniphagemids, but these VLPs are successfully formed in cells treated with the precursor phagemids encoding the same gene cassette. Overall, this study highlights the necessity for further investigation and optimization to enhance miniphagemid-mediated gene transfer by overcoming cellular barriers, paving the way for its application as a novel targeted gene therapy for cancer.
Use of Physiologically-Based Pharmacokinetic Modelling To Support Dose Optimization in The Critically Ill
(University of Waterloo, 2024-09-12) Dubinsky, Samuel; Edginton, Andrea
Critical care is a subspeciality of medicine that incorporates a multidisciplinary approach for patient care in a heterogeneous population. The wide array of etiologies to critical illness may result in disturbances to homeostasis and organ function. This variability in host-response may impact the pharmacokinetics (PK) of an individual, resulting in potential differences in drug exposure compared to a healthy counterpart. The high mortality rates of critically ill patients are thought in part to be due to altered drug exposure, resulting in suboptimal dosing or adverse effects. The complexity and heterogeneity of critical illness limits the feasibility of conventional PK studies for dose determination in this population. Furthermore, current “one-dose-fits-all” approaches in pharmacotherapy overlook the interplay between drug physicochemical properties and pathophysiology towards altered PK in the critically ill. Methods incorporating pharmacometrics have advanced our understanding of this relationship, however current approaches may require robust data sets to inform dose-exposure relationships and limit extrapolations to untested clinical scenarios. Methods incorporating physiologically-based pharmacokinetic (PBPK) modelling are encouraging to overcome some of the current challenges and knowledge gaps towards optimizing drug therapy in critically ill patients. PBPK models leverage mechanistic principles to incorporate knowledge of physiology and drug physicochemical properties to provide a simulation-based approach to drug PK. Knowledge of drug PK and physiology in healthy adults may be adjusted to account for differences in age (i.e adults to children), or disease (i.e. critical illness), to predict drug exposure. As these methods leverage known prior information, it enables a more proactive approach in predicting drug PK in clinical settings when data sampling is sparse, such as the critically ill. This thesis aims to apply state-of-the-art mechanistic modelling strategies to advance pharmacotherapy in critically ill adults and in children receiving extracorporeal life-saving technology. The objectives are to (1) analyze the PK data in critically ill children receiving continuous renal replacement therapy (CRRT), (2) evaluate the extraction of drugs via CRRT utilizing a closed-loop ex-vivo study design, and (3) develop PBPK models to optimize drug dosing in various presentations of critical illness. The first objective included a systematic review to evaluate our current understanding of drug PK in critically ill children receiving CRRT. Several knowledge gaps were identified, which were to be addressed within the second objective by incorporating a closed-loop study design to isolate the drug-CRRT circuit interaction. This ex-vivo¬ approach was the first of the holistic methodological solution towards which this thesis aims to achieve. Knowledge gained from the ex-vivo studies may be integrated into a PBPK model structure to provide a mechanistic understanding of drug PK, allowing for prospective dose predictions in untested scenarios. Within this work, it was identified that 40% of PK studies conducted in critically ill children receiving CRRT focused on antimicrobials. Furthermore, only 50% of studies included in the final analysis provided dosing guidance based upon PK findings in this population, representing several knowledge gaps to support clinicians in dosing guidance at the bedside. Anakinra and cefazolin were targeted as drugs of need for dosing guidance in patients receiving CRRT. Ex-vivo studies were conducted to investigate the drug-circuit interaction. Both anakinra and cefazolin, were efficiently removed from plasma upon the completion of the experiment, rendering a sieving coefficient of 0.34 and 0.31, respectively. A PBPK model for anakinra was developed to predict the PK in children receiving concurrent CRRT and extracorporeal membrane oxygenation (ECMO). Upon applying the results from the ex-vivo experiment, PBPK model predictions successfully described anakinra PK amongst critically ill children enrolled in a prospective, opportunistic study. Overall anakinra exposure was similar amongst children receiving ECLS compared to those who are not, supporting the hypothesis that renal dose adjustments in this population may not be required. The work herein this thesis seeks to advance the application of PBPK in supporting pharmacotherapy to a diverse group of critically ill patients. In doing so, a mechanistic representation of cefazolin drug concentrations in the central nervous system (CNS) through PBPK was performed. Cefazolin cerebrospinal fluid (CSF) concentrations were accurately predicted, and simulated CSF:plasma ratios fell within a 1.5 fold-error compared to observed values. Dosing simulations demonstrate that a continuous infusion of 10 g/day may be required to achieve pharmacodynamic response towards methicillin-susceptible Staphylococcus aureus in treating infections in the CNS. This thesis aims to articulate the multiplicity of PBPK to support dosing guidance in the critical care setting. Several knowledge gaps were identified, and the methods applied aim to address some of the current limitations of conventional PK studies in this population. PBPK modelling efforts such as these have the potential to improve generalizability, and efficiently conduct PK studies in a highly vulnerable patient population by reducing the number of participants required and applying a more proactive approach to dose determination in untested clinical scenarios.
Professional regulation in healthcare: Exploring trends, predictors, and the disciplinary action process for health professionals in Canada
(University of Waterloo, 2024-09-03) Foong-Reichert, Esther; Grindrod, Kelly; Houle, Sherilyn
Background: Health professionals in Canada are governed by provincial regulatory bodies, whose mandate is to protect the public. Regulatory bodies license and register professionals, and handle complaints and disciplinary action processes when warranted. In recent years, jurisdictions internationally and in Canada have been undergoing regulatory reform. More research is needed on current processes to inform future improvements. Objectives: The overall goal of this thesis was to describe the disciplinary action process for health professionals in Canada. Methods: This thesis is comprised of five studies. The first study was a scoping review to describe the research on disciplinary action outcomes for health professionals, and to describe the research on characteristics or predictors of health professionals subject to disciplinary action. The next three studies reviewed disciplinary action outcomes for Canadian pharmacists, dentists, and nurse practitioners, respectively. Characteristics of professionals subject to disciplinary action were also studied. The fifth study sought to compare and contrast disciplinary action processes across professions and jurisdictions in Canada and to describe regulatory body perspectives of the disciplinary action process. Results: The scoping review found that most research focuses on physicians, originates from the USA, and has been conducted from 2010 – 2020. A variety of demographic factors and predictors of disciplinary action have been studied, including gender, age, years in practice, practice specialty, license type/profession, previous disciplinary action, board certification, and performance on licensing examinations. The reviews of pharmacist, dentist, and nurse practitioner disciplinary found differences in reasons for disciplinary action between professions. All professions had low rates of disciplinary action, with nurse practitioners being the lowest. In the final study, interviews with regulatory bodies identified possible explanations for the differences observed in the reviews of disciplinary action. Conclusion: This thesis has generated new knowledge about disciplinary action for Canadian health professionals. This research will guide regulators and other stakeholders in improving health regulation and ensuring protection of the public.
Development and Characterization of Novel Cellulose-Based Soy Biopolymer for Sustainable 3D Printing
(University of Waterloo, 2024-08-21) Nodder, Ryan; Ho, Emmanuel
Most materials currently used in 3D printing are non-renewable petroleum thermoplastics, which will not support the anticipated growth of 3D printing as the applications and demand of the industry continue to rapidly evolve. In this study, a cellulose-based soy biopolymer (CSBP) feedstock for 3D printing applications is developed. With cellulose-based products accounting for one third of Canada’s municipal solid waste, this provides both a solution to the growing problem of environmental pollution and global warming, as well as the need for sustainable materials in 3D printing. A formulation of soy protein isolate, cellulose and additives has been developed to produce a print media to be used for extrusion 3D printing. A soy-protein component mixed with formulation additives allows for the creation of a self-curing binder, in combination with cellulose as a filler to improve the resulting mechanical properties. A synergistic combination of protein crosslinking, film formation and solvent evaporation is employed to create solid objects with CSBP. Most importantly, this formulation is composed entirely of generally regarded as safe (GRAS) formulation components to become an alternative for extrusion 3D printing that is biodegradable, non-toxic and has environmentally friendly synthesis. The material developed is paste-like in its uncured state and requires a syringe extrusion mechanism for applications in 3D printing. With CSBP established as a 3D printable material through a syringe-based mechanism, multiple physical characterizations are performed to characterize CSBP and provide insights on its overall properties. Furthermore, investigation of some preliminary end-user applications of CSBP were performed to provide insight into the future use of this new biomaterial. Collectively, this study developed and characterized the use of CSBP feedstock for 3D printing, capable of recycling paper into a new biomaterial. CSBP exhibited a variety of tunable properties as a function of the curing conditions and time and was found to be naturally biodegradable and biocompatible. Preliminary applications in the adhesive and packaging industries as well as the creation of drug delivery systems were achieved with encouraging initial results. This work highlights a solution to global issues in both the recycling of paper waste and provide a sustainable pathway for the ever-expanding applications of 3D printing.
Microfluidic development of a pneumolysin-responsive liposomal platform for selective treatment of Streptococcus pneumoniae
(University of Waterloo, 2024-08-12) Watt, Ethan; Ho, Emmanuel
The bacterium Streptococcus pneumoniae has become a leading cause of meningitis, sepsis, and bacterial pneumonia worldwide, with increased prevalence of antibiotic-resistant serotypes only serving to exacerbate the issue. This imposes a significant public health threat and growing economic burden, spurring research into alternative treatments. One effective route to bypass resistance development is through the targeting of virulence factors essential for bacterial pathogenesis, comprising cellular structures, regulatory systems, and molecules produced by the bacterium. The main factor responsible for colonization and immune response escape in pneumococcal infections is the secreted molecule pneumolysin, which is a subset within a family of related toxins that form transmembrane pores in biological membranes through cholesterol recognition and binding. The conserved activity and structure of pneumolysin between all observed S. pneumoniae serotypes, along with its requirement for pathogenicity, has made this molecule an attractive target for vaccination, diagnostic, and sequestration platforms, but not yet as a facilitative agent for therapeutic treatment. The present work aimed to take advantage of pneumolysin’s dependencies through the fabrication of cholesterol-rich liposomes, a natural mimic of the external surface of mammalian cells, to elicit pore formation and subsequent release of an encapsulated peptide. A custom microfluidic system was manufactured to enable rapid and consistent liposomal synthesis, with key formulation parameters optimized through a Box-Behnken response surface design. The vesicles themselves were comprised of naturally-derived phospholipids and varying levels of cholesterol (30/45/60/75 mol%), which displayed desirable physical properties and high encapsulation rates of the bacteriocin nisin. A liposomal cholesterol content above 45 mol% was determined necessary to facilitate interactions with both purified pneumolysin toxin and S. pneumoniae culture, as demonstrated through enhanced nisin release and a reduction in hemolytic rates upon exposure of the toxin with cholesterol-rich vesicles. Antibacterial testing highlighted the ability of the developed platform to elicit a potent and specific bactericidal response in vitro against S. pneumoniae when compared to a control strain, Staphylococcus epidermidis. It further improved viability of a fibroblast cell line upon S. pneumoniae challenge, outperforming free nisin via the synergistic impact of simultaneous bacterial clearance and pneumolysin neutralization. This study indicates that cholesterol-rich liposomes hold promise as a treatment platform against pneumococcal infections, with potential for expansion to other pathogenic bacteria known to produce similar cholesterol-dependent toxins.
Isoform-specific Roles of Prolyl-Hydroxylases in the Regulation of β-cell Insulin Secretion during Diet-Induced Obesity in Males
(University of Waterloo, 2024-06-13) Jentz, Emelien; Joseph, Jamie
Type 2 diabetes affects approximately 480 million individuals worldwide and is associated with impaired tissue insulin sensitivity and β-cell dysfunction. Although there has been much research into nutrient-regulated insulin secretion and the progression to β-cell dysfunction in type 2 diabetes, the story is still incomplete. Hypoxia-inducible factor prolyl 4-hydroxylases (PHDs) are α-ketoglutarate dioxygenases commonly known to regulate hypoxia-inducible factor-1α (HIF-1α). Unique expression profiles of PHD1, PHD2 and PHD3 isozymes suggest isoform-specific roles in α-ketoglutarate-sustained insulin secretion. Our laboratory recently showed a role for β-cell PHD1 and PHD3 in insulin secretion, and previous research suggests that PHD2 may play a role in obesity-induced metabolic dysfunction. This thesis focuses on possible roles that β-cell PHDs may play in moderating the interrelationship between defective nutrient-sustained insulin secretion and obesity-induced β-cell dysfunction. We placed β-cell-specific PHD1, PHD2 or PHD3 knockout mice on a high-fat diet to explore the roles of PHD isoforms in regulating β-cell function under diet-induced obesity. β-cell-specific PHD1 knockout mice did not display any unique obesity-induced metabolic phenotypes compared to high-fat diet-fed control mice. β-cell-specific PHD3 knockout mice on the high-fat diet experienced increased weight gain compared to high-fat diet-fed control mice. However, despite increased fasting blood glucose levels, they showed no exacerbated impairments to in vivo glucose homeostasis and plasma lipid profiles. β-cell-specific PHD2 knockout mice resisted high-fat diet-induced obesity and showed improved in vivo glucose homeostasis combined with minor alterations in their plasma lipid profile. The lack of obesity-induced metabolic dysfunction in β-cell-specific PHD2 knockout mice could be explained by enhanced β-cell mass and ex vivo glucose-stimulated insulin secretion. Overall, β-cell-specific PHD2 knockout mice have ameliorated glucose homeostasis and β-cell function during obesity, potentially due to PHD2’s role in discouraging HIF-1α stability during metabolic stress.
USER EXPERIENCE WITH MEDICATION ADHERENCE TECHNOLOGY: DETERMINING USABILITY BY CAPABILITIES
(University of Waterloo, 2024-05-27) Baby, Bincy; Patel, Tejal
Background: As populations worldwide grow older, the prevalence of chronic conditions and the complexity of managing multiple medications significantly increase. This challenge is further complicated by a range of barriers older adults face, including physical limitations, cognitive impairments, sensory issues, motivational challenges, and non-supportive environments. Such barriers can lead to a decline in capacity to self-manage medications, resulting in poor adherence to prescribed medication regimens, which in turn can cause increased hospitalizations and a decrease in quality of life. Medication Adherence Technologies (MATech), which range from simple electronic devices to more complex smart devices with connectivity and real-time monitoring capabilities, are recognized as one of the solutions to these challenges. However, the design and features of these technologies vary significantly, influencing how they are used by different users. Usability varies widely; some older adults may find certain features of these devices challenging to use due to their barriers. Hence, it is crucial to ensure that MATech are accessible and user-friendly for all older adults, regardless of their individual challenges. This study aims to identify the most suitable MATech for older adults with various physical, cognitive, sensory, motivational, and environmental limitations, tailored to their unique needs and abilities. Objectives: The primary objectives of this study were to evaluate the usability and user experience (UX) of thirteen MATech devices among older adults facing various barriers to medication self-management and to gather comprehensive feedback on the usability and features of these technologies. Secondary objectives included determining how different barriers affect the usability outcomes of these technologies and identifying design features that best meet the needs of this demographic to enhance their independence and well-being. Methods: The study used a mixed-methods approach to evaluate the usability of MATech. Eighty older adults, aged 60 and older, were recruited through convenience, purposive, and snowball sampling methods from various settings across Ontario, including academic and residential facilities. Data collection was conducted in three steps after obtaining informed consent from the participants. The first step involved measuring barriers to medication self-management using various scales such as the Self-Medication Assessment Tool (SMAT) for physical, cognitive, and vision barriers; the Whisper Test for hearing barriers; the Self-Efficacy for Medication Adherence Scale (SEAMS) for motivational barriers; and the Martin and Park Environmental Demands (MPED) Questionnaire for environmental barriers. The second step involved usability and user experience testing of three smart devices and ten electronic devices, to measure various performance-based metrics (task success rate, total task completion time, efficiency, error rate) and perception-based usability metrics (System Usability Scale (SUS) score, NASA-TLX workload score, Single Ease of Use Question (SEQ), and Subjective Mental Effort Question (SMEQ)). The third step consisted of in-depth qualitative interviews to explore feedback regarding the features of various MATech tested. Quantitative data were statistically analyzed using descriptive statistics and univariate and multivariate regression to assess usability across various devices, while qualitative responses were analyzed using inductive thematic analysis. Results: Quantitative Results: Cognitive impairments were identified in 20% of participants, physical limitations in 33.75%, hearing impairment (both ears) in 60%, and vision impairments in 11.25%. Backward stepwise multivariate regression analysis identified critical predictors for task success rates, including 'SEAMS score' (p<0.001) which measures motivational barrier positively influencing outcomes, whereas 'Low vision score' negatively affected success rates (p<0.001). Moreover, Old 'age' (p<0.001) and 'number of subtasks for product' (p<0.001) notably extended the total task completion times, and 'physical score' (p<0.001) increased error rates, suggesting necessary improvements in MATech design for better usability. While no predictors significantly impacted the SUS scores, the NASA TLX identified 'old age', 'vision impairment', and the ‘number of products tested’ as significant factors in perceived task load, particularly noting that using multiple products increased task load considerably, underscoring their profound impact on user experience and workload management. Predictive models were also developed to determine each participant's ability to successfully complete subtasks. For example, the model for a participant characterized by significant cognitive, physical, hearing, motivational and environmental impairments, but with high vision capacity, indicated high success probabilities for visually intensive subtasks such as "scroll the screen options" (92%) and "locate and touch an icon on a screen" (87%). Conversely, tasks requiring more physical interaction like "flip device" showed much lower success probabilities (45%). Qualitative Findings: Five themes were identified: (1) the practicality of device design, (2) the impact of technological complexity, (3) the necessity for inclusivity in device functionality, which includes considerations for impairments, security, and privacy, (4) the influence of socio-economic and environmental factors, and (5) the importance of feedback for iterative design. Discussion: The findings from this study underscore the critical importance of designing MATech that are not only functional but also tailored to the unique needs of older adults who face multiple barriers to effective medication management. Key findings from the regression analyses highlighted the importance of addressing physical and sensory impairments in MATech design, as these significantly influence user performance and error rates. Additionally, factors such as age and the complexity of device operations significantly influence usability and workload, suggesting the need for simpler, more intuitive designs that minimize cognitive and physical strain. Overall, the research emphasizes the need for a user-centered design approach in developing MATech, emphasizing simplicity, accessibility, and personalization to better support older adults in managing their medications effectively. This approach not only aids in improving medication adherence but also contributes to the broader goal of facilitating a more independent, quality life for older adults.
Arylbenzamide and Arylcarboxamide Derivatives as Modulators of Amyloid-Beta Aggregation
(University of Waterloo, 2024-03-11) Zhao, Yusheng; Nekkar Perampalli Rao, Praveen
Alzheimer’s disease (AD) is a complex neurodegenerative disease with increasing incidence and prevalence globally. The current AD therapies based on small molecules offer only symptomatic relief and are not curative therapies. The recently launched anti-amyloid monoclonal antibodies hold promise although these are new to the market and their long-term benefits and potential disease-modifying effects are unknown. The global increases in the aging population and increasing life span mandate the need to understand the mechanisms of AD and discover effective and safe therapies. Over the past several decades, few hypotheses have been proposed to explain the pathophysiology of AD, among which the amyloid beta (Aβ) cascade is now considered as one of the initiating factors that drives the progression and other pathological factors of AD. The aggregation of Aβ into oligomers and fibrils together with its downstream signaling pathway are neurotoxic. Thus, small molecule modulators that could reduce the overall toxic burden of Aβ aggregates are thought to be beneficial in treating AD. In this thesis, a library of 72 small molecule derivatives were designed based on the chemical structure of chalcone and curcumin, two bioactive natural compounds that are able to modulate Aβ aggregation and reduce their neurotoxicity. The derivatives reported in this thesis encompass four different templates, namely, N-benzyl (Chapter 2), N-phenethyl (Chapter 3), N-benzyloxy (Chapter 4), and N-phenyl (Chapter 5) benzamide and carboxamides. These compounds were synthesized by coupling the amine substrates with either acid halides or carboxylic acids to obtain the target compounds in 72-93.6% yields. A number of biophysical and biochemical experiments were carried out to determine the ability of these small molecules to modulate the aggregation properties of Aβ42. The experiments carried out include i) thioflavin T based fluorescence aggregation kinetics experiments; ii) transmission electron microscopy studies; iii) 8-anilino-1-naphthalenesulfonic acid based fluorescence spectroscopy; iv) antioxidant assay by fluorescence spectroscopy; iv) cell viability studies in mouse hippocampal HT22 neuronal cells and Aβ42-induced neurotoxicity assay; v) fluorescence microscopy studies to assess the neurotoxicity using Proteostat dye, and vi) computational modelling studies to determine the interactions of small molecules with Aβ42 aggregates. From this library, 51 aggregation inhibitors were identified (inhibition of Aβ42 ranging from 7-53.1% at 25 µM). These derivatives were able to provide significant neuroprotection from Aβ42-induced cytotoxicity in mouse hippocampal HT22 cells (cell viability ranging from 80.8-96.8% versus 38.7% for Aβ42-treated control). Molecular docking studies indicate that these derivatives were able to interact with the hydrophobic domains of the Aβ42 oligomer and fibril through hydrophobic interactions. In a striking and unusual finding, 8 derivatives were identified as Aβ42 aggregation promotors with the ability to promote the aggregation by 1.2-5.1 folds. Two lead promotors 14b (N-benzylbenzofuran-2-carboxamide) and 14c (N-benzylbenzo[b]thiophene-2-carboxamide) were identified. These two compounds were able to rescue HT22 cells from Aβ42-induced cytotoxicity (cell viability 73.8% and 73.9% for 14b and 14c versus 19.7% for Aβ42-treated control). These two compounds have the ability to increase the surface hydrophobicity of Aβ42 aggregates and promote fibrillogenesis. Molecular docking studies suggested that Aβ42 aggregates might undergo conformational change upon binding and thus transit to much more stable and less toxic/nontoxic fibrils. Further structure-activity relationship study indicated that the hydroxy- and methoxy-disubstituted phenyl moiety was required to possess Aβ42 inhibition activity, where the presence of bicyclic aromatic rings such as benzofuran and benzothiophene, and 4-methoxyphenyl moiety is required for pro-aggregation activity. The results show that these benzamides and carboxamides possessing N-benzyl, N-phenethyl, N-benzyloxy, and N-phenyl templates hold promise in the design and development of novel small molecules as Aβ42 aggregation modulators. Remarkably 14b (N-benzylbenzofuran-2-carboxamide) and 14c (N-benzylbenzo[b]thiophene-2-carboxamide) were able to accelerate Aβ42 aggregation and remodel the aggregation pathway to form less toxic/nontoxic aggregates suggesting their application as novel chemical tools to understand the mechanisms of Aβ42 aggregation cascade.
Enhancing the Ministring DNA (msDNA) Purification Using PI-Sce1 Homing Endonuclease/CRISPR-Cas3 Recombinant System
(University of Waterloo, 2024-02-05) Fernando, Merium; Slavcev, Roderick
In the generation of msDNA the recombinant E. coli cells are transformed by a msDNA generating precursor plasmid, whereupon expression of the Tel protelomerase enzyme, acting on the pal target sequence present in the precursor plasmid, generated linear covalently closed (LCC) msDNA. However, the in vivo recombinant platform to produce msDNA results in a mixture of plasmids including unprocessed precursor plasmid, unwanted LCC bacterial backbone, and their topological isoforms, which interferes with the purification of the target species. For larger scale synthesis, the plasmid extract needs to be pretreated with commercially available restriction enzymes before being purified through chromatographic columns. Meanwhile, at the laboratory scale, msDNA is purified from agarose gels based on their size. These purification processes are time-consuming and inefficient and therefore, there is a need to optimize the process. To address this issue, we developed two in vivo recombinant systems for digesting the unwanted prokaryotic backbone and unprocessed precursor plasmid. These systems are the PI-SceI homing endonuclease enzyme system and the clustered regularly interspaced short palindromic repeats-Cas3 (CRISPR-Cas3) system. Homing endonucleases are highly specific DNA cleaving enzymes. The homing endonuclease PI-SceI, encoding gene vma from Saccharomyces cerevisiae was successfully integrated into the tel integrated bacterial chromosome via site-specific recombination using conditional replication and integration (CRIM) plasmid. The double integrants, both vma and tel integrated recombinant bacteria, were transformed with msDNA synthesizing precursor plasmids and induced the msDNA synthesis and vma gene overexpression. Even though the double integrants were able to overexpress the homing endonuclease enzyme and digest the precursor plasmid, they were not able to synthesize msDNA. Therefore, the Tel protelomerase enzyme was expressed episomally inside the vma integrated recombinant bacteria. This vma gene is under the control of an inducible PBAD promoter. In the presence of L-arabinose in the media, the Tel protelomerase enzyme was episomally expressed and synthesized msDNA by acting on the precursor plasmid. Subsequently, the overexpressed PI-SceI homing endonuclease enzyme digested the undesired byproducts of msDNA synthesis as expected. Introducing homing endonuclease enzyme recognition sequences into the Tel protelomerase enzyme-expressing plasmid will further improve the purification process. The other recombinant system that was developed is the utilization of the CRISPR-Cas3 system which is naturally present in W3110 E. coli K-12 bacteria. A pre-crRNA targeting the origin of replication (ori) of the msDNA synthesizing precursor plasmid was successfully designed and cloned into the low copy number plasmid. The pre-crRNA expressing gene cassette was placed under the control of the PBAD promoter. Upon overexpression, crRNA was synthesized inside the W3110 E. coli K-12 bacteria. The crRNA bound to the expressed CRISPR-Cas3 protein cascade of the bacteria, guided the effector complex to the target sequence and successfully digested the targeted precursor plasmid. Even though the W3110 tel+ recombinant bacteria synthesized msDNA in a pre-crRNA expressing background, an efficient degradation of the unwanted by-products of msDNA synthesis was not observed. This could be due to the disruption of the CRISPR locus of W3110 tel+ recombinant bacteria. Episomal expression of the CRISPR-Cas genes inside W3110 tel+ recombinant bacteria will enhance the digestion of the non-msDNA species.

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