Pharmacy

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Design, Synthesis and Biological Evaluation of 2,4-Disubstituted Pyrimidine Derivatives: Multifunctional Candidates as Potential Treatment Options for Alzheimer’s Disease
(University of Waterloo, 2011-08-30T19:05:22Z) Mohamed, Tarek
Alzheimer’s disease (AD) is a highly complex and rapidly progressive neurodegenerative disorder characterized by the systemic collapse of cognitive function and formation of dense amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). AD pathology is derived from the cholinergic, amyloid and tau hypotheses, respectively. Current pharmacotherapy with known anti-cholinesterases, such as Aricept ® and Exelon ®, only offer symptomatic relief without any disease-modifying effects (DMEs). It is now clear that in order to prevent the rapid progression of AD, new therapeutic treatments should target multiple AD pathways as opposed to the traditional “one drug, one target” approach. This research project employed medicinal chemistry tools to develop multifunctional small organic molecules against three key targets of AD pathology – the cholinesterases (AChE and BuChE), AChE-induced and self-induced Aβ1-40 aggregation and generation (β-secretase). A chemical library composed of 112 derivatives was generated to gather structure-activity relationship (SAR) data. The derivatives were based on a novel, non-fused, 2,4-disubstituted pyrimidine ring (2,4-DPR) template with substituents at the C-2 and C-4 position varying in size, steric and electronic properties. Molecular modeling was utilized to investigate their binding modes within the target enzymes and along with the acquired SAR, the chemical library was screened to identify lead multifunctional candidates.
Lack of neuroprotective effects by platelet-derived growth factor against beta-amyloid induced toxicity uncovers a novel hypothesis of Alzheimer's disease pathology
(University of Waterloo, 2012-05-18T17:44:28Z) Liu, Hui
Aβ oligomer-induced neurotoxicity has become an important area of therapeutic development in treating Alzheimer’s disease. Platelet-derived growth factor (PDGF) has been shown to be able to protect neurons against several neuronal insults such as ischemia and HIV1 toxin induced cytotoxicity. These neuroprotective effects correlate well with our previous results that demonstrate the neuroprotective effects of PDGF-BB, one of the PDGF receptor ligand subtypes, against NR2B containing NMDA receptor induced excitotoxicity, a possible underlying cause of Aβ oligomer induced synaptic dysfunction and neuronal death. This project examines the neuroprotective effect of PDGF-BB against Aβ1-42 oligomer induced cytotoxicity in both SH-SY5Y cells and primary hippocampal neurons. Cell viability was monitored by MTT assay and the affected signaling pathways were examined using pharmacological methods and Western blotting. The results demonstrated that Aβ1-42 oligomer elicited a dose-dependent toxicity with a sign of saturation at higher dosages, PDGF-BB failed to protect neurons against Aβ1-42 oligomer induced cytotoxicity. In contrast, Aβ1-42 oligomers strongly inhibit PDGF-BB induced mitogenesis in both SH-SY5Y cells and primary neurons. Further investigation using Western blotting to measure PDGF receptor expression and phosphorylation in SH-SY5Y cells showed that Aβ1-42 oligomer can inhibit PDGF-BB induced phosphorylation of PDGF β-receptor on Tyr1021, a site that is crucial for PLCγ mediated mitogenesis. These findings not only explained the poor neuroprotective effect elicited by PDGF-BB against Aβ1-42 oligomers, but also led to a novel hypothesis that Aβ1-42 oligomer may interfere with neurotrophic factor induced neuronal survival, either selectively or perhaps globally. Further exploration on this hypothesis will be able to shed light on this potentially novel mechanism of pathogenesis in Alzheimer’s disease.
Non-viral gemini surfactant-phospholipid nanoparticles for topical gene delivery to the retina
(University of Waterloo, 2013-01-29T15:32:53Z) Alqawlaq, Samih
Glaucoma is a group of optic nerve degenerative diseases, which leads to gradual and permanent vision loss. Recent developments in the field of gene therapy have proposed increasingly promising treatments for glaucoma, in the form of delivery of neuro-protective or neuro-regenerative genes to the retina. Despite these developments, there are concerns related to the biocompatibility and invasiveness of common gene delivery systems, since they are commonly mediated by viral gene carriers and invasive administration methods. Non-viral gene delivery systems offer a safe and increasingly efficient alternative to deliver therapeutic genes to the retina. An example of these systems is gemini-phospholipid nanoparticles (GL-NPs), which have been successfully used to deliver genes in similarly challenging anatomical settings, such as the skin. The objective of this thesis is to demonstrate the potential of GL-NPs, as candidate gene delivery vehicles for topically administered genes, targeted to the retina. The dicationic gemini surfactant, 12-7NH-12 was used, along with the helper lipids, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), to prepare various types of GL-NPs, and assess their transfection efficiency in the rat retinal ganglion cell line (RGC-5). The transfection efficiency was evaluated using flow cytometry, as a function of several physical and chemical parameters of GL-NPs. These include a range of charge ratios (5:1 to 15:1 ρ±), helper lipid composition (several DOPE: DPPC ratios), order of assembly (plasmid-gemini + lipid versus gemini-lipid + plasmid), and manufacturing method of helper lipid vesicles (thin film versus high pressure homogenization method). Size and zeta (ζ) potential characterization of GL-NPs was carried out in parallel, using dynamic light scattering, to relate the physical parameters of GL-NPs to their respective transfection efficiency. A comprehensive toxicological evaluation was undertaken to assess the extent of GL-NP’s toxicity in RGC-5 cells, using the resazurin-based PrestoblueTM cell toxicity assay. Optimized GL-NPs were used to induce expression of the brain derived neurotrophic factor (BDNF) in RGC-5 cells, and were assessed in terms of their capacity to induce neurite outgrowth. Quantification of neurite outgrowth was carried out by measuring average neurite length in RGC-5 cells, by confocal microscopic imaging of immunostained neurites. Furthermore, confocal microscopic studies were carried out to assess the extent of GL-NP’s corneal permeation in a 3-D human corneal epithelial (HCE) model. A parallel toxicological evaluation was completed to ensure GL-NP’s biocompatibility with the corneal epithelial cells. Finally, GL-NP biodistribution pattern and gene transfer capacity was assessed in a mouse model, following topical and intravitreal administration. The transfection efficiency in RGC-5 cells, which ranged between 2.1 ± 0.3% and 14.5 ± 1.4%, was highly dependent on GL-NP’s charge ratio, helper lipid composition, order of assembly, and manufacturing technique. GL-NPs at 10:1 ρ± charge ratio, assembled with homogenized DOPE (25%)-DPPC (75%) helper lipid vesicles, in the plasmid-gemini + lipid order, mediated the highest transfection efficiency in RGC-5 cells. These GL-NPs had a size of 222.8 ± 4.2 nm and a ζ potential of +33.5±2.9 mV. Optimized GL-NPs were highly biocompatible with both RGC-5 and HCE model cells, with viability values ranging between 94.8 ± 6 % to 100 ± 3.4 %. Assessment of corneal permeation showed that GL-NPs were able to bind to the corneal epithelial surface and achieve a moderate permeation depth (35-40 μm), following topical application in the HCE model. Intravitreal injection of the non-viral GL-NPs in mice has successfully led to their localization within the nerve fiber layer (NFL) of the retina. Finally, GL-NPs were non-invasively delivered to several anterior chamber tissues, including the limbus, the iris and conjunctiva, following topical administration. GL-NPs offer several advantageous features critical to topical and intravitreal ocular administration of gene carriers, including in vitro corneal binding and effective biodistribution following in vivo topical and intravitreal administration, high biocompatibility, and a highly tunable transfection efficiency. The current data presents 12-7NH-12 GL-NPs as a promising candidate for ocular gene therapy applications.
Development of a correlation based and a decision tree based prediction algorithm for tissue to plasma partition coefficients
(University of Waterloo, 2013-04-29T20:52:11Z) Yun, Yejin Esther
Physiologically based pharmacokinetic (PBPK) modeling is a tool used in drug discovery and human health risk assessment. PBPK models are mathematical representations of the anatomy, physiology and biochemistry of an organism. PBPK models, using both compound and physiologic inputs, are used to predict a drug’s pharmacokinetics in various situations. Tissue to plasma partition coefficients (Kp), a key PBPK model input, define the steady state concentration differential between the tissue and plasma and are used to predict the volume of distribution. Experimental determination of these parameters once limited the development of PBPK models however in silico prediction methods were introduced to overcome this issue. The developed algorithms vary in input parameters and prediction accuracy and none are considered standard, warranting further research. Chapter 2 presents a newly developed Kp prediction algorithm that requires only readily available input parameters. Using a test dataset, this Kp prediction algorithm demonstrated good prediction accuracy and greater prediction accuracy than preexisting algorithms. Chapter 3 introduced a decision tree based Kp prediction method. In this novel approach, six previously published algorithms, including the one developed in Chapter 2, were utilized. The aim of the developed classifier was to identify the most accurate tissue-specific Kp prediction algorithm for a new drug. A dataset consisting of 122 drugs was used to train the classifier and identify the most accurate Kp prediction algorithm for a certain physico-chemical space. Three versions of tissue specific classifiers were developed and were dependent on the necessary inputs. The use of the classifier resulted in a better prediction accuracy as compared to the use of any single Kp prediction algorithm for all tissues; the current mode of use in PBPK model building. With built-in estimation equations for those input parameters not necessarily available, this Kp prediction tool will provide Kp prediction when only limited input parameters are available. The two presented innovative methods will improve tissue distribution prediction accuracy thus enhancing the confidence in PBPK modeling outputs.
Design, Synthesis, and Evaluation of Tacrine-Based Derivatives: Potential Agents to Treat Alzheimer’s Disease
(University of Waterloo, 2013-08-01T17:26:40Z) Osman, Wesseem
With the incidence of Alzheimer’s disease (AD) growing worldwide and in Canada along with the growing economic and social burdens, the need for more effective therapies becomes of great importance. Since the discovery of AD, a number of proposed theories have arisen to explain the pathophysiology including the i) cholinergic theory, ii) oxidative stress pathways, and iii) metal ion imbalance. The major class of drug therapies to treat AD are cholinesterase inhibitors; however, the “one drug, one target” approach has not proven fruitful and generally becomes ineffective in later stages of disease progression. In this project, we synthesized a library of 1,2,3,4-tetrahydroacridine derivatives (10a-d, 11a-e, 12a-e, and 13a-f) as potential agents to target the cholinergic and oxidative stress pathways of AD. Chapter I provides background information on the role of AChE and BuChE enzymes in AD. Furthermore, this chapter describes the neurotoxicity of reactive oxygen species (ROS) and metals in AD. Chapter II provides a summary of project hypothesis and rationale. Chapter III describes the synthetic details regarding the synthesis of target small molecules. It further describes the principles involved in carrying out biological evaluation such as AChE and BuChE inhibition, antioxidant properties via DPPH stable radical scavenging, iron chelation capacity using ferrozine and in vitro cell viability data in neuroblastoma cells. Chapter IV describes the SAR details on ChE inhibition, antioxidant activities, iron chelation and cell viability profiles and molecular modeling details. A brief conclusion and future directions are included in Chapter V and the final section, Chapter VI provides experimental details for synthetic chemistry including analytical data of synthesized compounds and protocols for biological evaluations. This study identified novel tetrahydroacridine derivatives with nanomolar inhibition of both human AChE and human BuChE enzymes that were more potent relative to the reference agent tacrine. Compound 10d[N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified as a potent inhibitor of BuChE (IC50 = 24.0 nM) and compound 13c [6-chloro-N-(pyridine- 2-ylmethyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified as a potent inhibitor of AChE (IC50 = 95.0 nM) with good inhibition of BuChE (IC50 = 1.61 μM) whereas compound 11e [6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified with an optimum combination of dual AChE and BuChE inhibition (AChE IC50 = 0.9 μM; BuChE IC50= 1.4 μM). In conclusion, our studies provide new insight into the design and development of novel tetrahydroacridine derivatives to target multiple pathological routes of AD.
Effect of Gemini Surfactants on Amyloid Beta Aggregation
(University of Waterloo, 2013-09-27T15:17:33Z) Bahmani, Mehrnoosh
Alzheimer’s disease (AD) is a progressive dementia affecting cognition, behavior, and functional status and there is no cure which exists for it. In AD, Amyloid Beta (Aβ) peptides form aggregates that are neurotoxic in the brain. Hence, molecules that are able to prevent Aß aggregation could be effective in AD treatment. Gemini surfactant (GS) molecules consist of two hydrophilic heads separated by a covalently bound spacer and two hydrophobic tails. Their structure gives rise to a number of unique properties, including low critical micelle concentrations, the ability to form multiple types of aggregates (governed primarily by the nature of the spacer group) and enhanced ability to bind to polymers. These properties make gemini surfactant a good choice for solubilizing very hydrophobic materials such as Aß. The aim of this study was to examine various GS structures to help us to understand their interaction with Aβ and the influence of spacer group in Aβ disassembly. We employed 12-carbon tail GS with varying spacer groups of different hydrophilicities, such as: (-CH2-CH2-O)m, (-CH2)m, N(CH2)m, OH(CH2)4 and (OH)2(CH2)4. Surface tension measurement, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) have been employed to observe the gemini-Aβ interaction. Surface tension measurements did not show a typical surfactant-polymer interaction; rather, the presence of Aß induced aggregate formation at concentrations well below the cmc. Headgroup areas were observed to decrease for some of the surfactants in the presence of Aß, which may result from partial neutralization of the surfactant headgroups and a relaxation of electrostatic repulsion resulting in decreased head group areas. ITC results suggest substantial reorganization of Aß/gemini surfactant aggregates, with distinct difference seen depending upon the nature of the headgroup. It was observed that in 12-(CH2)n-12 (n=2,3,4,7) shorter spacer gemini surfactants have stronger interaction with Aß than the ones with longer spacers. In the 12-4(OH)n-12 series, a stronger interaction was observed in the GS with 2 hydroxyl groups compared to one hydroxyl group GS. For 12-(EO)n-12 GS, a stronger interaction was observed in that GS with two ethoxy groups. In the 12-XN-12 series, although the 8N spacer is more hydrophilic than 5N, the interaction of 12-5N-12 with Aß was stronger than that of 12-8N-12. The particle size data also revealed that there is an interaction between gemini surfactant and Aß. It appeared that mixed micelles formed when the surfactant concentration increased in the Aß solution. Overall, it was observed that changes in the length and hydrophilic character of the gemini surfactant spacer influenced the type of interaction and gemini-Aβ conformation.
Construction and Characterization of a Robust in vivo Technology for the Production of Superior DNA Vectors for Gene Therapy and Vaccination
(University of Waterloo, 2013-09-27T20:29:57Z) Nafissi, Nafiseh
Plasmid DNA (pDNA) vectors are the current conventional technology driving therapeutic gene transfer, whether for use toward mal/nonfunctional gene replacement, DNA vaccination, or production of therapeutic proteins in mammalian cells. However, the conventional pDNA vector suffers from several safety and efficiency limitations: 1) it imparts adverse immune responses to bacterial sequences required for maintenance and amplification in prokaryotes; 2) its bioavailability can be compromised due to size; and 3) it may be genotoxic due to its potential to integrate into the host chromosome and yield an oncogenic event. In this study we have constructed a robust in vivo bacterial platform for the production of bacterial sequence-free linear covalently closed (LCC) DNA vectors, termed DNA Ministrings, through the manipulation and application of bacteriophage-encoded recombination systems. Phage N15 and PY54 lysogenize their bacterial hosts as a linear plasmid with covalently closed ends (LCC plasmid). LCC morphology is conferred by the phage-encoded telomerase via a single cleaving-joining reaction of the perfect palindrome target site. This system was exploited to generate DNA Ministring vectors, encoding only the gene(s) of interest and necessary complementary eukaryotic expression/enhancement genetic elements that are devoid of unwanted bacterial sequences and are linearized through a single in vivo enzymatic reaction. The tel and telN prokaryotic telomerase (protelomerase) genes were amplified from PY54 and N15 lysates, respectively, and cloned into a bacterial vector that expresses the gene under control of the temperature sensitive bacteriophage λ CI857 repressor that confers conditional expression from λ pL/pR promoters. This regulatory circuit was integrated into a RecA+ lacZ+ E. coli K-12 strain via homologous recombination, where successful recombinants were disrupted for the lacZ gene. Recombinant cells are capable of conditional expression of the phage-derived telomerase enzymes by shifting the temperature to >37 °C. Phage P1-derived Cre recombinase was applied as a positive control, since its functionality in generating DNA minicircle vectors has been previously shown. A multi-purpose 342 bp target site termed Super Sequence (SS) that possesses the Cre, Flp, Tel, and TelN target sites in addition to two flanking SV40 enhancer sequences was cloned into two different sites of a GFP expression eukaryotic pDNA vector. The amplification of this DNA vector through telN / tel or cre expressing Recombinant E. coli cells (R-cells) generated bacterial sequence-depleted (LCC) DNA Ministring and (CCC) Minicircle vectors, respectively, as evidenced by digestion patterns of the purified vector. Transfection efficiency of these vectors was assessed in rapidly dividing human ovarian cancer and in relatively slowly dividing human embryonic kidney cell lines. In vitro experiments with DNA Ministrings in human cells lines resulted in significantly higher transfection efficiency, bioavailability, and cytoplasmic diffusion levels compared to the parental plasmid precursor and isogenic DNA Minicircle counterparts. The safety of the LCC DNA vector conformation, with respect to insertional genotoxicity, was assessed by forcing LCC pDNA vectors into bacterial and human genomic DNA. The integration of LCC DNA into bacterial and human host genomic DNA resulted in chromosomal DNA disruptions at site of integration, loss of genome stability, and subsequent cell death. LCC integration-induced apoptotic cell death and natural elimination of the integrant from human cell population improves the safety profile of DNA Ministrings by eliminating integrants following the potential genotoxic side effects of undesired vector integration into the host genome.
Transactivation of platelet-derived growth factor receptor type β: Mechanisms and potential relevance in neurobiology
(University of Waterloo, 2013-12-23) Kruk, Jeffrey Stephen
In the absence of ligand, certain growth factor receptors can be activated via G protein-coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate the receptor tyrosine kinase (RTK) platelet-derived growth factor (PDGF) β receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here, it is shown that 5-HT can transiently increase the phosphorylation of PDGFβ receptors in a time- and concentration-dependent manner in SH-SY5Y neuroblastoma cells. This transactivation pathway was pertussis-toxin sensitive, and was dependent on phospholipase C activity, intracellular calcium signaling and subsequent protein kinase C activation. Exogenous application of non-lethal concentrations of H2O2 induced the phosphorylation of PDGFβ receptors in a concentration-dependent fashion, similar to that observed with 5-HT. Further investigation revealed reactive oxygen species (ROS) production as a necessary component in the transactivation pathway, as scavenging ROS eliminated PDGFβ receptor phosphorylation. NADPH oxidase was determined to be the likely source of ROS given that the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin abrogated PDGFβ receptor transactivation. The role of Src tyrosine kinase was also investigated, and its location in this signaling cascade was determined to be downstream of calcium signaling, but upstream of NADPH oxidase activity. In addition, the activation of ERK1/2 in this system was elucidated to be independent of PDGFβ receptor transactivation. Interestingly, 5-HT also transactivated TrkB receptors, another RTK whose function is implicated in clinical depression. Expectedly, the enzymes in this mechanism were consistent with those revealed in 5-HT-to-PDGFβ receptor signaling. This cross-talk between 5-HT and RTKs such as TrkB and PDGFβ receptors identifies a potentially important signaling link between the serotonergic system and neurotrophic factor signaling in neurons that could have implications in mental health disorders including depression. Furthermore, although transactivation pathways are commonly initiated by a GPCR, recent reports have demonstrated that selective serotonin reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of PDGFβ receptors, suggesting that in addition to GPCRs, monoamine transporters may also be involved in RTK transactivation. SH-SY5Y cells pretreated with the SSRI fluoxetine blocked 5-HT-induced transactivation of the PDGFβ receptors, but not PDGF-induced PDGFβ receptor activation. Upon further examination, it was discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGFβ receptor via 5-HT2 receptors. By the end of the pretreatment period, the effects of fluoxetine on PDGFβ receptor phosphorylation had returned to baseline, and a subsequent transactivating stimulus (5-HT) failed to “re-transactivate” the PDGFβ receptor. Additional investigations demonstrated that 5-HT pretreatment can block dopamine-induced PDGFβ receptor transactivation, but not PDGF-induced PDGFβ receptor activation. This is the first demonstration of the heterologous desensitization of an RTK via a transactivation pathway, and this phenomenon is specific for transactivation pathways because in all cases the PDGFβ receptor ligand PDGF-BB was able to directly stimulate receptor activity in spite of GPCR agonist pretreatment. Heterologous desensitization in transactivation signaling reveals a previously unknown short-term “blackout” period wherein no further transactivation signaling can occur to potentially exploit the mitogenic effects of RTK activation.
Characterization of gemini nanoparticle assembly by fluorescence correlation spectroscopy
(University of Waterloo, 2014-01-23) Dong, Chilbert
Research in the field of non-viral gene delivery has demonstrated that a deeper understanding of the fundamental processes of nanoparticle assembly is required in order to improve their efficacy. While gemini nanoparticles (gemini NPs) and other non-viral delivery systems have been vigorously characterized using several techniques, our knowledge is still incomplete. The first objective of this study was the development of new methodology using fluorescence correlation spectroscopy (FCS) to investigate the stages of gemini NPs assembly. It was demonstrated that by labeling the plasmid, different stages of gemini NP assembly from the gemini-plasmid pre-complex (GP) to the final gemini nanoparticle (or gemini-plasmid-lipid complex; GPL), could be studied. Based on diffusion coefficients and particle numbers extrapolated from the autocorrelation function (ACF), FCS was able to determine that each phase of assembly had distinct characteristics. The FCS study using 12-3-12 gemini surfactant showed that both the diffusion coefficient and particle number of GPs (0.98±0.31 x 10-12 m2/s) was significantly lower than the final GPL (3.11±0.41 x 10-12 m2/s). Based on the Stokes-Einstein equation the particle size was calculated to be 300-500 nm for GP and 200-300 nm for GPLs. The raw intensity histograms showed that both GPs and GPLs are composed of multiple plasmids. Furthermore the study showed that the final GPLs contain fewer plasmids compared to the intermediate GP. FCS results were validated by using existing characterization methods including dynamic light scattering (DLS), zeta potential and dye exclusion assays. The second objective involved the detailed characterization of gemini NP. Nine different gemini surfactants and two different phospholipids were used in a systematic study to assess the effect of gemini surfactant and lipid structure on the final morphology of gemini NP. The study revealed that gemini surfactant structure had a strong effect on structure of GP intermediates, but addition of phospholipids resulted in the formation of uniform gemini NPs. Based on the results of this study a new model for GP and GPL assembly is proposed based on the formation of supramolecular aggregates of gemini-plasmids, governed by gemini surfactant chemical structure, and dispersed by phospholipids to form GPLs.
Identifying the mechanism of Aβ42 inhibition of PDGF-BB signalling
(University of Waterloo, 2014-01-23) Saffi, Golam Tanjib
Alzheimer’s disease is a late-onset neurological disorder characterized by extracellular aggregates of Aβ plaques and neurofibrillary tangles of hyperphosphoryated tau protein resulting in neuronal dysfunction, cognitive decline and death. Some of the molecular mechanisms which cause neuronal dysfunction in Alzheimer’s disease are oxidative stress, excitotoxicity and hypoxia. PDGF-BB is a neurotrophic factor which is neuroprotective against these molecular events. However, PDGF-BB failed to be neuroprotective against Aβ42 toxicity in SH-SY5Y neuroblastoma cells. Rather, Aβ42 actually inhibited PDGF-BB signalling and reduced the phosphorylation level at multiple phosphotyrosine sites on the PDGFβ receptor. Aβ42 inhibition of PDGF-BB signalling also inhibited a downstream effector, Akt, a neuroprotective protein. Thus, Aβ42 inhibition of PDGF-BB signalling could worsen oxidative stress, excitotoxicity and hypoxia observed in Alzheimer’s disease. Indeed, Aβ42 treatment prevented PDGF-BB neuroprotection against excitotoxicity. Aβ42 mediated inhibition of PDGF-BB signalling was not due to Aβ42 interaction with PDGFβ receptor. However, it remains inconclusive whether Aβ42 binds to PDGF-BB to prevent PDGF-BB binding to PDGFβ receptor.
Pluronic-Based Nanoparticles for Gene Therapy Applications
(University of Waterloo, 2014-01-24) Madkhali, Osama
Non-viral delivery vectors have potential advantages over the viral systems that currently are used extensively for delivering therapeutic genes of interest. However, non-viral gene therapy has low efficiencies in vivo, in part due to the aggregation of the particles in the delivery system associated with serum proteins and other components of the blood. An effective technique for overcoming this problem to use PluronicTM block copolymers to cover the surfaces of the particles in the delivery system with polyethylene oxide, which decreases their charge density and reduces their interactions with the serum proteins. The objectives of this project were to characterize a Pluronic-gemini surfactant system to be used as non-viral vectors for gene therapy. Five Pluronics (L44, F68, F87, F108, and F127) were evaluated by studying their physiochemical properties, including particle size and zeta potential. Also, these systems were evaluated in OVCAR-3 cell culture for gene expression and cell viability. The in vitro systems showed small particle sizes (approximately 200 nm) for all Pluronics. The particle sizes in the systems were increased dramatically (up to 2000 nm) by adding dioleylphosphatidylethanolamine (DOPE) to the systems. The zeta potential of these systems shifted the negative zeta potential of DNA (-43 mV) to a positive value (+35 mV). The addition of DOPE had very little effect on zeta potential. The in vitro transfection efficiency in OVCAR-3 showed that all of the Pluronics were able to transfect OVCAR-3 at various DNA/gemini surfactant ratios. The highest transfection efficiency was obtained with Pluronics L44, F87 and F108. PluronicF127 demonstrated the lowest transfection efficiency among the five Pluronics. Adding DOPE did not improve the transfection efficiency in any of the pluronic-gemini surfactant systems. The viabilities of the cells in these systems were high, and there were greater than the positive control (Lipofectamine 2000). The greatest cell viability (about 60%) was observed when the DNA to gemini surfactant ratio was 1:2. After adding DOPE, the cell viability decreased in all of the Pluronics except for Pluronic F68. The results of this investigation indicated that Pluronic block copolymers can transfect OVCAR-3 cell cultures in vitro and that they had a low level of cytotoxicity.
Optimized Production and Purification of LCC DNA Minivectors for Applications in Gene Therapy and Vaccine Development
(University of Waterloo, 2014-01-28) Sum, Chi Hong
Linear covalently closed (LCC) DNA minivectors serve to be superior to conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors due to enhancements to both transfection efficiency and safety. Specifically, LCC DNA minivectors have a heightened safety profile as insertional mutagenesis is inhibited by covalently closed terminal ends conferring double-strand breaks that cause chromosomal disruption and cell death in the low frequency event of chromosomal integration. The development of a one-step, E. coli based in vivo LCC DNA minivector production system enables facile and efficient production of LCC DNA minivectors referred to as DNA ministrings. This novel in vivo system demonstrates high versatility, generating DNA ministrings catered to numerous potential applications in gene therapy and vaccine development. In the present study, numerous aspects pertaining to the generation of gene therapeutics with LCC DNA ministrings have been explored with relevance to both industry and clinical settings. Through systematic assessment of induction duration, cultivation strategy, and genetic/chemical modifications, the novel in vivo system was optimized to produce high yields of DNA ministrings at ~90% production efficiency. Purification of LCC DNA ministrings using anion exchange membrane chromatography demonstrated rapid, scalable purification of DNA vectors as well as its potential in the separation of different DNA isoforms. The application of a hydrogel-based strong Q-anion exchange membrane, with manipulations to salt gradient, constituted effective separation of parental supercoiled CCC precursor pDNA and LCC DNA. The resulting DNA ministrings were employed for the generation of 16-3-16 gemini surfactant based synthetic vectors and comparative analysis, through physical characterization and in vitro transfection assays, was conducted between DNA ministring derived and CCC pDNA derived lipoplexes. Differences in DNA topology were observed to induce differences in particle size and DNA protection/encapsulation upon lipoplex formation. Lastly, the in vivo DNA minivector production system successfully generated gagV3(BCE) LCC DNA ministrings for downstream development of a HIV DNA-VLP (Virus-like particle) vaccine, thus highlighting the capacity of such system to produce DNA ministrings with numerous potential applications.
Design, construction and characterization of LysK endolysin display phage against Staphylococcus aureus
(University of Waterloo, 2014-02-04) El-Zarkout, Farah
The growing threat of drug- resistant Staphylococcus aureus (S. aureus) infections mandates the need to develop novel, effective and alternative antibacterial therapeutics. Despite infection prevention and control measures, methicillin resistant S. aureus (MRSA)-associated deaths reached 11,285 in 2011 in the USA (CDC, 2013). To counteract the threat of drug resistant S. aureus, we sought to construct and characterize a novel therapeutic based on the display of lytic antibacterial enzymes, termed endolysins. These endolysins were displayed on the surface of a specific bacterial virus, bacteriophage (phage), to generate lytic antibacterial nanoparticles. Endolysins are encoded individually by a variety of double-stranded DNA phage and act to direct host lysis and escape. These lytic enzymes confer a high degree of host specificity that could potentially substitute for, or be combined with, antibiotics in the treatment of gram-positive drug resistant bacterial infections such as MRSA. In this study, modular domains of the phage-encoded endolysin K enzyme, specific to S. aureus, were displayed on the capsid surface of phage lambda () via fusion with the λ major head (capsid) protein, gpD. The constructs of displayed endolysins were prepared in various combinations to maximize the functional display of gpD::X fusions on the phage. Phage lysates were generated, collected and purified and lysis was investigated by adding to fresh lawns of MRSA, vancomycin resistant S. aureus (VRSA) and bovine S. aureus. Phage preparations did not readily confer cell lysis, likely due to poor incorporation of the fusions onto the functional phage capsid. We purified the fusion proteins (gpD::X) and tested them for their lytic activity. We noted that the activity of the gpD::LysK protein was not impaired by the fusion and demonstrated lysis on live and dead (autoclaved) bovine S. aureus. In contrast to gpD::LysK, the gpD::CHAP protein fusion, expressing only the CHAP catalytic domain of endolysin K showed variable results in the lysis assays that we performed. In the zymogram assay, gpD::CHAP did not elicit any observable lysis on live bovine S. aureus cells, but did effectively lyse dead cells of the same S. aureus species; however, it was highly lytic in the inhibition assay on bovine S. aureus. The CHAP::gpD protein fusion, which is the CHAP domain fused to the N terminus of gpD only showed its ability to inhibit bovine S. aureus growth on the inhibition assay. The fusion of endolysin K or its CHAP domain to gpD protein does not seem to interfere with lytic activity, but may result in recalcitrant gpD fusions that compromise the ability to efficiently decorate the phage capsid. Suggestions for improved fusion capsid integration are discussed.
Identifying signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation
(University of Waterloo, 2014-04-01) Kouchmeshky, Azita
Growth factor receptors have significant effects on various normal function of body such as cell proliferation, differentiation and apoptosis. They are also involved in neuronal function and dysfunction, cardiovascular diseases, and malignancies. Recently, multiple G protein-coupled receptors (GPCRs) have been shown to transactivate receptor tyrosine kinases (RTKs). Since both classes of receptors have complicated downstream cascades individually, understanding the signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation is an important challenge. To clarifying this phenomenon we investigated the phosphorylation profile and downstream effectors of ligand-activated vs. transactivated PDGFβ receptors. Dopamine receptors (one of the receptors of the GPCRs family) were used to compare the PDGFβ receptor phosphorylation and activity during direct activation and transactivation. Dose-response and time-course data between these two stimuli were evaluated. Furthermore, the phosphorylation site profiles and the intracellular signaling pathways of PDGFβ receptor after direct activation and transactivation were examined. In addition, possible synergic effects between transactivation and direct activation were explored. The results of this project showed that the phosphorylation profile and downstream effectors of ligand activated receptors versus transactivated receptors are different. Our data indicated that transactivation-induced pathways are more involved in survival and proliferation effects compared to ligand activation. This research answered basic questions about transactivation phenomena and proposes that these transactivation pathways could be exploited as a therapeutic approach for neurodegenerative diseases.
5-HT7 Receptor Neuroprotection against Excitotoxicity in the Hippocampus
(University of Waterloo, 2014-04-11) Vasefi, Seyedeh Maryam
Introduction and Objectives: The PDGFβ receptor and its ligand, PDGF-BB, are expressed throughout the central nervous system (CNS), including the hippocampas. Several reports confirm that PDGFβ receptors are neuroprotective against N-methyl-D-asparate (NMDA)-induced cell death in hippocampal neurons. NMDA receptor dysfunction is important for the expression of many symptoms of mental health disorders such as schizophrenia. The serotonin (5-HT) type 7 receptor was the most recent of the 5-HT receptor family to be identified and cloned. 5-HT receptors interact with several signaling systems in the CNS including receptors activated by the excitatory neurotransmitter glutamate such as the NMDA receptor. Although there is extensive interest in targeting the 5-HT7 receptor with novel therapeutic compounds, the function and signaling properties of 5-HT7 receptors in neurons remains poorly characterized. Methods: The SH-SY5Y neuroblastoma cell line, primary hippocampal cultures, and hippocampal slices were treated with 5-HT7 receptor agonists and antagonists. Western blotting was used to measure PDGFß receptor expression and phosphorylation as well as NMDA receptor subunit expression and phosphorylation levels. Real-time RT-PCR was used to measure mRNA level of PDGFß receptor in neuronal cultures. Cell death assays (MAP2, MTT) were used to measure the neuroprotective effects of 5-HT7 and PDGFß receptor activation. Results: My research involved elucidating the molecular mechanisms of neuroprotection after 5-HT7-induced PDGFß receptor upregulation. I demonstrated that 24 h treatment with the selective 5-HT7 receptor agonist, LP 12, increased not only the expression but also the activation of PDGFß receptors as measured by the phosphorylation of tyrosine 1021, the phospholipase Cγ binding site. Activation of the 5-HT7 receptor also selectively changed the expression and phosphorylation state of the NR2B subunit of the NMDA receptor. Activation of 5-HT7 receptors was neuroprotective against NMDA-induced toxicity in primary hippocampal neurons and this effect required PDGFß receptor kinase activity. Thus, long-term (24 h) activation of 5-HT7 receptors was neuroprotective via increasing the expression of a negative regulator of NMDA activity, the PDGFß receptor. In contrast, acute activation (5-30 min) of 5-HT7 receptor increased NMDA evoked current and altered NMDA receptor subunit phosphorylation in hippocampal neurons in a manner that was different from what we observed in our 24 h experiments. Conclusions: I identified two 5-HT7 receptor to NMDA receptor pathways: acute activation of the receptor increased NMDA-evoked currents whereas long-term 5-HT7 agonist treatment prevented NMDA-induced excitotoxicity in a PDGFß receptor-dependent manner. This research is significant in the ongoing advances for the treatment of mental heath disorders, such as schizophrenia and depression, that involve the 5-HT, glutamate, and neuronal growth factor systems.
Pain Management within the Long-term Care Setting: An Inquiry into Staff-perceived Contemporary Pain Management Practices
(University of Waterloo, 2014-05-06) Weber, Haley
Background: Chronic pain is a frequent and undertreated ailment within the long-term care community (Herman et al, 2009). The likelihood of experiencing pain increases with age and failure to treat this condition may expose individuals to prolonged and unnecessary suffering (Ramage-Morin, 2008). Furthermore, undertreated pain can lead to a life of inactivity and a failure to carry out normal social and vocational roles which in term may result in higher rates of depression, anxiety and sleep disorders (Clark, 2000). The present study aimed to explore staff perceptions on current pain management within long-term care including insights to future needs in optimizing pain management. This work will contribute to the overall awareness surrounding possible reasons that current pain management within long-term care is viewed as suboptimal (Herman et al, 2009). Methods: A qualitative, post-positivist grounded theory study was carried out in order to investigate staff-perceived strengths, weaknesses and barriers surrounding the topic of pain-management within the long-term care setting. Semi-structured interviews with 17 long-term care staff members from a variety of vocations were conducted with a focus on identifying and clarifying properties surrounding the notion that pain management is currently suboptimal. A focus group session was implemented as a method to further develop the emerging grounded theory. Results: Nine themes surrounding pain management within the long-term care setting were identified in the present study. These themes gave rise to the core concept of creating an environment supportive of optimal pain management. The nine themes were integrated into the theory of optimization of pain management within long-term care through thematic interpretation. The focus group session further developed and confirmed themes identified throughout the one-on-one interviews as well as expanded the discussed theory. Discussion: The developed theory of optimization of pain management within the long-term care setting provides a comprehensive overview of the current barriers facing adequate pain management as well as outlines future suggestions for improvement of managing pain within the long-term care setting.
Construction and Characterization of a Fine-Tuned Lytic Phage Display System
(University of Waterloo, 2014-08-15) Nicastro, Jessica
Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our current understanding of molecular genetics. The lytic nature of this bacterial virus along with the conformation of its wild-type capsid protein (gpD) assembly offer many advantages for the virus for a phage display platform. Protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and persevere, or even increase, solubility. In this study, the exploitation of gpD for the design of a dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. In this system, gpD expression is encoded by mutant  infecting phage particles, λDam, that can only produce a wild type length gpD allele within specialized strains of E. coli that can suppress the mutation. However, the functionality of gpD alleles, produced by passage of λDam through various suppressor strains, varies dramatically in their ability to restore functional packaging to the λDam phage, imparting a first dimension of decorative control. As a second dimension of decorative control, a D::eGFP translational fusion on a multicopy plasmid, encoding gpD::eGFP, complements the Dam mutation in trans and is regulated by the temperature-labile  CI[Ts]857 repressor, allowing for the conditional expression of D::eGFP by thermoregulation. In combination, the effective exploitation of these two variables has permitted the effective development of a fine-tuned λ lytic phage display system. Of the suppressor-imparted alleles, gpDQ68S, gpDQ68Y, and gpDwt: the allele with the poorest functionality, gpDQ68S (SupD), in combination with submaximal expression of gpD::eGFP conferred the highest incorporation of the fusion into the λDam phage capsid in all combinations. Differences in size, fluorescence, and absolute protein decoration between phage preparations was achieved by varying the temperature of the suppressor host carrying the D::eGFP fusion plasmid. The effective preparation with these two variables provides a simple means to manage fusion decoration on the surface of phage λ for a variety of fusion partners and applications.
Glucopsychosine increases cytosolic calcium to induce calpain-mediated apoptosis of acute myeloid leukemia cells.
(University of Waterloo, 2014-08-20) Angka, Leonard
To identify novel anti-cancer agents, we created and screened a unique nutraceutical library for activity against acute myeloid leukemia (AML) cells. From this screen, we determined that glucopsychosine was selectively toxic toward AML cell lines and primary AML patient samples with no effect toward normal hematopoietic cells. It delayed tumor growth and reduced tumor weights in mouse xenograft models without imparting toxicity. Glucopsychosine increased cytosolic calcium and induced apoptosis through calpain enzymes. Extracellular calcium was functionally important for glucopsychosine-induced AML cell death and surface calcium channel expression is altered in AML cells highlighting a unique mechanism of glucopsychosine’s selectivity.
Perceptions of Community-Dwelling Patients on the Impact of the Discontinuation of OxyContin® on their Pain Management: A Mixed Methods Study
(University of Waterloo, 2014-09-02) Ibrahim, Sara
Chronic pain is considered a major health problem in older adults. OxyContin®, one of the most commonly used medications for treating pain in Canada, was recently discontinued, and delisted from the Ontario Drug Formulary due to high rates of abuse associated with its use. A tamper-resistant formulation was developed by the manufacturing company to replace it. Inadequacies in the available body of literature reporting on the discontinuation process prompted this study of patient perceptions on the impact of the discontinuation of OxyContin® on personal pain control. A qualitative descriptive study was carried out to provide evidence on patients’ lived-experiences, placing particular focus on identifying any flaws within the medical infrastructure through sharing discontinuation experiences and investigating the efficacy of alternative pain medications. Chronic pain patients 45 years or older were recruited from three sites and interviewed using a semi-structured guide. Interviews were conducted with physicians to obtain their perceptions on discontinuation to develop a more comprehensive understanding of the experience from both patient and professional levels. Findings of the current study provided preliminary data illuminating several aspects of patients’ pain and medical care through the discontinuation process. The emergent themes represented both convergences and divergences between patients and their clinicians. For example, areas of divergence included the motive for discontinuation, which was condemned by most patients, but supported by all physicians. Another discrepancy was reflected in the perceived impact of discontinuance on pain control, with the majority of patients reported experiencing a negative impact while most physicians described it as being insignificant within their practice. On the other hand, perceptions of patients and physicians on their experiences overlapped giving rise to a prominent theme of the need to optimize current pain management practices. Further research is warranted to build upon the foundation generated by this study to improve understanding of shortcomings in pain management and to optimize care for patients.
“I gained a skill and a change in attitude”: A Case Study Describing How an Online Continuing Professional Education Course for Pharmacists Supported Achievement of Its Transfer-to-Practice Outcomes
(The Canadian Association for University Continuing Education, 2014-11-27) Marks, Pia Zeni; Jennings, Brad; Farrell, Barbara; Kennie-Kaulbach, Natalie; Jorgenson, Derek; Pearson Sharpe, Jane; Waite, Nancy
The convenience and flexibility of online learning clearly make it an attractive option for learners in professional development contexts. There is less clarity, however, about how it fares as a vehicle for enabling the applied, practice-oriented outcomes typically associated with professional development learning.This paper presents a case study describing how transfer-of-learning strategies were employed in a continuing professional education (CPE) course developed for practicing pharmacists, called ADAPT (ADapting pharmacists’ skills and Approaches to maximize Patients’ drug Therapy effectiveness).To gain insight into the extent to which learning was transferred to practice as a result of participation in the course, qualitative data were collected over a 12-month period from participants of the 2010 pilot offering of ADAPT. Participants reported making changes to their practice as a result of participating in the course, and they identi- fied three course features as being particu- larly useful in facilitating practice transfer: providing learners with (i) a vision of targeted outcomes and skills, (ii) support to enable them to attain targeted outcomes and skills, and (iii) explicit preparation for action.

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