VictorJuiz/thesis huggingface dataset:
Regulation of Hepatic Drug Transporters Expression in Mice During Acute Inflammation

Inflammation generated by many disease conditions is known to alter the expression and activity of several hepatic ABC and SLC transporters and drug-metabolizing enzymes. These pronounced changes in hepatic transporters contribute to altered drug disposition; therefore, understanding the molecular mechanisms would aid in identifying potential sources of drug variability in diseases associated with inflammation. Pregnane X receptor (PXR) and nuclear factor-κB (NF-κB) are regarded as key transcriptional factors that are modulated in response to endotoxin and cytokine signaling. This work assesses the role of NF-κB and PXR in the transcriptional regulation of hepatic transporters and elucidate the signaling pathways behind changes in transporter expression. We hypothesized that the regulatory mechanism of hepatic transporter expression following inflammatory stimuli would depend primarily on the NF-κB signaling pathway and that PXR might play a role in this regulation. Inflammation was induced by either endotoxin or proinflammatory cytokine interleukin (IL)-6 administration in wild type PXR (+/+) and knockout PXR (-/-) mice. The mRNA expression of most transporters was downregulated to a similar extent for PXR (+/+) and PXR (-/-) mice following endotoxin administration. NF-κB inhibition attenuated the impact of endotoxin on transporter expression, suggesting that NF-κB is the major signaling pathway involved in endotoxin-mediated downregulation of hepatic transporter, and it appears to act independent of PXR. Conversely, IL-6-mediated downregulation of Abcb11, Abcc2, Slco2b1 and Slc10a1 was seen in PXR (+/+) but not PXR (-/-) mice, suggesting PXR-dependent regulation. Our IL-6 studies demonstrated that STAT3 inhibition attenuated IL-6-mediated downregulation of almost all transporters. However, IL-6-mediated activation of STAT3 stimulated the release of secondary cytokines that further activated NF-κB. Interestingly, NF-κB target genes (IL-6 and IL-1β) showed (35%-100%) higher basal expression in PXR (-/-) versus PXR (+/+) mice, and NF-κB inhibition attenuated IL-6-mediated changes of transporters in PXR (+/+) mice. Since NF-κB inhibition following IL-6 administration demonstrates indirect involvement of NF-κB in transporter expression via secondary cytokines, findings from these studies indicate that NF-κB is an essential regulator of hepatic transporter expression under inflammatory conditions. Thus, targeting NF-κB in diseases associated with inflammatory conditions could provide a strategy for therapeutic intervention.

Keywords: drug transporters
Inflammation
Liver
signaling pathways
Advisor: Piquette-Miller, Micheline
3D Microscopy of Nanoparticles in Tumours

The structure of solid tumours is a major factor contributing to poor treatment outcomes because it can prevent therapeutics from reaching cancer cells. Nanoparticles are able to increase the accumulation of therapeutics in the tumour as a whole but are not able to deliver therapeutics to cancer cells because of structural barriers within the tumour. However, researchers have been unable to visualize these structures within the tumour and how it impacts nanoparticle distribution because tumours are opaque. Here, I present a series of techniques that allow the structure of the tumour and the distribution of nanoparticles within to be visualized and quantified. I developed methods to 1) render tumour tissues transparent at the scale of 100s of tissues per month needed for biological experiments, 2) quantify and improve the retention of nanoparticles in the tumours after process, 3) label and visualize nanoparticles within the tumours. These methods can now be used to gain a deeper understanding of how the structure of the tumour is organized, how it impacts nanoparticle distribution and how it can be altered to achieve optimal cancer therapy.

Keywords: 3D microscopy
Cancer cells
Nanoparticles
Tissue clearing
Tumour
Advisor: Chan, Warren CW
Ray-tracing Propagation Models of Complex Guideway Geometries for the Deployment of Train Control Systems

This thesis presents the work carried out in modeling propagation in complex guideway geometries. The primary motivation of this work is to develop high-fidelity models that can enable improvements in the deployment and performance of train control systems. To that end, a 3-D image-based ray tracing tool is developed. It is validated in a variety of scenarios, including an indoor office hallway, an outdoor open environment with an undulating terrain, and an arched road tunnel. The limitations of the basic image-based algorithm are thoroughly investigated, especially with respect to their failings in modeling segmented approximations of circular and arched tunnels. Due to a lack of any rigorous solution that addresses these issues, a heuristic approach is developed to modify the Fresnel reflection coefficients to systematically weaken the contribution of reflected ray-paths that lead to an overestimation of RSS. This approach is inspired by the scattering of an incident wave by a perfectly conducting finite sized rectangular plate. Enabled with these modifications, the ray tracing tool is used to study propagation in a 3.5 km long real, complex tunnel environment in the London Underground. The estimated RSS is within a mean absolute deviation of 4-6 dB compared to the measurements. The input geometries required for these simulations are derived from point cloud data obtained from a laser survey conducted by Thales Canada, by developing a semi-automated error driven framework which enables a systematic study of the relationship between the accuracy of the simulated RSS and the accuracy of the estimation of the modeled geometry. Finally, physics-based models such as the developed ray tracing tool and others are used to characterize and enhance the performance of train control systems. It is shown that such modeling tools can be quite useful as they enable optimization of access points driven by the estimated network level performance indicators, thereby integrating propagation modeling with network-level system design leading to potential efficiency gains, instead of carrying out these tasks sequentially as is done conventionally.

Keywords: Geometrical Optics
Intelligent Transportation Systems
Radiowave Propagation
Ray-Tracing
Wireless Channel Modeling
Advisor: Sarris, Costas D
Ion Permeation and Selectivity in Voltage-gated Sodium Channels

Voltage-gated sodium channels are responsible for the initiation and propagation of electrical signals in living organisms. Though the function of these channels are well-described in humans, many questions remain at the microscopic level with respect to the relationship between their structure, function, and connection to human health and disease. The elucidation of high-resolution structures of sodium channels enables the use of computational models to connect microscopic biophysical measurements to macroscopic experimental observables. In this work, we present extensive molecular dynamics studies of the bacterial voltage-gated sodium channel NavAb to examine the molecular basis for four key mechanisms underlying the generation of action potentials in excitable cells; ionic conduction, selectivity, gating, and leakage. Our simulations support a molecular mechanism for ionic conduction involving a strong coupling between the conformational isomerization (dunking) of glutamic acid sidechains and ionic conduction. We determined single-point mutations which modulate dunking and ionic conduction and enable experimental validation of this hypothesis. Building on this mechanism, we present a model for Na+ selectivity over K+ in NavAb that results from differences in ionic desolvation penalties within the selectivity filter (SF) and the capability of channel fluctuations to facilitate multi-ion occupancy. The presence of three ions within the SF results in a diffusive free-energy landscape for Na+ whereas K+ conduction is comparatively restricted. We describe a mechanism for Na+ permeation at the intracellular gate wherein fluctuations between a wetted and dewetted state modulate the free-energy of conduction in this region. Lastly, we studied the molecular basis of Na+ leakage in the voltage-sensor of NavAb with analogous mutations linked to the hereditary disease periodic paralysis. Molecular simulations reveal a decrease in the free-energy barrier opposing Na+ permeation within the voltage sensor consistent with leakage, providing a structural basis for this disease that may facilitate treatment. Throughout this work, we quantify and compare the relationship between ionic desolvation and free-energy of ion conduction. Our studies have broad implications for understanding ion transport and permeation across the larger family of ion channels and have relevance to the treatment of patients suffering from diseases that result from abnormal function of channels.

Keywords: computer simulation
electrophysiology
free energy
ion solvation
molecular dynamics
sodium channel
Advisor: Pomès, Régis
Comparative Efficacy and Safety of Pharmacological and Non-pharmacological Interventions in Persons Living with Dementia

Background: Neuropsychiatric symptoms of dementia are treated with pharmacological and non-pharmacological interventions; however, their comparative efficacy and safety is largely unknown. As practice patterns shift because of concerns regarding antipsychotic safety, we need to understand the efficacy and safety of alternative interventions. Methods: Outcomes for this thesis were chosen by dementia care partners. Three studies were conducted: a systematic review and network meta-analysis (NMA) of randomized controlled trials (RCTs) comparing the efficacy of pharmacological and non-pharmacological interventions for treating aggression and agitation in PLWD, a systematic review and NMA of RCTs and non-randomized studies (NRSs) comparing the safety (i.e. fracture, cerebrovascular event, fall, or mortality risk) of pharmacological interventions used to treat neuropsychiatric symptoms in PLWD, and a retrospective cohort study comparing the risk of major osteoporotic fractures, falls, and death associated with trazodone or atypical antipsychotic use in PLWD. Results: Non-pharmacological interventions were as, or more, efficacious than pharmacological interventions for treating aggression and agitation in PLWD. No pharmacological interventions were associated with increased odds of fracture among PLWD. Compared to placebo, cholinesterase inhibitors were associated with decreased odds of death (odds ratio (OR) 0.82, 95% credible interval (CrI) 0.67 to 1), antipsychotics were associated with increased odds of cerebrovascular events (OR 2.19, 95% CrI 1.3 to 3.79), and dextromethorphan-quinidine was associated with increased odds of falls (OR 4.16, 95% CrI 1.47 to 14.22) in NMAs of RCT data. These associations were not seen in NRS data. Compared to atypical antipsychotics, trazodone was associated with a similar rate of falls and major osteoporotic fractures (weighted hazard ratio (HR) 0.89, 95% confidence interval (CI) 0.73 to 1.07); however, it was associated with a lower rate of mortality (weighted HR 0.75, 95% CI 0.66 to 0.85) in PLWD. Conclusion: Non-pharmacological rather than pharmacological interventions should be used for treating aggression and agitation in PLWD. Comparable rates of falls and fractures associated with trazodone or atypical antipsychotic use suggests a shift in prescribing to trazodone may not be entirely approproriate. Data comparing the safety of antipsychotics to other interventions was sparse. More studies comparing antipsychotics to alternative interventions are needed.

Keywords: cohort study
dementia
interventions
network meta-analysis
neuropsychiatric symptoms
systematic review
Advisor: Straus, Sharon
Strategies for Interfacing Digital Microfluidics with Nuclear Magnetic Resonance Spectroscopy

Digital microfluidics (DMF) is a fluid handling paradigm wherein minute droplets (with volumes in the range of 100 nL - 1 mL) are actuated through electrostatic forces over an electrode array. The discrete nature of DMF makes it a perfect fit for delivery of samples and other reagents to microcoil sensors used for high field Nuclear Magnetic Resonance (NMR) spectroscopy. This thesis describes the outcome of several years of work towards the development of a DMF-NMR interface. Chapter 2 considers a one-plate DMF design with a 1 mm outer diameter (OD) planar microcoil and demonstrates how the system can be used to manipulate, mix, and sense droplets within the NMR spectrometer. Chapter 3 is motivated by the need to ensure reliable droplet movement in an NMR spectrometer (where there is no visualization of droplet movement). This section evaluates how to determine best practices for droplet manipulation on the basis of the relationship between applied force and droplet velocity. Chapter 4 describes a two-plate DMF device configuration, enabled by altering the microcoil substrate to include a copper grounding plate to serve as the DMF counter-electrode. The increased droplet control of this design allows for better spectral resolution and better time resolution when studying reacting droplets. Finally, chapter 5 proposes a method to overcome the space limitations within the NMR spectrometer that limit the DMF device size by mindfully engineering the electrodes to allow for continuous forward motion using only two electrical channels. I propose that these innovations establish DMF as a useful new tool sample handling in conjunction with micro-NMR detectors like the planar microcoils described here, with potential extension to microslots, microstriplines, and other geometries in the future.

Keywords: Digital Microfluidics
Force Velocity
Nuclear Magnetic Resonance
Advisor: Wheeler, Aaron R
Patient Readmissions After Hospital Discharge

Many readmissions after hospital discharge may be preventable through improved transitional care. This thesis seeks to inform clinical practice and policy development to reduce avoidable readmissions. The three included projects use health administrative data to examine the post-discharge care processes and outcomes for patients hospitalized in Ontario, Canada. In the first study, we compared the outcomes for patients discharged during the extended December holiday to outcomes for patients discharged from hospital at other times, over a 14 year period. We found that December holiday-discharged patients were at greater risk of 30-day death or readmission, while also being less likely to have outpatient physician follow-up within 14 days of discharge. The second study evaluated the effects of a physician financial incentive (an additional billing code) on timely follow-up after discharge. Despite physician uptake of the incentive code, there was no change in 14-day follow-up rates after incentive introduction, suggesting that it was not effective in changing physician behavior. In our third study, we compared the outcomes of post-discharge patients receiving a community pharmacy-based medication review to those not receiving one. Among older adults filling a prescription in a community pharmacy, receipt of a medication review was associated with a reduced rate of 30-day death or readmission. These thesis findings provide evidence to support decision- and policymaking relating to the clinical care of patients transitioning home from hospital.

Keywords: Health services research
Pharmacy services
Primary care
Readmissions
Transitional care
Advisor: Bell, Chaim M
Dynamic Cross-Talk between Macrophages and Contractile Fibroblasts in Fibrosis

Accumulation and contraction of collagen extracellular matrix (ECM) by myofibroblasts (MFs) is a hallmark of fibrosis that can culminate in loss of organ function. In contrast to the sequential appearance and disappearance of macrophages (Mϕ) and MFs in normal tissue repair, Mϕ-MF pairs co-exist in excessive healing. Spatial proximity of Mϕ supports persistent activation of MFs. The Hinz lab has shown that Mϕ-MF contacts are mediated by strong homotypic cadherin 11 (Cad11) junctions. However, the initial guidance cues and mechanisms directing migratory Mϕ towards contracting MFs are not clear. We hypothesize that local remodelling of collagen ECM by contracting MFs provides mechanical and physical cues in the ECM that act as attraction signals for distant migratory Mϕ. To test this hypothesis, we used collagen type I ECM to investigate Mϕ adhesion and migratory mechanism in response to MF-induced deformation fields in in vitro co-cultures. My data show that MF-generated deformations in collagen ECM extended far beyond the cell diameter. When positioned within deformation fields, Mϕ sense and migrate towards mechanical forces exerted by contracting MFs and actuated microneedles. Mϕ chemotaxis is excluded as attraction mechanism by eliminating possible cytokines using fluid flow. Microneedle experiments identified the presence of a dynamic force source as the critical signal initiating and directing Mϕ migration in the ECM. In contrast, collagen fiber alignment resulting from MF remodelling activities is neither required nor sufficient to guide Mϕ migration. We show that Mϕ perceive changes in the displacement of their local fibrillar collagen substrate. The ability of Mϕ to follow actuated microneedles is impaired in the presence of α2β1 integrin antibodies and stretch-activated membrane channel blockers, suggesting the functional requirement of them in Mϕ force navigation in the ECM. We propose a novel mechanism of far-ranged Mϕ mechanosensing that integrates mechanical changes in the ECM to facilitate MF-Mϕ attraction and forming a pro-fibrotic niche. Our study identified stretch-activated ion membrane channels and fibrillar collagen receptors required for Mϕ mechanosensation as potential molecular targets for anti-fibrotic strategies.

Keywords: Biomechanics
collagen
intracellular communication
macrophages
myofibroblast
wound repair
Advisor: Hinz, Boris
The Design of Optogenetic Constructs for the Regulation of Translation Initiation

The precise spatiotemporal regulation of protein synthesis (translation) is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods for studying protein synthesis offer a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. This thesis presents several novel optogenetic constructs designed to control protein synthesis via well-known binding interactions of the translation initiation machinery. Chapter 2 presents a yeast system capable of screening optogenetic designs for the light-dependent down-regulation of protein synthesis. Using this system, we discovered cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of the LOV2 domain from Avena sativa (AsLOV2) and 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. This construct was improved, producing cLIPS2. We show that these constructs can inhibit translation in yeast harboring human eIF4E and bind human eIF4E in vitro in a light-dependent manner. Chapter 3 presents the characterization of LITRs (LOV2 insertion in Translational Regulators), a group of constructs where AsLOV2 is inserted into flexible loops of eIF4E (eukaryotic initiation factor 4E). With a different yeast-based screen, we discovered LITUP1, a light-dependent up-regulator of protein synthesis. We showed that the linker sequences between AsLOV2 and eIF4E were coupled to the activity of LITUP1. This led to the discovery of LITUP2, an improved version, and LITDOWN1 a light-dependent down-regulator of protein synthesis. Chapter 4 presents two photocaging strategies using Dronpa and RsLOV. These were initial attempts at designing optogenetic 4EBP2 and eIF4E constructs, which encountered difficulties with in vitro characterization. Altogether, the constructs presented in this work expand the current optogenetic toolbox and will hopefully allow researchers to dissect the dynamic molecular events underlying translation.

Keywords: AsLOV2
eIF4E
optogenetics
protein engineering
protein synthesis
translation
Advisor: Woolley, Andrew
Exploring Maternal Birthplace and Child Gender Disparities in Markers of Neglect and Maltreatment among Young Children of Immigrants

Significant immigration to Canada has brought many cultures and parenting practices together, highlighting differences in child health and wellbeing. Given the increasingly large amount of migration from countries with high gender inequality, it is unknown how parental immigration interacts with child gender to affect healthcare and wellbeing in early childhood. In this dissertation, I present four studies regarding variation in routine preventive health care and maltreatment in very early childhood by maternal birthplace and child gender. The first study is a scoping review where I mapped the use of gender-based analysis in research on the health of children in immigrant families. I found that child gender is an understudied aspect of immigrant children’s health, thereby presenting an opportunity for further research. Next, in three population-based retrospective cohort studies, I compared the risk of three markers of child health care and well-being across immigrant maternal birthplaces in comparison to mothers born in Canada—immunizations, well-child visits at 24 months, and early child maltreatment at five years of age. To explore whether son preference affects child routine preventive care and maltreatment, I also compared outcomes between daughters and sons within families. I demonstrate that children of immigrants are well cared for concerning routine immunizations and are less likely to experience maltreatment in early childhood than children of non-immigrants. Maternal birthplaces associated with high levels of gender inequity do not seem to place daughters at risk of adverse outcomes compared to sons, except for a select case. Results support addressing vaccine hesitancy and child maltreatment in the general population to promote well-being in early childhood, as well as select targeted approaches among specific immigrant groups. My studies provide a model (including data sources, study design, and analytic techniques) to monitor and detect gender inequality in the general population as well as among minority groups. My research adds to the evidence around gender equity, which will hopefully ensure girls continue to achieve the same level of health care and well-being as boys.

Advisor: Urquia, Marcelo L