CRISPR/Cas9-Mediated Gene Editing of ARG1 in Cell Lines and Primary Cells

Arginase 1 deficiency, a urea cycle disorder resulting from an inability of the body to convert arginine into urea, results in hyperargininemia and sporadic episodes of hyperammonemia. Arginase 1 deficiency can lead to a range of developmental disorders and progressive spastic diplegia in children, and current therapeutic options are limited. Clustered regularly interspaced short palindromic repeat (CRISPR) /CRISPR associated protein (Cas) 9 gene editing systems serve as a novel means of treating genetic disorders such as Arginase 1 (ARG1) deficiency, and must be thoroughly examined to determine their curative capabilities. In these experiments numerous guide RNAs and CRISPR/Cas9 systems targeting the ARG1 gene were designed and observed by heteroduplex assay for their targeting capabilities and cleavage efficiencies in multiple cell lines. The CRISPR/Cas9 system utilized in these experiments, along with a panel of guide RNAs targeting various locations in the arginase 1 gene, successfully produced targeted cleavage in HEK293, MCF7, A549, K562, HeLa, and HepG2 cells; however, targeted cleavage in human dermal fibroblasts, blood outgrowth endothelial cells, and induced pluripotent stem cells was not observed. Additionally, a CRISPR/Cas system involving partially inactivated Cas9 was capable of producing targeted DNA cleavage in intron 1 of ARG1, while a Cas protein termed Cpf1 was incapable of producing targeted cleavage. These results indicate a complex set of variables determining the CRISPR/Cas9 systems’ capabilities in the cell lines and primary cells tested. By examining epigenetic factors and alternative CRISPR/Cas9 gene targeting systems, the CRISPR/Cas9 system can be more thoroughly considered in its ability to act as a means towards editing the genome of arginase 1-deficient individuals.

Petri Net Siphon Analysis and Network Centrality Measures for Identifying Combination Therapies in Signaling Pathways

Aberrant behavior of biological signaling pathways has been implicated in diseases such as cancers. Therapies have been developed to target proteins in these networks in the hope of curing the illness or bringing about remission. However, identifying targets for drug inhibition that exhibit good therapeutic index has proven to be challenging since signaling pathways have a large number of components and many interconnections such as feedback, crosstalk, and divergence. Unfortunately, some characteristics of these pathways such as redundancy, feedback, and drug resistance reduce the efficacy of single drug target therapy and necessitate the employment of more than one drug to target multiple nodes in the system. However, choosing multiple targets with high therapeutic index poses more challenges since the combinatorial search space could be huge. To cope with the complexity of these systems, computational tools such as ordinary differential equations have been used to successfully model some of these pathways. Regrettably, for building these models, experimentally-measured initial concentrations of the components and rates of reactions are needed which are difficult to obtain, and in very large networks, they may not be available at the moment. Fortunately, there exist other modeling tools, though not as powerful as ordinary differential equations, which do not need the rates and initial conditions to model signaling pathways. Petri net and graph theory are among these tools. In this thesis, we introduce a methodology based on Petri net siphon analysis and graph network centrality measures for identifying prospective targets for single and multiple drug therapies. In this methodology, first, potential targets are identified in the Petri net model of a signaling pathway using siphon analysis. Then, the graph-theoretic centrality measures are employed to prioritize the candidate targets. Also, an algorithm is developed to check whether the candidate targets are able to disable the intended outputs in the graph model of the system or not. We implement structural and dynamical models of ErbB1-Ras-MAPK pathways and use them to assess and evaluate this methodology. The identified drug-targets, single and multiple, correspond to clinically relevant drugs. Overall, the results suggest that this methodology, using siphons and centrality measures, shows promise in identifying and ranking drugs. Since this methodology only uses the structural information of the signaling pathways and does not need initial conditions and dynamical rates, it can be utilized in larger networks.

Subfornical organ neurons integrate cardiovascular and metabolic signals

The subfornical organ (SFO) is a critical circumventricular organ involved in the control of cardiovascular and metabolic homeostasis. Despite the abundant literature clearly demonstrating the ability of SFO neurons to sense and respond to a plethora of circulating signals that influence various physiological systems, investigation of how simultaneously sensed signals interact and are integrated in the SFO is lacking. In this study, we use patch clamp techniques to investigate how the traditionally classified ‘cardiovascular’ hormone angiotensin II (ANG), ‘metabolic’ hormone cholecystokinin (CCK) and ‘metabolic’ signal glucose interact and are integrated in the SFO. Sequential bath-application of CCK (10nM) and ANG (10nM) onto dissociated SFO neurons revealed that: 63% of responsive SFO neurons depolarized to both CCK & ANG; 25% depolarized to ANG only; and 12% hyperpolarized to CCK only. We next investigated the effects of glucose by incubating and recording neurons in either hypo-, normo- or hyperglycemic conditions for a minimum of 24 hours and comparing the proportions of responses to ANG (n=55) or CCK (n=83) application in each condition. A hyperglycemic environment was associated with a larger proportion of depolarizing responses to ANG (X2, p<0.05), and a smaller proportion of depolarizing responses along with a larger proportion of hyperpolarizing responses to CCK (X2, p<0.01). These data demonstrate that SFO neurons excited by CCK are also excited by ANG, suggesting that CCK may influence fluid intake or blood pressure via the SFO, complementary to the well-understood actions of ANG at this site. Additionally, the demonstration that glucose environment affects the responsiveness of neurons to both these hormones highlights the ability of SFO neurons to integrate multiple metabolic and cardiovascular signals to affect transmission of information from the circulation to the brain, which has important implications for this structure’s critical role regulation of autonomic function.

Supporting Run-time Monitoring of UML-RT through Customizable Monitoring Configurations in PapyrusRT

Model Driven Engineering uses the principle that code can automatically be generated from software models which would potentially save time and cost of development. By this methodology, a systems structure and behaviour can be expressed in more abstract, high level terms without some of the accidental complexity that the use of a general purpose language can bring. Models are the actual implementation of the system unlike in traditional software development where models are often used for documentation purposes only. However once the code is generated from the model, testing and debugging activities tend to happen on the code level and the model is not updated. We believe that monitoring on the model level could potentially facilitate quality assurance activities as the errors are detected in the early phase of development. In this thesis, we create a Monitoring Configuration for an open source model driven engineering tool called PapyrusRT in Eclipse. We support the run-time monitoring of UML-RT elements with a tracing tool called LTTng. We annotate the model with monitoring information to be used by the code generator for adding tracepoint statements for the corresponding elements. We provide the option of a timing specification to discover latency errors on the model. We validate the results by creating and tracing real time models in PapyrusRT.

IMPROVING THE PERFORMANCE OF DATABASE-CENTRIC APPLICATIONS THROUGH PROGRAM ANALYSIS

Modern software applications are becoming more dependent on database management systems (DBMSs). DBMSs are usually used as black boxes by software developers. For example, Object-Relational Mapping (ORM) is one of the most popular database abstraction approaches that developers use nowadays. Using ORM, objects in Object-Oriented languages are mapped to records in the database, and object manipulations are automatically translated to SQL queries. As a result of such conceptual abstraction, developers do not need deep knowledge of databases; however, all too often this abstraction leads to inefficient and incorrect database access code. Thus, this thesis proposes a series of approaches to improve the performance of database-centric software applications that are implemented using ORM. Our approaches focus on troubleshooting and detecting inefficient (i.e., performance problems) database accesses in the source code, and we rank the detected problems based on their severity. We first conduct an empirical study on the maintenance of ORM code in both open source and industrial applications. We find that ORM performance-related configurations are rarely tuned in practice, and there is a need for tools that can help improve/tune the performance of ORM-based applications. Thus, we propose approaches along two dimensions to help developers improve the performance of ORM-based applications: 1) helping developers write more performant ORM code; and 2) helping developers configure ORM configurations. To provide tooling support to developers, we first propose static analysis approaches to detect performance anti-patterns in the source code. We automatically rank the detected anti-pattern instances according to their performance impacts. Our study finds that by resolving the detected anti-patterns, the application performance can be improved by 34% on average. We then discuss our experience and lessons learned when integrating our anti-pattern detection tool into industrial practice. We hope our experience can help improve the industrial adoption of future research tools. However, as static analysis approaches are prone to false positives and lack runtime information, we also propose dynamic analysis approaches to further help developers improve the performance of their database access code. We propose automated approaches to detect redundant data access anti-patterns in the database access code, and our study finds that resolving such redundant data access anti-patterns can improve application performance by an average of 17%. Finally, we propose an automated approach to tune performance-related ORM configurations using both static and dynamic analysis. Our study shows that our approach can help improve application throughput by 27--138%. Through our case studies on real-world applications, we show that all of our proposed approaches can provide valuable support to developers and help improve application performance significantly.

Study of Liquid Desiccant Air-Conditioning for High Performance Greenhouses

Greenhouses have become an invaluable source of year-round food production. Further development of viable and efficient high performance greenhouses is important for future food security. Closing the greenhouse envelope from the environment can provide benefits in space heating energy savings, pest control, and CO2 enrichment. This requires the application of a novel air conditioning system to handle the high cooling loads experienced by a greenhouse. Liquid desiccant air-conditioning (LDAC) have been found to provide high latent cooling capacities, which is perfect for the application of a humid greenhouse microclimate. TRNSYS simulations were undertaken to study the feasibility of two liquid desiccant dehumidification systems based on their capacity to control the greenhouse microclimate, and their cooling performance. The base model (B-LDAC) included a natural gas boiler, and two cooling systems for seasonal operation. The second model (HP-LDAC) was a hybrid liquid desiccant-heat pump dehumidification system. The average tCOPdehum and tCOPtotal of the B-LDAC system increased from 0.40 and 0.56 in January to 0.94 and 1.09 in June. Increased load and performance during a sample summer day improved these values to 3.5 and 3.0, respectively. The average eCOPdehum and eCOPtotal values were 1.0 and 1.8 in winter, and 1.7 and 2.1 in summer. The HP-LDAC system produced similar daily performance trends where the annual average eCOPdehum and eCOPtotal values were 1.3 and 1.2, but the sample day saw peaks of 2.4 and 3.2, respectively. The B-LDAC and HP-LDAC results predicted greenhouse temperatures exceeding 30°C for 34% and 17% of the month of July, respectively. Similarly, humidity levels increased in summer months, with a maximum of 14% of the time spent over 80% in May for both models. The percentage of annual savings in space heating energy associated with closing the greenhouse to ventilation was 34%. The additional annual regeneration energy input was reduced by 26% to 526 kWhm-2, with the implementation of a heat recovery ventilator on the regeneration exhaust air. The models also predicted an electrical energy input of 245 kWhm-2 and 305 kWhm-2 for the B-LDAC and HP-LDAC simulations, respectively.

ENVIRONMENTAL INFLUENCES AND EPIGENETIC MECHANISMS IN RISK FOR DEPRESSION

Genetic and environmental factors interact to influence vulnerability for internalizing psychopathology, including Major Depressive Disorder (MDD). The mechanisms that account for how environmental stress can alter biological systems are not yet well understood yet are critical to develop more accurate models of vulnerability and targeted interventions. Epigenetic influences, and more specifically, DNA methylation, may provide a mechanism by which stress could program gene expression, thereby altering key systems implicated in depression, such as frontal-limbic circuitry and its critical role in emotion regulation. This thesis investigated the role of environmental factors from infancy and throughout the lifespan affecting the serotonergic (5-HT) system in the vulnerability to and treatment of depression and anxiety and potential underlying DNA methylation processes. First, we investigated the contributions of additive genetic vs. environmental factors on an early trait phenotype for depression (negative emotionality) in infants and their stability over time in the first 2 years of life. We provided evidence of the substantial contributions of both genetic and shared environmental factors to this trait, as well as genetically- and environmentally- mediated stability and innovation. Second, we studied how childhood environmental stress is associated with peripheral DNA methylation of the serotonin transporter gene, SLC6A4, as well as long-term trajectories of internalizing behaviours. There was a relationship between childhood psychosocial adversity and SLC6A4 methylation in males, as well as between SLC6A4 methylation and internalizing trajectory in both sexes. Third, we investigated changes in emotion processing and epigenetic modification of the SLC6A4 gene in depressed adolescents before and after Mindfulness-Based Cognitive Therapy (MBCT). The alterations from pre- to post-treatment in connectivity between the ACC and other network regions and SLC6A4 methylation suggested that MBCT may work to optimize the connectivity of brain networks involved in cognitive control of emotion as well as also normalize the relationship between SLC6A4 methylation and activation patterns in frontal-limbic circuitry. Our results from these three studies strengthen the theory that environmental influences are critical in establishing early vulnerability factors for MDD, driving epigenetic processes, and altering brain processes as an individual undergoes treatment, or experiences relapse.

The application of PCA as a movement pattern recognition technique: A proof of principle

Quantitative methods can help us understand how underlying attributes contribute to movement patterns. Applying principal components analysis (PCA) to whole-body motion data may provide an objective data-driven method to identify unique and statistically important movement patterns. Therefore, the primary purpose of this study was to determine if athletes’ movement patterns can be differentiated based on skill level or sport played using PCA. Motion capture data from 542 athletes performing three sport-screening movements (i.e. bird-dog, drop jump, T-balance) were analyzed. A PCA-based pattern recognition technique was used to analyze the data. Prior to analyzing the effects of skill level or sport on movement patterns, methodological considerations related to motion analysis reference coordinate system were assessed. All analyses were addressed as case-studies. For the first case study, referencing motion data to a global (lab-based) coordinate system compared to a local (segment-based) coordinate system affected the ability to interpret important movement features. Furthermore, for the second case study, where the interpretability of PCs was assessed when data were referenced to a stationary versus a moving segment-based coordinate system, PCs were more interpretable when data were referenced to a stationary coordinate system for both the bird-dog and T-balance task. As a result of the findings from case study 1 and 2, only stationary segment-based coordinate systems were used in cases 3 and 4. During the bird-dog task, elite athletes had significantly lower scores compared to recreational athletes for principal component (PC) 1. For the T-balance movement, elite athletes had significantly lower scores compared to recreational athletes for PC 2. In both analyses the lower scores in elite athletes represented a greater range of motion. Finally, case study 4 reported differences in athletes’ movement patterns who competed in different sports, and significant differences in technique were detected during the bird-dog task. Through these case studies, this thesis highlights the feasibility of applying PCA as a movement pattern recognition technique in athletes. Future research can build on this proof-of-principle work to develop robust quantitative methods to help us better understand how underlying attributes (e.g. height, sex, ability, injury history, training type) contribute to performance.

‘Black Settlers’: Hybridity, Neoliberalism and Ecosystemic Change Among Tonga Farmers of Southern Zambia, 1964-2008

This dissertation examines a process of indigenous accumulation among Tonga farmers in Zambia’s Southern Province. In the 1970s multiple authors concluded that capitalist farmers had emerged among Tonga agro-pastoralists, predominantly within private titled holdings. Relying on archival research, newspapers, secondary sources and extensive oral testimony this thesis fills a 35-year gap on the topic, providing insights into the social and environmental impacts of neoliberal policy among African peasants and capitalist farmers. In contrast to dominant narratives of the post-independence period, this study argues that Zambia did experience a developmental process post-independence, which saw significant achievements made in the agricultural sector, including the doubling of national cattle stocks. The data reveals a painful process of disarticulation beginning in the late 1980s. Following neoliberal adjustment, we observe significant heterogeneity in production systems, some regional specialization, and processes of migration. Most importantly, the thesis uncovers processes of overwhelming ecosystemic change that contributed to livestock epidemics of severe scale and scope. Amazingly, this went largely undocumented because of the simultaneous crisis of the state, which left the national statistics office and other state bodies incapable of functioning from the late 1980s into the 2000s. In response, the Zambian state has introduced a number of neodevelopmental initiatives in the sector, yet the lack of animal traction remained up to 2008 and agricultural production declined, while more capitalized farmers (largely white, and/or with foreign direct investment) have become more significant players in the country. This thesis provides compelling evidence to challenge dominant economic thinking of the Washington institutions as well as many of the common Marxian formulations.

THE DESIGN AND SYNTHESIS OF SILOXANE PRECURSORS FOR CHIRAL PERIODIC MESOPOROUS ORGANOSILICA MATERIALS

The development of cost-effective and reliable methods for the synthesis and separation of asymmetric compounds is paramount in helping to meet society’s ever-growing demand for chiral small molecules. Of these methods, chiral heterogeneous supports are particularly appealing as they allow for the reuse of the chiral source. One such support, based on the synergy between chiral organic units and structurally stable inorganic silicon scaffolds are periodic mesoporous organosilicas (PMOs). In the work described herein, I examine some of the factors governing the transmission of chirality between chiral dopants and prochiral bulk phases in chiral PMO materials. In particular, the exploration of 1,1’-binaphthalene-bridged chiral dopants with a focus on the point of attachment into the materials. Moreover, the effects of ordering in the materials are examined and reveal that chirality transfer is more facile in materials with molecular-scale order then those containing amorphous walls. Secondly, the issues surrounding the synthesis and purification of aryl-triethoxysilanes as siloxane precursors are addressed. Both the introduction of a two-carbon linker and the direct attachment of allyl and mixed allyldiethoxysilane species are explored. This work demonstrates that allyldiethoxysilanes are ideal, in that they are stable enough to permit facile synthesis, while still being able to hydrolyze completely to produce well-ordered materials. Lastly, the production of new bulk phases for chiral PMO materials is examined by introducing new prochiral nitrogen-containing siloxane precursors. Biphenyldiamine and bipyridine-bridged siloxane precursors are readily synthesized on reasonable scales. Their use as the bulk siloxane precursor in the production of PMO materials however, is precluded by insufficient gelation and additional siloxane precursors are necessary for the production of ordered materials. In addition to the research detailed above that forms the body of this thesis, two short works are appended. The first details the production of polythiophene assemblies mediated through coordination nanospaces, while the second explores the production of N-heterocyclic carbene functionalized gold nanoparticles through ligand exchange.

ROCK OUTCROPS IN THE CANADIAN SHIELD: AN INVESTIGATION OF CONTAMINANT TRANSPORT FROM SURFACE SOURCES IN FRACTURED ROCK AQUIFERS

Flow, recharge and transport dynamics in fractured rock aquifers with low lying rock outcrops is a largely unexplored area of study in hydrogeology. The purpose of this thesis is to examine these topics in an agricultural area in Eastern Ontario. The study consists of a regional scale groundwater quality study, an infiltration experiment that considers bacteria transport from the ground surface to a well, and a numerical modelling study that tests the parameters that affect surface infiltration of a tracer from a rock outcrop to a deeper horizontal fracture. In the water quality study, approximately 65% of the samples contained total coliform, 16% contained E. coli, and 1% contained nitrate-N at greater than 5 mg/L. Occurrence of E. coli increased when considering seasonality, where wells were drilled on rock outcrops, and for shallow well intervals. Nitrate-N did not occur above the Guidelines for Canadian Drinking Water Quality (Health Canada, 2012) of 10 mg/L. Rapid arrival times were observed in the infiltration study for both the microspheres (30 minutes) and a dye tracer (45 minutes) in a well approximately 6.0 m in horizontal and 2.8 m in vertical distance from the tracer source. Transport velocities were approximately 38.9 m/day for the dye tracer and 115.2 m/day for the colloidal tracer. Results of the model runs indicate that overburden can provide an effective protective layer to transport in fractures, that high groundwater velocities occur in larger fracture apertures and higher gradients dilute tracer concentrations, and that lower groundwater velocities occur with smaller fracture apertures and lower gradients result in elevated tracer concentrations. Lower rainfall rates, larger fracture apertures, early tracer time, larger gradients, and lower water levels maintained unsaturated conditions for longer time periods such that tracer transport was delayed until saturated conditions were attained. The overall heterogeneity of this aquifer environment creates a source water protection conundrum where the water quality is generally good, while transport can occur very quickly in proximity to rock outcrops and in areas with limited overburden.

Characterization of New Carbosilane Liquid Crystalline Monomers and Dimers

‘De Vries-like’ smectic liquid crystals exhibit low layer contraction of approximately 1% on transitions from the SmA to the SmC phase. These materials have received considerable attention as potential solutions for problems affecting liquid crystal displays using surface-stabilized ferroelectric liquid crystals (SSFLC). In SSFLCs, layer contraction of 710% is normally observed during the SmA to SmC phase transition. A study by the Lemieux group has shown that liquid crystals with nanosegregating carbosilane segments exhibit enhanced ‘de Vries-like’ properties through the formation of smectic layers and by lengthening the nanosegregating carbosilane end-groups from monocarbosilane to tricarbosilane. This observed enhancement is assumed to be due to an increase in the cross-section of the free volume in the hydrocarbon sub-layer. To test this hypothesis, it is assumed that dimers with a tricarbosilane linking group have smaller cross-sections on time average. In his thesis, this hypothesis is tested through the characterization of new liquid crystalline monomers (QL39-n) and dimers (QL40-n) with 2-phenylpyrimidine cores and tricarbosilane end-groups and spacers, respectively. The thesis describes the synthesis of two homologous series of liquid crystals and their characterization using a variety of techniques, including polarized optical microscopy, differential scanning calorimetry and X-ray diffraction. The results show that the monomers QL39-n form a tilted SmC phase only, whereas the dimers QL40-n form an orthogonal SmA phase. These results are discussed in the context of our hypothesis.

RELIGIOUS SCULPTURE OF THE CHILOÉ ARCHIPELAGO, FROM THE 17TH TO THE 21ST CENTURY

Despite its central role in religious life of the region, the sculptural tradition of the Southern Chilean Chiloé Archipelago, ranging from the 17th century to the present day, has been vastly understudied. Isidoro Vázquez de Acuña’s 1994 volume Santeria de Chiloe: ensayo y catastro remains the only catalogue of Chilote sculpture. Though the author includes photographs of a vast array of works, he does not attempt to place the sculptures within a chronology, or consider their place within the greater Latin American context. My thesis will place this group of works within a chronological and geographical context that reaches from the 16th century to the present day, connected to the artistic traditions of regions as far afield as Paraguay and Lima. I will first consider the works brought to the Archipelago by religious orders – the Jesuits and Franciscans – as well as influences on artistic style and religious culture throughout the 17th, 18th and 19th centuries. I will focus in particular on three works generally considered to be from the 17th and 18th centuries – the Virgin of Loreto at Achao, the Saint Michael at Castro, and the Jesus Nazareno of Caguach – using visual analysis and sifting through generations of primary and secondary sources to determine from where and when these sculptures came. With this investigation as a foundation, I will consider how they inspired vernacular sculptural expression and trace ‘family trees’ of vernacular works based on these precedents. Vernacular artistic traditions are often viewed as derivative and lacking in skill, but Chilote sculptors in fact engaged with a variety of outside influences and experimented with different sculptural styles. I will conclude by considering which aspects of these styles Chilote artists chose to incorporate into their own work, alter or exclude, artistic decisions that shed light on the Archipelago’s religious and cultural fabric.

Lost in Translation? Russian Media Portrayals and Laypersons’ Interpretations of Angelina Jolie’s Breast Cancer Discourse

In May 2013, Angelina Jolie revealed that because she had a family history of breast and ovarian cancer and carried a rare BRCA gene mutation, she had undergone a preventive double mastectomy. Media coverage has been extensive around the world, including in Russia, not an English-language country, where all global news is inevitably filtered by translation. After examining the reactions of Russian mass media and members of the public to Jolie’s disclosure, I consider what transformations have occurred with Jolie’s message in the process of cross-cultural transfer. I explore the mass media portrayal of Jolie’s announcement, laypersons’ immediate and prolonged reactions, and the reflections of patients involved directly in the field of hereditary breast cancer. To my knowledge, this multifaceted and bilingual project is the first conceptualization of Jolie’s story as it has been translated in a different sociocultural environment. I start with examination of offline and online publications that appeared in Russia within two months after Jolie’s announcement. In this part of my analysis, I conceptualize the representation of Jolie’s case in Russian mass media and grasp what sociocultural waves were generated by this case among general lay audiences. Another part of my study contains the results of qualitative in-depth interviews. Eight women with a family history of hereditary breast cancer were recruited to participate in the research. The findings represent Jolie’s case through the eyes of Russian women with the same gene mutation as Jolie. Consolidating my findings, I argue that Jolie’s announcement was misinterpreted and misrepresented by Russian mass media, as well as misunderstood by a considerable part of the media audience. Jolie’s perspective on hereditary breast cancer mostly remained unheard among members of the Russian public. I make suggestions about the reasons for such a phenomenon, and demonstrate how Jolie’s case is implicated in politics, economics, and the culture of contemporary Russia.

Investigating the potential magnetic origin of wind variability in OB stars

In this thesis, the origin of large-scale structures in hot star winds, believed to be responsible for the presence of discrete absorption components (DACs) in the absorption troughs of ultraviolet resonance lines, is constrained using both observations and numerical simulations. These structures are understood as arising from bright regions on the stellar surface, although their physical cause remains unknown. First, we use high quality circular spectropolarimetric observations of 13 well-studied OB stars to evaluate the potential role of dipolar magnetic fields in producing DACs. We perform longitudinal field measurements and place limits on the field strength using Bayesian inference, assuming that it is dipolar. No magnetic field was detected within this sample. The derived constraints statistically refute any significant dynamical influence from a magnetic dipole on the wind for all of these stars, ruling out such fields as a cause for DACs. Second, we perform numerical simulations using bright spots constrained by broadband optical photometric observations. We calculate hydrodynamical wind models using three sets of spot sizes and strengths. Co-rotating interaction regions are yielded in each model, and radiative transfer shows that the properties of the variations in the UV resonance lines synthesized from these models are consistent with those found in observed UV spectra, establishing the first consistent link between UV spectroscopic line profile variability and photometric variations and thus supporting the bright spot paradigm (BSP). Finally, we develop and apply a phenomenological model to quantify the measurable effects co-rotating bright spots would have on broadband optical photometry and on the profiles of photopheric lines in optical spectra. This model can be used to evaluate the existence of these spots, and, in the event of their detection, characterize them. Furthermore, a tentative spot evolution model is presented. A preliminary analysis of its output, compared to the observed photometric variations of xi Persei, suggests the possible existence of “active longitudes” on the surface of this star. Future work will expand the range of observational diagnostics that can be interpreted within the BSP, and link phenomenology (bright spots) to physical processes (magnetic spots or non-radial pulsations).