Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.

<div>As the soft tissue that transmits muscular forces to the bony skeleton, tendons play a key role in the human musculoskeletal system and must adapt over time to repeated mechanical loads to maintain functionality. Resistance exercise is one of the primary stimuli for increases in tendon size and strength in healthy, young individuals, but similar benefits are not observed in healthy, aged tendon. This failure in the elderly to adapt, along with the fact that tendons inevitably decline in morphology and function with age, puts older individuals at an increased risk of poor tendon health, subsequent injury, and a compromised quality of life. Alternative strategies to preserve and strengthen aged tendon has gone largely unexplored, highlighting a critical need to determine an effective stimulus for tendon adaptations in aging populations</div><div>The purpose of this study was to determine if age impacts the delivery of orally-consumed amino acids (AA) to the peritendinous Achilles space. If so, this investigation could serve as the foundation for future studies to evaluate the efficacy of supplemental amino acids for inducing positive adaptations in tendon during exercise. Furthermore, an enzyme-linked immunosorbent assay (ELISA) was performed to quantitively measure procollagen, a precursor of collagen, in the samples to evaluate the impact on supplemental amino acids on collagen synthesis. </div><div>To assess amino acid delivery, a microdialysis fiber was inserted into the peritendinous space anterior to the Achilles tendon in healthy young (n = 7, 21-30 years) and elderly (n = 6, 60-75 years) men and women after a twelve-hour fast. After baseline collection, subjects consumed a non-caloric, noncaffeinated AA beverage (16.65 g). Microdialysis samples were collected every fifteen minutes for four hours and analyzed using reverse-phase high-performance liquid chromatography. </div><div>Amino acid delivery to the peritendinous space was not compromised with age, and the administration of amino acids upregulated procollagen synthesis significantly more in healthy, elderly subjects than in those that are healthy and young. Though preliminary, these findings provide a strong foundation for future studies assessing the impact of amino acid supplementation as novel impetus for tendon adaptations in the elderly. </div><div><br></div>

This study aims to examine participation in the digital campaign #BlackoutTuesday to gain an understanding of the campaign's impact on university students’ awareness and participation in Black Lives Matter. Based on this, the study has two research questions: How have the students experienced that the #BlackoutTuesday campaign has affected their awareness of Black lives matter, and, for what reasons did the students feel that they participated in #BlackoutTuesday? With the help of slacktivism, networked publics, collective and connective action as the study's theoretical framework, we hope to gain a good interpretation of the results. To gather data, the study has used semi-structured interviews and to interpret the data, a thematic analysis has been used. The results that the study came to were that the students did not experience that their awareness was effected by #BlackoutTuesday. However, they still believed that they made a difference and that other people in their surrounding were affected in regard to awareness about Black Lives Matter. An unexpected discovery was also that many of the students connected Black Lives Matters, as an movement, solely to USA and not Sweden. The reasons for participation varied, but in this study, three main reasons were noted for the students participation. The reasons for participating were about the collective power, dissemination of information and taking a stand. With the results of the study we hope to create a better understanding of participation in similar campaign's and its effect on movements possibly society. However, more research is needed in this area to get a more thoroughly view of the field.

In the beginning of 2021, the stock of strained and heavily shorted company GameStop rose by almost 2,000 percent. The meteoric rise was caused after users of the subreddit r/wallstreetbets mobilized to buy up stock of the business, hoping that short- sellers would be forced to close their positions, leading to further upside. The purpose of this study is to describe the culture of the subreddit, using Stuart Hall’s (1997) theory of representation to explain how users create meaning. Furthermore, we attempt to explain how the digital nature of the movement affected its structure and organisation, for which Bennett and Segerbergs (2012) framework The logic of connective action is applied. The study used a netnographic approach for data-collection, which was later analyzed using qualitative text- analysis and semiotic-image- analysis. In total, 50 different posts were analyzed which included both texts and memes. Our results indicate that the movement is characterized by the lack of hierarchy and a substantial amount of user- generated content. Narratives and motives have been naturally constructed collectively through the interaction of users rather than enforced by a central authority. A unique feature of the movement is its exclusively digital presence, allowing for fluid participation and individualized framing. The premises and boundaries of activism change as activism becomes more dependent on the digital world. This study offers insight into how digital movements can be constructed and how the digital fundamentally changes the preconditions of activism and mobilization.

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.

Data from the McMaster Occupational Biomechanics Laboratory were consolidated to evaluate overall trends relating to tissue motion and blood flow in the carpal tunnel. Regarding tissue motion, displacements of the flexor digitorum superficialis (FDS) tendon and its subsynovial connective tissue (SSCT) were found to decrease with greater movement speed and a flexed wrist posture. Notably, changes to shear outcomes including relative tendon-SSCT displacement, the shear strain index (SSI), and maximum velocity ratio (MVR) demonstrate that greater movement speed contributes to SSCT damage according to the shear strain mechanism of injury theorised to promote carpal tunnel syndrome (CTS). Median nerve blood flow was also found to be implicated by wrist flexion, and appeared to decrease with greater CTS severity status. Finally, induced blood flow alteration of the carpal tunnel was found to elicit a median nerve blood flow response similar to the level found in CTS subjects, confirming its effectiveness as an intervention to study tissue motion in a CTS-like state. The influence of altered blood flow on tissue motion was differential, where the higher supradiastolic condition altered FDS displacement, and the lower subdiastolic condition affected SSCT displacement and SSI. These findings provide valuable evidence for changes in median nerve blood flow—and by extension, the local fluid environment within the carpal tunnel—not only being a consequence of SSCT fibrosis characteristic of CTS, but potentially also acting as a cause for said changes in carpal tunnel tissue motion. / Thesis / Master of Science in Kinesiology / This thesis aimed to evaluate and summarize key findings from the McMaster Occupational Biomechanics Laboratory relating to tissue motion and blood flow in the carpal tunnel. Performing repetitive finger movements faster and with a flexed wrist posture were found to decrease the distance travelled of the underlying finger tendon. Blood flow of the median nerve, which is implicated in carpal tunnel syndrome (CTS), is higher with forceful exertion and flexed wrist posture, and lower with greater severity of CTS. Finally, altering blood flow to the carpal tunnel was found to create a CTS-like environment, affected tissue motion in the carpal tunnel, and promoted movement disparity between these tissues that is associated with injury. This suggests that fluid/blood flow changes affecting the carpal tunnel is a plausible mechanism for increasing the likelihood of developing CTS.

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Carpal tunnel syndrome is characterized by non-inflammatory fibrosis of the subsynovial connective tissue next to the tendons in the carpal tunnel, suggesting a shear injury owing to repetitive wrist and finger motion at work. I tested the effects of several well-established biomechanical predictors of injury on tendon and subsynovial connective tissue motion and shear in the carpal tunnel. These included non-neutral finger and wrist posture, speed of work, and forceful exertion. A cadaveric paradigm was used to directly measure tendon gliding characteristics, which showed that concurrent exposure to multiple biomechanical risk factors disproportionately increased tendon frictional work (Chapter 2). Given that tendon shear cannot be directly measured in vivo, colour flow ultrasound was used to assess relative motion between tendon and subsynovial connective tissue as a metric of shear potential (Chapter 3 − 5). Healthy participants completed middle finger movements while colour flow ultrasound imaged carpal tunnel structures and optical motion capture recorded finger joint kinematics. From the data, I developed regression equations to predict both tendon and subsynovial connective tissue displacements as a function of finger joint angles, which can be used as an ergonomic method to calculate the relative displacement (Chapter 3). Furthermore, relative motion between tendon and subsynovial connective tissue increased with wrist flexion angle, suggesting a greater susceptibility to shear injury during repetitive work when the wrist is flexed (Chapter 4). Using colour flow imaging, electrogoniometry, and fine-wire EMG, relative displacement was found to increase with tendon velocity and force (Chapter 5). Relative displacements in Chapters 3 to 5 were combined into a prediction model, and further compared to a tendon friction model derived from Chapter 2. The relative displacement model showed an additive relationship with combined physical exposures, including finger and wrist position, tendon velocity, and force (Chapter 6). The relative displacement model was more responsive to lower physical exposures whereas the friction model produced greater overall changes (with higher exposures). While both models infer a greater risk of shear injury due to repetitive and forceful wrist/finger movement, future studies will aim to set protective guidelines based on tendon motion and shear during hand-intensive work. Overall, this thesis showed that tendon friction and relative motion between tendon and subsynovial connective tissue both increased in response to well-established biomechanical risk factors. We propose the current models for use in ergonomics, representing a move towards mechanistic-based injury risk assessment of the wrist and hand. / Thesis / Doctor of Philosophy (PhD)

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