The role of skeletal muscle PLIN proteins at rest and following lipolytic stimulation

This thesis investigated the subcellular location of skeletal muscle PLIN proteins (PLIN2, PLIN3, and PLIN5) as well as protein interactions with ATGL and HSL at rest and following lipolytic stimulation. In addition, the serine phosphorylation state of PLIN2, PLIN3, and PLIN5 was determined at rest and following lipolytic stimulation. An isolated whole muscle technique was used to study the effects of contraction and epinephrine-induced lipolysis. This method allowed for the examination of the effects of contraction and epinephrine alone and in combination. Further, the soleus was chosen for investigating the role of PLIN proteins in skeletal muscle lipolysis due to its suitability for isolated incubation, and the fact that it is primarily oxidative in nature (~80% type I fibres). It has also been previously shown to have the greatest reliance on lipid metabolism and for this reason is ideal for investigating the role of PLIN proteins in lipolysis. Immunofluorescence microscopy revealed that skeletal muscle lipid droplets are partially co-localized to both PLIN2 and PLIN5 and that contraction does not affect the amount of colocalization, indicating that PLIN5 is not recruited to lipid droplets with contraction (PLIN2 ~65%; PLIN5 ~56%). Results from the immunoprecipitation studies revealed that with lipolysis in skeletal muscle the interaction between ATGL and CGI-58 is increased (study 2: 128% with contraction, p<0.05; study 3: 50% with contraction, 25% epinephrine, 80% contraction + epinephrine, p>0.05). Further PLIN2, PLIN3, and PLIN5 all interact with ATGL and HSL, while only PLIN3 and PLIN5 interact with CGI-58. Among these interactions, the association between PLIN2 and ATGL decreases with lipolytic stimulation (study 2: 21% with contraction, p<0.05). Finally our results demonstrate that PLIN3 and PLIN5 are serine phosphorylated at rest and that the level of phosphorylation remains unchanged in the face of either contractile or adrenergic stimulation. In summary, the regulation of skeletal muscle lipolysis is a complex process involving multiple proteins and enzymes. The skeletal muscle PLIN proteins likely play a role in skeletal muscle lipid droplet dynamics, and the data from this thesis indicate that these proteins may work together in regulating lipolysis by interaction with both ATGL and HSL.

Using Effective Teaching Strategies and Personality Type to Enhance the Mathematics Classroom: A Handbook for Intermediate Math Teachers

This project addressed the need for more insightful, current, and applicable resources for intermediate math teachers in Canadian classrooms. A need for a handbook in this division seemed warranted by a lack of government resource support. Throughout an extensive review of the literature, themes and topics for the handbook emerged. The handbook was designed to not only provide educators with examples of effective teaching strategies within the mathematics classroom but to also inform them about the ways in which their personal characteristics and personality type could affect their students and their own pedagogical practices. Three teaching professionals who had each taught in an intermediate math class within the past year evaluated the handbook. The feedback received from these educators was directly applied to the first draft of the handbook in order to make it more accessible and applicable to other math teachers. Although the handbook was written with teachers in mind, the language and format used throughout the manual also make it accessible to parents, tutors, preservice education students, and educational administrators. Essentially, any individual who is hoping to inspire and educate intermediate math students could make use of the content within the handbook.

Sport Values of Bantam, Midget and Intermediate Female Hockey Players and Their Minor Hockey Associations

The sport values of female hockey players and their minor hockey associations were explored to better inform a values-based approach for adult-managed minor hockey. Data were collected from 294 participatory HL and competitive Rep players (12-22 years of age) using the Youth Sport Values Questionnaire-2 and from 30 hockey association board members using a modified YSVQ-2. Results indicated player importance (VI) ratings for Moral (M = 4.08) and Competence (M = 4.15) values were not significantly different but were significantly higher than Status (M = 2.11) value. Significant weak relationships between age and competition level versus VI ratings were found. There were medium/ high Moral, medium/ low Competence and high/ high Status value congruence between Rep and HL Player-Board Members, respectively. Based upon the findings, girls’ minor hockey associations need to recognize the values female youth players prioritize, and ensure each is considered within a values-based decision-making approach to governance.

Working Memory in the Junior/Intermediate Classroom

This research project explored the connection between working memory and children’s learning. The project created a resource titled Working Memory Strategies for the Junior/ Intermediate Educator: A Handbook based on a literature review, the deconstruction of theoretical and empirical studies, teacher resources, and findings from a needs assessment completed by teachers that together show there is insufficient support for teachers working with students who have deficits in working memory along with other common classroom learning disabilities. As learning disabilities become more common in the classroom that increasingly affect working memory in a majority of cases, teachers must be prepared not only to address specific symptoms of the conditions, but also to help students learn how to navigate and become aware of their working memory ability. The handbook thus was developed as a useful resource for teachers looking to expand their knowledge about how learning occurs. A needs assessment completed by junior and intermediate division teachers in Ontario helped determine what educators found most important for inclusion in the handbook, and the same teachers were offered the opportunity to review the completed handbook. Teacher participants provided constructive feedback and indicated that the handbook would be a valuable resource for them and their colleagues when working with students who have working memory issues. It was suggested that the handbook would be useful when creating students’ Individual Education Plans and that the assessment checklist included in the handbook would be an excellent resource for teachers collecting data regarding students’ working memory and ability to learn.

The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)

Human Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITPα and PITPβ. PITPs are believed to be lipid transfer proteins based on their capacity to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named retinal degeneration B (RdgBα). The PITP domain of RdgBα shares 40 % sequence identity with PITPα and has been shown to possess PI and PC binding and transfer activity. The detailed molecular mechanism of ligand transfer by the human PITPs and the Drosophila PITP domain remains to be fully established. Here, we investigated the membrane interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in comparison to PITPα as determined by the FRET assay. Interestingly, DPI analysis also demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound PITPs. In addition, the FRET studies demonstrated the significance of membrane curvature in the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA) which have smaller head groups, the transfer rate increased. In contrast, when the accepting vesicles contained phosphoinositides which have larger head groups, the transfer rate was diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not appear to influence the ligand transfer rate of PITPs. Both DPI and FRET examinations revealed that the PITP domain of RdgBα was able to bind to membranes. However, the RdgBα PITP domain appears to be a poor binder and transporter of PC.