The zebrafish belladonna (bel) mutation was identified in a large-scale mutagenesis screen to identify genes involved in retino-tectal pathfinding in Tubingen, Germany. In bel mutants, after exiting the eye, retinal axons grow ipsilaterally instead of crossing the midline to form optic chiasm. bel mutants are semi-viable and live bel embryos at 5 days show a "dilated pupil" phenotype after which the mutation was named. Later work showed that bel mutants have functional eyes although the optokinetic response is reversed in the mutants. Previous work in our lab showed that most retinal axons in the mutants initially grow towards the midline but later turn ipsilaterally. Also, two major forebrain commissures, the anterior commissure (AC) and the post-optic commissure (POC) also failed to form in bel mutants. These studies showed that bel defects are restricted to forebrain. Detailed analysis of eye sections showed defects in bel eye morphology during embryonic and adult stages. Initial work also mapped the bel locus on chromosome 8 and finer mapping linked one z-marker on either side of bel locus (z24272 and z44909). My dissertation project was to clone the bel gene and understand its role in forebrain patterning and axon guidance. I identified that bel locus encodes a zebrafish lim-homeodomain transcription factor, Lhx2. To further understand how bel(lhx2) might affect axon guidance, I first showed that bel mutants have subtle defects in forebrain patterning in the regions where axons cross the midline. I also showed that these forebrain patterning defects lead to defects in expression of proper cellular and molecular axon guidance cues at the midline in bel mutants. Finally, I showed that bel(lhx2) is required for cell proliferation in the diencephalon. Thus my detailed analysis of bel mutants has revealed new roles for lhx2 in diencephalon patterning and axon guidance.

The ability of established synaptic connections to strengthen and weaken (synapse plasticity) underlies higher order behavior such as learning and memory. Several gaps remain in our understanding of the cellular and molecular changes in pre- and postsynaptic cells associated with synapse plasticity. Using the Drosophila larval neuromuscular junction as a convenient model system throughout my dissertation work, I have concentrated on understanding various molecular mechanisms that underlie this phenomenon. Specifically, I have addressed fundamental issues governing the postsynaptic cell in these processes, such as: (i) what are the crucial proteins required to scaffold the postsynaptic apparatus during plasticity? (ii) what are the molecules that are brought together at this scaffold and how are their levels regulated during plasticity? and finally (iii) what are the signals that initiate the formation of these scaffolds during plasticity? More specifically, I have uncovered a mechanism by which a novel synaptic protein, Guk-holder (Gukh), coordinates the formation of a tripartite complex at the synapse with two proteins containing multiple protein-protein interaction domains: Discs Large (DLG) and Scribble (Scrib) (Mathew et al., 2002). By bringing DLG and Scrib together at the synapse, Gukh likely coordinates the function of these two complexes. As stated above Fasciclin II (FasII) is one of several molecules that is localized to the synapse by Dig (Thomas et al., 1997a), and its levels and symmetric distribution at the synapse have been found to be important for regulating synapse plasticity (Ashley et al., 2005; Schuster et al., 1996b). I showed that FasII also undergoes fast recycling at the synaptic membrane and that this recycling is regulated by the Drosophila homolog of Amphiphysin (Amph)-(Mathew et al., 2003). Such a mechanism would be compatible with a potential activity dependent regulation of FasII levels during plasticity. To understand the initial signaling mechanisms by which synaptic scaffolds are organized during synapse formation, I further investigated the role of the Wingless receptor Dfrizzled2 (DFz2) in this process. Wg signaling has been previously implicated in synapse development and proper localization of postsynaptic proteins (Packard et al., 2002). In these studies I uncovered an unconventional mechanism involved in transducing the Wg signal in the muscle cell. I show that DFz2 is cleaved at the synaptic plasma membrane, and the c-terminus of the molecule traffics to the nucleus. This nuclear import of DFz2 is dependent upon Wg signaling and is important to transduce the downstream effects of Wg signaling at synapses (Mathew et al., 2005 in press). (Abstract shortened by UMI.)

The peripubertal period is a period during which significant brain re-organization occurs, and during which changes in the neuroendocrine milieu normally lead to the onset of fertility. Shipping female mice during the peripubertal period causes a long-lasting behavioral defeminization of feminine sexual behavior. Because shipping is a stressor, it was suggested that peripubertal stress exposure might interfere with the regulation of feminine sexual behaviors in adult female mice. As a result, the goal of this research was to examine the effects of peripubertal stress exposure on neuroendocrine regulation of the hypothalamo-pituitary-gonadal (HPG) and the hypothalamo-pituitary-adrenal (HPA) axis. However, although peripubertally exposing female mice to shipment stress caused a significant decrease in sexual receptivity, peripubertal exposure to restraint stress, food deprivation, social stress and instability, or multiple stressors (heat, light, and restraint) did not decrease sexual receptivity during adulthood. Only exposure to a high dose of bacterial endotoxin during the peripubertal period duplicated the defeminizing effect of shipping during the peripubertal period on adult mouse feminine sexual behavior. Interestingly, significant long-term effects of peripubertal stress exposure were also observed in the regulation of the HPA axis, where exposure to peripubertal shipping and LPS led to blunted stress-induced corticosterone responses in adult female mice. Peripubertal exposure to shipping and LPS had long-lasting effects on steroid receptor expression in brain areas relevant to female reproduction. Nevertheless, peripubertal exposure to LPS did not interfere with the ability of estradiol to induce the release of LH. Moreover, although peripubertal exposure to shipping decreased masculine sexual behavior in male, but not female, mice, peripubertal exposure to LPS was without effect on masculine sexual behavior. These results suggest that exposure to some, but not all, peripubertal stressors can induce long-lasting changes in reproductive behaviors, as well as changes in the expression of sex steroid receptors known to regulate these behaviors.

Background  Patients in the intensive care unit often receive broad spectrum antibiotic (e.g., gentamicin) treatment that are mostly eliminated through the renal route. Therefore, the kidney function is a crucial factor to establish a good dosage regimen especially in patients with acute kidney injuries. Old-fashioned estimation formulas are used today to estimate the kidney function and there is room for improvement. A publication from 2009 have presented that clearance for gentamicin is a good enough estimation compared to the golden standard measurement methods.  Aim  The aim of the study was to first find and investigate already developed equations to estimate non-steady-state kidney function. Thereafter, the aim was to identify which equations, both suited for steady-state and non-steady-state situations, that could best correlate to CLg in adult ICU patients.  Methods  PubMed was used to gather data about non-steady-state kidney function. The equations were then applied on Excel and a dataset containing 100 patients was used to calculate kidney function estimates. Clearance for gentamicin were then compared to the estimates using linear regression, Bland-Altman plots and P30 values.  Results and discussion  The Cockcroft-Gault formula had the highest P30-value, while Grubb’s cystatin C formula generated the best Bland-Altman plot as well as the best linear regression. However, neither of these were good enough to be used in the clinical work. Surprisingly, the Jelliffe equation was the poorest to estimate kidney function.  Conclusion  None of the evaluated estimations were clinically good enough to replace the gentamicin clearance method.

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People with stroke may experience losses of muscle strength and physical function. The two are related, whereby a threshold of strength is often needed to execute activities of daily living. Strength training (ST) is a potent stimulus to elicit gains in muscle mass, strength, and function. However, it is underutilized in clinical practice due to ambiguity around how to prescribe it. It is also unclear to which extent people with stroke are currently engaging in these activities. The purpose of this thesis was to advance the understanding and application of ST in stroke rehabilitation. Study 1 was a systematic review and meta-analysis of 39 trials (n=1,928), which found that ST improved both muscle strength, as well as outcomes rated as important by a community advisory group of adults with stroke, such as balance, walking capacity and speed. We also found that traditional ST programs using free weights and machines, power-focused intensities (i.e., exercises performed at lower external resistance and high speeds), and more frequent ST were more beneficial for some aspects of stroke recovery. Study 2 examined the feasibility and effects of a 10-week power-focused ST program on muscle strength, power, physical function, and health-related quality of life in 15 people living with mild to moderate chronic stroke. We found that the study was feasible with respect to participant recruitment, sex and gender distribution, attendance, attrition, safety, exercise tolerance, and satisfaction with the program. Moreover, the program showed potential for clinically important improvements in physical function and health-related quality of life. Finally, Study 3, was a cross-sectional study (n=2,094) using data from the Canadian Longitudinal Study on Aging which found that fewer than a quarter of Canadians with stroke engage in muscle strengthening activities and are not likely to meet moderate to vigorous physical activity (MVPA) guidelines. Moreover, people with lower intrinsic capacity, functional mobility, and negative environments had lower physical activity levels. These findings suggest that ST is effective and may be feasibly optimized through power-focused intensities. However, very few people with stroke reported engaging in ST, indicating that health promotion should be a key focus for future research and practice. / Thesis / Doctor of Philosophy (PhD) / After a stroke, people often lose muscle mass and strength, which can impact their physical abilities. Strength training can help improve muscle strength and function, but rehabilitation experts are often unclear how to prescribe it. We also do not know whether people with stroke participate in this form of exercise. This thesis contains 3 studies that explored the role of strength training for impacting stroke recovery and the extent of strength training participation in Canadians with stroke. In the first study, we reviewed 39 studies and found that strength training improved muscle strength, balance, and walking abilities for stroke survivors. Performing strength training more frequently, using free-weights and machines, and power-focused strength training (exercises designed to improve speed and strength together) was especially beneficial. In the second study, we tested a 10-week power-focused strength training program for 15 people living with stroke. We showed that our program was safe and showed potential for improving strength, power, physical function, and quality of life. Our third study looked at data from over 2,000 Canadians with stroke. We found that less than 25% were doing ST, and 94% were not meeting physical activity guidelines. In summary, strength training, especially power-focused strength training, can enhance stroke recovery, but not enough stroke survivors are doing it. We need more research on promoting strength training for better recovery.

Call number: LD2668 .T4 1978 P38 / Master of Science