The HIV-1 surface protein, Envelope (Env), is covered in asparagine-linked glycans, which interact with the human immune system and are thus important as potential vaccine targets. Laboratory studies have shown that the glycan type and form can differ substantially at each glycan site on Env clones. However, these studies are limited by time and cost and rely on biosynthetic assumptions to elucidate the structure of branched glycans. Furthermore, glycan heterogeneity creates challenges when determining the three-dimensional structure of Env, which has resulted in the use of methods that restrict glycan processing to produce uniform glycans for these studies. Computational methods are used to complement the laboratory studies; however, due to the limitations of modelling software, even computational studies have focussed on uniformly glycosylated Env models using a limited set of high-mannose glycans, rather than a mix of glycan types. To bridge this gap, this study set out to examine the structural differences of two computationally glycosylated HIV-1 Env trimers, one uniformly glycosylated, and the other based on the heterogeneous glycosylation of a laboratory determined gp160 strain. A secondary aim was to estimate whether the type of glycan is predictable using computational techniques, since these are less expensive and time consuming than laboratory studies. Using 500 ns molecular dynamics (MD) simulations, it was found that the heterogeneously glycosylated trimer had 64% greater stability, likely due to the presence of 25% more hydrogen bonds, as well as stabilising bonds which appeared to prevent asymmetrical movements. Furthermore, by focussing on the heterogeneously glycosylated trimer, a computational method based on surface area was explored to estimate the accessibility to enzymes involved in glycan processing, and to use this measure as a predictor of the glycan type. The results of this study highlight the differences between a uniformly, and a heterogeneously, glycosylated trimer, and suggest that previous MD studies, which used uniformly glycosylated trimers, may not sufficiently describe the structural dynamics of HIV-1 Env. Notably, complex glycans appear to stabilise the trimer to a greater extent than the high-mannose glycans used in previous studies. Thus, it is evident that research on Env models should incorporate a more diverse set of glycans in order to deepen our understanding of the dynamics of Env, which will, in turn, further our understanding of its interactions with antibodies and anti-HIV compounds.

This study examined the anthropometry and anthropometric fit of a group of ward and theatre nurses in Western Cape private hospitals. Anthropometric variables were measured using a sample of nurses and a correlation matrix generated. All nurses were given a questionnaire concerned with operational problems in the work environment and musculoskeletal pain. The questionnaire was also completed by a group of sedentary nurses. The ward and theatre nurses reported numerous problems in the working environment, including lumbar backache, inadequate space and equipment that caused bodily discomfort. There were consistent, statistically significant associations between the frequency of occurrence of these problems and the anthropometric data indicating that the problems were caused or amplified by body size variability and were not simply general usability problems which would affect all nurses irrespective of their body dimensions. Further studies testing specifically for the consequences of mismatches and body size variability are recommended.

In 1985, a cohort of 14 children with cerebral palsy underwent a neurosurgical procedure - selective posterior rhizotomy - in an effort to reduce their spasticity. They were part of a prospective study in which their gait was analysed before surgery and at one and three years' post-operatively. In 1995, ten years after surgery, there were important questions that needed to be addressed: would their gait be different from the findings before surgery and three years after surgery and how would they compare with age-matched normal subjects? Three of the original subjects were lost to follow-up and 11 were invited to participate in this long-term study. Their ages ranged from 12 to 24 years at this time of evaluation. Their gait was studied in the sagittal plane by taping retro-reflective markers onto the greater trochanter, lateral femoral epicondyle and lateral malleolus and having the subjects walk in a direction perpendicular to a video camera recording their gait. The data were digitised and analysed making use of custom written software and all relevant gait parameters were calculated. The parameters evaluated were the ranges of motion and midrange values of the knee and thigh, and the spatial-temporal parameters, namely cadence, stride length and velocity. Data were also obtained from twelve age-matched normal control subjects. The ten-year results were compared to the pre-operative data, the data obtained from the three-year study and the normal controls. A statistical analysis was performed on these parameters by using multiple analysis of variance (MANOVA) and post hoc comparisons were performed with a Scheffe test and a significance level set at p < 0.05. Results indicated that after ten years, the joint ranges of motion and mid-range values did not differ significantly from the normal values. The values also did not vary significantly from the values obtained in the three-year follow-up study, suggesting that functional changes acquired due to rhizotomy were of long term duration. The spatial-temporal parameters, however, did differ significantly from the normal range. Although this finding may have been influenced by the environment and subject motivation, the reduced cadence and stride length meant that the cerebral palsy subjects walked slower than their normal counterparts. This project, while confirming the results of the previous subjective studies, provides the first long-term objective data that establishes the effectiveness of selective posterior rhizotomy in improving and maintaining locomotor function for children with spastic cerebral palsy.

During murine development, retinal ganglion cell (RGC) axons are presented with multiple navigational choices as they exit the eyes and follow a complex path to targets in the thalamus and superior colliculus of the brain. The optic chiasm is a major choice point, positioned at the ventral midline of the hypothalamus, where the majority of retinal axons cross to the contralateral side of the brain whilst only 3% remain uncrossed and project ipsilaterally. Identifying the cellular and molecular processes involved in retinal axon divergence at the chiasm is an intense area of study and knockout mice have proved useful tools. Foxg1 is a winged helix transcription factor that is expressed in the nasal retina, nasal optic stalk and anterior ventral hypothalamus, which are all structures that retinal axons encounter as they project out of the RGC layer towards the chiasm. The coincidence between the expression pattern of Foxg1 and the route followed by retinal axons led to the hypothesis that Foxg1 plays a role in guiding retinal axons at the optic chiasm. Previous experiments in this laboratory lent support to this idea by revealing an increase in the number of ipsilateral projections in Foxg1-/- mouse embryos from both nasal and temporal retina. Since Foxg1 is expressed in both the nasal retina and at the optic chiasm midline, the main hypotheses for this thesis are that Foxg1 influences retinal axon divergence by transcriptionally regulating the expression of cell surface molecules on (1) growth cones from the nasal retina or (2) guidance molecules on chiasm cells. In order to address these possibilities, the key aims of this thesis were (i) to investigate whether Foxg1 is primarily required in the nasal retina or at the chiasm for retinal axon divergence, (ii) to determine whether the Foxg1 null retina and chiasm are patterned differently from those of wild types and (iii) to investigate the expression of candidate molecules in the retina or chiasm known to influence retinal axon navigation.

Medulloblastomas represent the most frequent malignant brain tumour in children, and are thought to develop in the posterior fossa of the cerebellum. Some patients with medulloblastomas have a deficiency in the tumour suppressor gene Apc (Turcot’s syndrome). Although the majority of medulloblastomas arise sporadically, people with Apc mutations are 92 times more likely to develop medulloblastomas. Apc encodes a very large protein known to function as a regulator of the Wnt signalling pathway. Activation of the canonical Wnt pathway leads to the stabilisation of beta-catenin. In response to Wnt signals, beta-catenin translocates to the nucleus where it interacts with the LEF/TCF family of transcription factors to activate transcription of target genes such as c-myc and cyclinD1. Mutations of Apc that cause an increase in beta-catenin are found to be tumourgenic, whereas other mutations are not. Therefore it is thought that the main tumour suppression function of Apc is in its ability to destabilise and hence reduce cytoplasmic beta-catenin. The central hypothesis of this thesis is that the loss of Apc can lead to the development of medulloblastoma. Work from other groups has reported activation of Wnt signalling in a proportion of primary medulloblastomas. We undertook a study to assess this by using the cre-loxP recombination system to mutate Apc in a temporal and spatial manner. This approach is necessary as Apc has many functions in development and Apc mutant mice (Apcmin) do not develop past embryonic day 6.5 (E6.5). To date, there are no known cre-strains available to mutate Apc specifically in the cerebellum at early postnatal stages, so we combined the creloxP method with an avian retrovirus mediated method for tissue specific gene delivery (RCAS/tv-a system), in an attempt to create a strain of mice which carried the genotype Ntv-a +;ApcLoxP/LoxP. This would allow us to infect an RCAS-cre virus directly into the hindbrain at postnatal day 4 (P4). However subsequent genotyping of these animals showed that none carried the desired genotype of Ntv-a +;ApcLoxP/LoxP, making it impossible for both copies of Apc to be mutated in a mouse most likely because both the Ntv-a and Apc transgenes were located on the same chromosome. Consistent with this, out of a total of 265 mice none were found to have the Ntv-a +;ApcLoxP/LoxP genotype. We then adopted an alternative method for mutating Apc by infecting ApcLoxP/LoxP mice directly with an AdCre virus. PCR analysis showed that Apc was mutated, however the AdCre virus did not infect cells of the cerebellum, and instead only infected the choroid plexus. In these animals, 7 of 94 (7%) developed hydrocephalus indicating that losing Apc in the choroid plexus may promote hydrocephalus. Finally, to address the role of Apc in normal hindbrain development, we crossed our ApcLoxP/LoxP mice to an En1cre strain which caused mutation in Apc from E8.5 in the midhindbrain region. The resulting En1cre+;ApcLoxP/LoxP mutants displayed hydrocephalus in all ventricles and an in-growth of mesenchyme tissue at the mid-hindbrain border, closely associated with a tumour-like area of cells showing activated Wnt signalling. No mice were found to live past E18.5. In conclusion, the role of Apc in medulloblastoma remains unclear. Future studies could use a different technique to mutate Apc such as crossing ApcLoxP/LoxP mice to the new nestin-creER strain and inducting cre with administration of tamoxifen.

Regulation of the early rise in gonadotropin secretion in bull calves and its involvement in the reproductive development of bull calves and ram lambs were studied. Development of the testes, prostate and seminal vesicles was assessed from birth to puberty by ultrasonagraphy and image intensity analysis in bull calves and ram lambs. A GnRH superagonist ( Leuprolide acetate) was used to suppress early gonadotropin secretion in bull calves and ram lambs, to study the importance of early gonadotropin secretion for testicular development. GnRH treatment was used to induce LH secretion in the early postnatal period in bull calves and its effects on testicular and endocrine development were studied. Histology and flowcytometry were used to evaluate testicular development in these studies. The role of estradiol in the negative feedback regulation of early gonadotropin secretion was studied in bull calves by using an aromatase inhibitor (ICI 16949A) and a nonsteroidal antiestrogen (ICI 182,780). The involvement of opioidergic and dopaminergic neuronal systems in the regulation of the early rise in gonadotropin secretion was studied in bull calves by using their receptor antagonists (naloxone and pimozide, respectively). Suppressing the early increase in gonadotropin secretion stressed its importance for testicular development in bull calves. The timing of the early increase in secretion of gonadotropins is also important; by inducing higher gonadotropin secretion early in the life of bull calves, reproductive development was enhanced. In ram lambs, high gonadotropin secretion up to 11 weeks of age did not appear critical for reproductive development. We were unable demonstrate a role for estradiol in the negative feedback regulation of early gonadotropin secretion in bull calves. Opioidergic and dopaminergic neuronal systems appeared to be important in the regulation of gonadotropin secretion in bull calves towards the end of the early increase in LH secretion; opioids may be critical for termination of the early rise in LH secretion. Ultrasonography and computer assisted image intensity analysis appeared to be very useful techniques in assessment of reproductive development in bull calves and ram lambs. Image intensity analysis showed promise for identifying critical stages in development of the reproductive system.

The activity patterns of 5-HT-releasing neurons can be positively correlated with behavioural state and motor function and the central 5-HT system modulates motor activity at the cellular level. The rat facial motor nuclei are densely innervated by 5- HT releasing afferents and 5-HT-mediated modulation of ion channels on the soma and dendrites can markedly influence the excitability of facial motoneurones and their integration of excitatory postsynaptic potentials (EPSPs). 5-HT facilitates facial motoneuron excitation by inhibiting a ‘leak’ potassium (K+) conductance (gK+ Leak) and enhancing the hyperpolarisation-activated cation current, Ih. These actions of 5- HT have been confirmed using whole-cell voltage-clamp recordings from visually identified facial motoneurones in an acute brainstem slice preparation. Pharmacological approaches have been used to identify the receptors which mediate the actions of 5-HT in facial motoneurones. The inhibition of gK+ Leak by 5-HT can be blocked by the 5-HT2A receptor antagonist, R96544 (0.3 – 1μM) and the enhancement of Ih by 5-HT is sensitive to the 5-HT7 receptor antagonist, SB269970 (0.3 – 10 μM). Noradrenaline was also found to inhibit gK+ Leak, via activation of a1 adrenoceptors, and the molecular identity of the amine-sensitive ‘leak’ K+ channels has been investigated. TASK-1 and TASK-3 are pH-sensitive two-pore domain K+ channels that can be modulated by amines and provide ‘leak’ K+ conductances in several central neurones. The mRNAs for these channels have been reported to be present in the rat facial motor nucleus. The gK+ Leak in facial motoneurones is sensitive to changes in external pH and has a pK of ~7.1, which is intermediate between the v values for homomeric TASK-1 and TASK-3 channels (7.5 and 6.8 respectively). The TASK-1 selective inhibitor anandamide (10 μM), its stable analogue methanandamide (10 μM), the TASK-3 selective inhibitor ruthenium red (10 μM) and Zn2+ (100-300 μM) all failed to alter the actions of noradrenaline or changing external pH. These findings argue against principal contributions to gKLeak by homomeric TASK-1 or TASK-3 channels. Isoflurane, a volatile anaesthetic that enhances heteromeric TASK-1 / TASK-3 currents, potentiated gKLeak supporting a predominant role for heterodimeric TASK-1 / TASK-3 channels in the gKLeak in facial motoneurones. Evoked fast excitatory synaptic transmission in the facial motor nucleus has been characterised and NMDA and non-NMDA receptor-mediated components of this synaptic transmission have been identified. Through a combination of analysis of the paired pulse ratio, rate of failure to generate a response and the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) this study provides evidence to suggest that glutamate release from pre-synaptic terminals in the facial motor nucleus is depressed by 5-HT. This action of 5-HT is mediated by activation of presynaptic 5-HT1B receptors as this effect is mimicked by the 5-HT1B receptor agonist, CP93129 (10 μM) and can be blocked by the 5-HT1B receptor antagonist, isamoltane (1 μM). These studies indicate that the modulation of synaptic integration in the facial motor nucleus involves activation of distinct pre- and post-synaptic 5-HT receptor subtypes. These findings not only increase our understanding of the cellular mechanisms for vi the 5-HT modulation of motor activity but may also be relevant to the role of 5-HT in the control of other central neurones.

Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references (p. 25).