In the common fruit fly, Drosophila melanogaster, neural cell fate specification is triggered by a group of conserved transcriptional regulators known as proneural factors. Proneural factors induce neural fate in uncommitted neuroectodermal progenitor cells, in a process that culminates in sensory neuron differentiation. While the role of proneural factors in early fate specification has been described, less is known about the transition between neural specification and neural differentiation. The aim of this thesis is to use computational methods to improve the understanding of terminal neural differentiation in the Peripheral Nervous System (PNS) of Drosophila. To provide an insight into how proneural factors coordinate the developmental programme leading to neural differentiation, expression profiling covering the first 3 hours of PNS development in Drosophila embryos had been previously carried out by Cachero et al. [2011]. The study revealed a time-course of gene expression changes from specification to differentiation and suggested a cascade model, whereby proneural factors regulate a group of intermediate transcriptional regulators which are in turn responsible for the activation of specific differentiation target genes. In this thesis, I propose to select potentially important differentiation genes from the transcriptional data in Cachero et al. [2011] using a novel approach centred on protein interaction network-driven prioritisation. This is based on the insight that biological hypotheses supported by diverse data sources can represent stronger candidates for follow-up studies. Specifically, I propose the usage of protein interaction network data because of documented transcriptome-interactome correlations, which suggest that differentially expressed genes encode products that tend to belong to functionally related protein interaction clusters. Experimental protein interaction data is, however, remarkably sparse. To increase the informative power of protein-level analyses, I develop a novel approach to augment publicly available protein interaction datasets using functional conservation between orthologous proteins across different genomes, to predict interologs (interacting orthologs). I implement this interolog retrieval methodology in a collection of open-source software modules called Bio:: Homology::InterologWalk, the first generalised framework using web-services for “on-the- fly” interolog projection. Bio::Homology::InterologWalk works with homology data for any of the hundreds of genomes in Ensembl and Ensembgenomes Metazoa, and with experimental protein interaction data curated by EBI Intact. It generates putative protein interactions and optionally collates meta-data into a prioritisation index that can be used to help select interologs with high experimental support. The methodology proposed represents a significant advance over existing interolog data sources, which are restricted to specific biological domains with fixed underlying data sources often only accessible through basic web-interfaces. Using Bio::Homology::InterologWalk, I build interolog models in Drosophila sensory neurons and, guided by the transcriptome data, find evidence implicating a small set of genes in a conserved sensory neuronal specialisation dynamic, the assembly of the ciliary dendrite in mechanosensory neurons. Using network community-finding algorithms I obtain functionally enriched communities, which I analyse using an array of novel computational techniques. The ensuing datasets lead to the elucidation of a cluster of interacting proteins encoded by the target genes of one of the intermediate transcriptional regulators of neurogenesis and ciliogenesis, fd3F. These targets are validated in vivo and result in improved knowledge of the important target genes activated by the transcriptional cascade, suggesting a scenario for the mechanisms orchestrating the ordered assembly of the cilium during differentiation.

Competitive environment exacts from companies to be flexible; solving the redundancy becomes commonplace for them. This work has for object outplacement programs in two companies. Based on analysis of these programs there are defined principles for formation and realization of outplacement program in general.

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Thesis (MCur)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: There is no formal succession plan for paediatric professional nurses (PNs) in academic, tertiary hospitals in the Western Cape. A lack of a succession plan could have major implications for the sustainability of effective and efficient health care services (Department of Health, DoH, 2010:1). Therefore, a systematic scientific investigation is required to determine the opinions of PNs regarding the characteristics or criteria for a succession plan in a paediatric organization. The aim of this study was to determine the opinions of paediatric PNs regarding succession or career planning in academic, tertiary hospitals in the Western Cape. A quantitative approach with an exploratory, descriptive, non-experimental design was applied by means of a questionnaire survey which consisted of closed and openended questions. Reliability and validity were assured by means of a pilot study and consultation with nursing experts and a statistician. Cronbach’s alpha test was used to test for internal consistency between the responses to the 3-point Likert scale and dichotomous questions on the characteristics of an ideal succession plan. The data was collected by means of a self-administered, structured questionnaire to elicit opinions regarding the characteristics of an ideal succession plan that includes a career plan. Ethical approval was obtained from the Health Research Ethics Committee of the University of Stellenbosch. Permission for access to the hospitals was obtained from the hospital and nursing managers. Informed written consent was obtained from the participants. The questionnaires were distributed personally by the researcher at two hospitals and via the assistant manager in nursing at one hospital. Data was analysed by the statistician and descriptive statistics were presented by means of frequency distribution tables and histograms. Furthermore, the existence of relationships between variables was compared by means of a t-test or when assumptions of the t-test were not fulfilled an appropriate non-parametric test was considered. The results were evidence of the need for the development of a succession plan based on Benner’s Novice to Expert Model for paediatric PNs in academic, tertiary hospitals in the Western Cape. In addition, participants’ opinions on the value of a succession plan, including a career plan showed multiple benefits that will outweigh its challenges once developed and implemented. Recommendations are based on the scientific evidence that show the urgent need for the development and implementation of a formal five level skill-based clinical training programme that includes a 360-degree feedback system for paediatric PNs by means of an integrated, collaborative approach. The development and implementation of a formal succession plan will strengthen and enhance the retention of the various levels of competent, proficient and expert paediatric PNs. In addition, a formal succession plan will attract and motivate the novice and advance beginners to progress to competent, proficient and expert levels. / AFRIKAANSE OPSOMMING: Daar is geen formele opvolgplan vir pediatriese professionele verpleegpersoneel in akademiese tersiêre hospitale in die Wes-Kaap nie. ’n Gebrek aan ’n opvolgplan kan ernstige gevolge vir die volhoubaarheid van effektiewe en doeltreffende gesondheidssorgdienste inhou (DoH, 2010:1). Dus, is ’n sistematiese wetenskaplike ondersoek nodig om die opinies van professionele verpleegpersoneel te bepaal, ten opsigte van die eienskappe of kriteria vir ’n opvolgplan in ’n pediatriese organisasie. Die doel van hierdie studie was om die opinies van pediatriese professionele verpleegpersoneel te bepaal ten opsigte van ’n opvolg- of beroepsplan in akademiese, tersiêre hospitale in die Wes-Kaap. ’n Kwantitatiewe benadering met ’n ondersoekende, beskrywende, nie-eksperimentele ontwerp is toegepas deur gebruik te maak van ’n vraelysopname wat bestaan het uit geslote en ope-vrae. Betroubaarheid en geldigheid is verseker deur gebruik te maak van ’n loodsondersoek en raadpleging van verpleegkundiges en ’n statistikus. Cronbach se alpha-toets is gebruik om te toets vir interne konsekwentheid tussen die response tot die 3-punt Likertskaal en tweeledige vrae oor die kenmerke van ’n ideale opvolgplan. Die data is gekollekteer deur gebruik te maak van ’n selfgeadministreerde, gestruktureerde vraelys om opinies te onthul ten opsigte van die kenmerke van ’n ideale opvolgplan, wat ’n beroepsplan insluit. Etiese goedkeuring is verkry van die Gesondheidsnavorsing se Etiese Komitee van die Universiteit van Stellenbosch. Toestemming vir toegang tot die hospitale is verkry van die hospitaal en verpleegbestuurders. Ingeligte skriftelike toestemming is van die deelnemers verkry. Die vraelyste is persoonlik versprei deur die navorser by twee hospitale en via die assistentbestuurder wat by een van die hospitale verpleeg. Data is geanaliseer deur die statistikus en beskrywende statistiek is aangebied by wyse van frekwensie verspreidingstabelle en histogramme. Voorts, is die bestaan van verwantskappe tussen veranderlikes vergelyk, deur gebruik te maak van ’n ttoets of waar veronderstellings van die t-toets nie bereik is nie, is ’n gepaste nieparametriese toets oorweeg. Die resultate is bewys van die behoefte vir die ontwikkeling van ’n opvolgplan wat gebaseer is op Benner se Novice to Expert Model vir pediatriese professionele verpleegpersoneel in akademiese, tersiêre hospitale in die Wes-Kaap. Daarbenewens, het deelnemers se opinies die waarde van ’n opvolgplan wat ’n beroepsplan insluit, die veelvoudige voordele wat dit inhou getoon wat die uitdagings sal oortref, sodra dit ontwikkel en geïmplementeer word. Aanbevelings is gebaseer op die wetenskaplike bewys wat dui op die dringende behoefte vir die ontwikkeling en implementering van ’n formele vyfvlak vaardigheidsgebaseerde kliniese opleidingsprogram wat ’n 360-grade terugvoersisteem insluit vir pediatriese professionele verpleegpersoneel deur middel van ’n geïntegreerde, medewerkende benadering. Die ontwikkeling en implementering van ’n formele opvolgplan sal die retensie van die verskeie vlakke van bekwame, vaardige en kundige pediatriese professionele verpleegpersoneel versterk en bevorder. Boonop sal ’n formele opvolgplan nuwelinge en gevorderdes trek en motiveer om te ontwikkel tot bekwame, vaardige en kundige vlakke.

Peripheral nerve stimulation (PNS) is commonly used to promote use-dependent cortical plasticity for rehabilitation of motor function in spinal cord injury. Pairing transcranial magnetic stimulation (TMS) with PNS has been shown to increase motor evoked potentials most when the two stimuli are timed to arrive in the cortex simultaneously. This suggests that a mechanism of timing-dependent plasticity (TDP) may be a more effective method of promoting motor rehabilitation. The following thesis is the result of applying a brain-computer interface to apply PNS in closed-loop simultaneously to movement intention onset as measured by EEG of the sensorimotor cortex to test whether TDP can be induced in incomplete spinal cord injured individuals with upper limb motor impairment. 4 motor incomplete SCI subjects have completed 12 sessions of closed-loop PNS delivered over 4-6 weeks. Benefit was observed for every subject although not consistently across metrics. 3 out of 4 subjects exhibited increased maximum voluntary contraction force (MVCF) between first and last interventions for one or both hands. TMS-measured motor map volume increased for both hemispheres in one subject, and TMS center of gravity shifted in 3 subjects consistent with studies in which motor function improved or was restored. These observations suggest that rehabilitation using similar designs for responsive stimulation could improve motor impairment in SCI.

Several factors contribute to the failure of the central nervous system (CNS) to regenerate after injury. These include inhibition of axonal growth by myelin and glial scar associated molecules, as well as the intrinsic inability of adult CNS neurons to grow long axons in environments that are permissive for younger neurons. Neurons in the peripheral nervous system (PNS) display a much higher capacity to regenerate after injury than CNS neurons, as shown by conditioning lesion experiments and by microtransplantation of dorsal root ganglia neurons into CNS white matter tracts. Our central hypothesis is that neurons of the PNS express specific regeneration associated genes that mediate their enhanced growth response after injury. We have employed a combination of subtractive hybridization, microarray comparison and promoter analysis to probe for genes specific to neurons of the dorsal root ganglia (DRG), using cerebellar granule neurons (CGN) as a reference. We have identified over a thousand different genes, many of whose products form interaction networks and signaling pathways. Moreover, we have identified several dozen transcription factors that may play a role in establishing DRG neuron identity and shape their responses after injury. One of these transcription factors is Signal Transducer and Activator of Transcription 3 (STAT3), previously known to be upregulated in the PNS after a conditioning lesion but not known to be specific to the PNS. Using a real time PCR and immunochemical approaches we have shown that STAT3 is constitutively expressed and selectively active in DRG neurons both in culture and in vivo. We show that the overexpression of wild type STAT3 in cerebellar granule neurons leads to the formation of supernumerary neurites, whereas the overexpression of constitutively active STAT3-C leads to a 20% increase in total neurite outgrowth. It is hoped that the genetic delivery of STAT3-C, potentially combined with co-activators of transcription, will improve functional regeneration of CNS axons in vivo.

Neural stem and progenitor cells (NSPC) can differentiate to neurons and glial cells. NSPC are easily propagated in vitro and are therefore an attractive tool for tissue regeneration. Traumatic brain injury (TBI) is a common cause for death and disabilities. A fundamental problem following TBI is tissue loss. Animal studies aiming at cell replacement have encountered difficulties in achieving sufficient graft survival and differentiation. To improve outcome of grafted cells after experimental TBI (controlled cortical impact, CCI) in mice, we compared two transplantation settings. NSPC were transplanted either directly upon CCI to the injured parenchyma, or one week after injury to the contralateral ventricle. Enhanced survival of transplanted cells and differentiation were seen when cells were deposited in the ventricle. To further enhance cell survival, efforts were made to reduce the inflammatory response to TBI by administration of ibuprofen to mice that had been subjected to CCI. Inflammation was reduced, as monitored by a decrease in inflammatory markers. Cell survival as well as differentiation to early neuroblasts seemed to be improved. To device a 3D system for future transplantation studies, NSPC from different ages were cultured in a hydrogel consisting of hyaluronan and collagen. Cells survived and proliferated in this culturing condition and the greatest neuronal differentiating ability was seen in cells from the newborn mouse brain. NSPC were also used in a model of peripheral nervous system injury, and xeno-transplanted to rats where the dorsal root ganglion had been removed. Cells survived and differentiated to neurons and glia, furthermore demonstrating their usefulness as a tool for tissue regeneration.

Chronic denervation and chronic axotomy present independent barriers for axonal regeneration. Chronic denervation occurs when nerves are no longer connected to their neuronal cell bodies; chronic axotomy occurs when neurons are not connected to their targets for prolonged periods of time. The harmful effects of chronic denervation can be addressed by the side-to-side bridge surgical technique. Additionally, the negative effects of chronic axotomy can be reversed by GDNF delivery to the nerve. The experiments in this thesis were designed to evaluate nerve regeneration in a rat model of chronic injury after treatment with local GDNF delivery, side to-side bridge protection, or both. The GDNF delivery system consisted of poly(lactic-co-glycolic acid) microspheres embedded in fibrin for controlled delivery of GDNF. Overall, the side-to-side bridges technique was effective in protecting against the negative effects of chronic denervation regardless of treatment with or without GDNF. Local delivery of GDNF did not increase axonal regeneration or functional recovery.