Computational methods in neuroscience have advanced our understanding of neuronal regulation of motor behavior and locomotion and have been applied to identify encoding of behavioral features in circuits. The cerebellum has an established role in sensorimotor processing during coordinated movements, referred to as the “head ganglion of the proprioceptive system” (Sherrington, 1906). Increasing evidence also highlights its role in the processing of behaviorally meaningful stimuli that have the potential of guiding adaptative movements relevant to the task and priming downstream targets for action. Yet the extent to which these diverse encodings of signals in complex motor tasks are present in the cerebellar nuclei and their influence on behavior remains unknown. To shed new light on the role of this subcortical region using computational approaches, this thesis begins with an introduction that reviews the circuity of the mammalian cerebellum, highlights its proposed functions in motor behavior, and explores our understanding of its role in locomotion. In the first chapter, I analyze electrophysiological recordings from cerebellar nuclei in a locomotor obstacle avoidance task in mice that involves a rich and diverse set of task relevant features. Given the complexity of and correlations between the behavioral features, statistical modeling is required to attribute the firing rates to the correct combinations. This model enables identifying the encoding of these signals and reporting on the prevalence and degree to which they are present across individual cells in the nuclei. Additionally, this model allows investigation into the encoding of groups of cells that are selective for specific features. Chapter 2 uses network modeling to generate hypotheses about population level activity in two cortical areas, the primary and supplementary motor areas, and differentiate their computations in monkeys performing a cycling task. Finally, in chapter 3 I concentrate on a specific class of recurrent network models in the balanced state and investigate the linkage between connectivity distribution and firing sparsity, which has the potential to further our understanding on the emergence of feature selectivity in excitatory/inhibitory circuits.

No description available.

Thesis (MSCMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2005. / There is an urgent need for a safe, effective, affordable human immunodeficiency virus type 1 (HIV-1) vaccine that induces both cellular and humoral immunity. A popular strategy for vaccine design is the use of plasmid DNA encoding HIV-1 genes for priming vaccinations followed by either viral vector or recombinant protein boosting. DNA-based vaccines are attractive because they are safe, easily administered and can induce both cellular and humoral immune responses. In order for DNA vaccination to induce a potent immune response it is necessary for plasmid-encoded genes to be targeted to dendritic cells (DCs) as these are the key antigen presenting cells in natural HIV infection. The immunogenicity of all potential vaccine candidates needs to be assessed in animal models prior to entry into human trials. Nonhuman primates are the best alternative to humans for assessment of vaccine immunogenicity and protective efficacy. In order to clearly understand how DNA vaccines interact with DCs, suitable in vitro DC culture systems for nonhuman primates need to be developed. This study investigated the culture and characterisation of chacma baboon DCs in vitro, and was the first to assess the effect of various transfection methods on baboon DC maturation and function. The study also evaluated the efficacy of a candidate HIV-1 subtype C DNA vaccine at the level of baboon DC transfection, gene transcription and antigen presentation. Generation of immature DCs (iDCs) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) was accompanied by a loss in the monocyte marker CD14. Expression of the markers CD80 and CD83 was observed on a minority of iDCs, whereas CD86 was expressed on almost all iDCs. Following maturation, all these markers were expressed on an increased number of cells, a pattern of marker expression and upregulation that is similar to that observed in both human and macaque DCs. Transfection of baboon DCs by passive pulsing, lipofection and electroporation was evaluated and compared in several ways. Transfection efficiency, cytotoxicity, the effect of the transfection on DC maturation and subsequent presentation of plasmidencoded antigen to memory T lymphocytes was examined. Baboon DCs lipofected with pDNA efficiently took up HIV-1 subtype C plasmid DNA, transcribed plasmid-encoded genes into mRNA, translated the mRNA into protein, processed the protein and presented peptide antigens to antigen-specific memory T cells. The other methods of transfection were less effective than lipofection due to either decreased transfection efficiency or increased cell cytotoxicity. However, neither lipofection nor passive pulsing in any way negatively impacted on DC marker, CD83, or costimulatory molecule, CD80 and CD86, upregulation. Both methods were found to be as effective as a standard cytokine maturation cocktail in inducing DC maturation. Transfected DCs were also found to be more potent inducers of allogeneic T cell stimulation than their untransfected counterparts, which would appear to indicate enhanced major histocompatibility complex (MHC) expression concurrent with DC maturation marker expression. Lipofection with candidate HIV-1 subtype C vaccine plasmid DNA constructs led to antigen-specific expansion of autologous memory T cells, a finding which indicates the effective expression of plasmid-encoded HIV genes in baboon DCs. This study highlights the functional activity of in vitro generated baboon DCs and provides the groundwork for future studies addressing targeting of plasmid DNA to DCs and enhancement of expression of plasmid-encoded antigens in DCs. A more detailed evaluation of baboon DC interaction with simian immunodeficiency viruses/chimeric simian human immunodeficiency viruses (SIVs/SHIVs) may also reveal how the course of infection in this primate differs from that seen in the macaque or chimpanzee and also how it relates to HIV-1 infection in humans.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Diabetes Mellitus is a metabolic disease characterized by the loss of beta cells from the islets, thereby disrupting islet composition and architecture which are important components that influence islet function. The experimental technique of pancreatic duct ligation (PDL), which is thought to induce the regeneration of beta cells within the adult pancreas, was investigated as a novel treatment strategy for diabetes. This study aimed at investigating the possibility that the PDL model may have the capacity to restore normal islet composition and architecture in diabetic animals, which could make it an effective approach to reverse diabetes. Male Wistar rats (n=55) were divided into three study groups: the normal control (NC) group, the diabetic control (DC) group consisting of five subgroups (day 0, 3, 5, 10 and 30) and the experimental (EX) group consisting of four subgroups (day 3, 5, 10 and 30). The experimental group was exposed to PDL. All pancreata were divided into a P1 portion (proximal to the point of ligature) and P2 portion (distal to the point of ligature) for histological assessment. Animals’ non-fasting blood glucose levels (BGLs) and body weights were monitored. The general morphology of the tissue was studied, while an immunohistochemical (IHC) study was performed to determine insulin, pancreatic polypeptide, glucagon and somatostatin protein expression in the P1 and P2 portions of the pancreas. From the IHC slides hormone fractions, staining intensity and distribution were determined as indication of islet composition and architecture. Despite apparent morphological recovery in the islet 30 days post-PDL, islet composition and architecture remained disrupted. Compared to diabetic animals, the proximal portion of the pancreas in experimental animals had a decreased beta cell fraction and increased delta cell fraction thirty days following PDL. These observed changes in islet composition in the part of the pancreas proximal to the ligature are novel findings. There was no change in the diabetic islet composition in the portion of the pancreas distal to the ligature thirty days following PDL. Furthermore, pancreatic duct ligation did not restore body weight or normoglycemia. We conclude that STZ disrupts islet composition and architecture and this could not be restored using PDL; we therefore suggest that a comparative study using a Type 2 diabetic model, where there is limited damage to pre-existing beta cells, may yield different results. / AFRIKAANSE OPSOMMING: Diabetes Mellitus is ʼn metaboliese siekte wat deur die verlies van beta selle uit die eilande van Langerhans gekarakteriseer word. Hierdie verlies van beta selle ontwrig eiland komposisie en argitektuur, twee belangrike komponente van eiland funksie. Die eksperimentele tegnieke van pankreatiese buisafbinding (in Engels PDL), wat moontlik beta sel regenerasie in die volwasse pankreas kan induseer, is ondersoek as behandelings-strategie vir diabetes. Hierdie studie het ten doel gehad om die moontlikheid te ondersoek dat die PDL model die kapasiteit het om normale eiland komposisie en argitektuur te herstel in diabetiese diere, wat dit ʼn effektiewe benadering vir die omkeer van diabetes kan maak. Manlike Wistar rotte (n=55) was in 3 studie groepe verdeel: die normale kontrole (NC) groep, die diabetiese kontrole (DC) groep wat uit vyf subgroepe bestaan (dag 0, 3, 5, 10 en 30) en die eksperimentele (EX) groep wat uit vier subgroepe bestaan (dag 3, 5, 10 en 30). Die eksperimentele groep is aan PDL blootgestel. Alle pankreata is verdeel in ʼn P1 porsie (proksimaal tot die afbinding) en ʼn P2 porsie (distaal tot die afbinding) vir histologiese assessering. Die diere se nie-vastende bloed glukose vlakke en liggaamsgewig is gemonitor. Die algemene morfologie van die pankreas weefsel is bestudeer, terwyl ’n immunohistochemiese (IHC) studie gedoen is om insulien, pankreatiese polipeptied, glukagon en somatostatien proteïen uitdrukking in die P1 en P2 porsies van die pankreas te bepaal. Vanaf die IHC snitte is hormoon fraksie, kleur intensiteit en verspreiding bepaal as aanduidings van eiland komposisie en argitektuur. Ten spyte van ooglopende morfologiese herstel in die eilande op dag 30 na PDL, het eiland komposisie en argitektuur versteur gebly. In vergelyking met die diabetiese diere, het die proksimale deel van die pankreas van eksperimentele diere verlaagde beta sel fraksie en verhoogde delta sel fraksie getoon dertig dae na PDL. Die waarneming van veranderde komposisie in die deel van die pankreas proksimaal tot die afbinding is nuut. Daar was geen verandering in diabetiese eiland komposisie in die deel van die pankreas distaal tot die afbinding dertig dae na PDL nie. Verder het PDL nie liggaamsgewig of bloedsuiker genormaliseer nie. Ons gevolgtrekking is dat STZ eiland komposisie en argitektuur ontwrig en dat dit nie met PDL herstel kon word nie; daarom stel ons ʼn vergelykende studie in ʼn tipe 2 diabetes model voor, waar die skade aan reeds bestaande beta selle beperk is, wat ander resultate mag lewer.

Dissertation (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Introduction Despite advances in insulin therapy, the side effects associated with diabetes mellitus still remain. Pancreas transplantation has benefited diabetics with end-stage renal failure by reversing the diabetic state and preventing or reversing the progression of diabetes associated diseases. Currently the side effects associated with lifelong immunosuppression preclude pancreas transplantation as a viable treatment option for both type I and II diabetics. In the laboratory, transplanted rat foetal pancreata have been shown to be able to reverse the clinical signs of streptozotocin-induced diabetes in an isogeneic model. Reversal of diabetes by allogeneic foetal rat pancreas transplantation, although possible has proved to be more difficult due to fierce rejection of the grafts and the diabetogenic effects of conventional immunosuppressants. Aims One of the goals, focus and intentions of this laboratory study in rodents, is to contribute new information to the scientific literature. The potential to “reverse” the diabetic state by allogeneic foetal pancreatic transplantation, was the main stimulus for this study. Methods Foetal pancreata of 16-18 days gestation were transplanted into a surgically prepared renal subcapsular space. Immunosuppressive protocols used to prevent rejection of the allogeneic foetal rat pancreata included donor specific transfusion (DST), cyclosporine [a calcineurin inhibitor (CsA)], mycophenolate mofetil [a purine syntase inhibitor (MMF)], and a mouse anti-rat CD4 monoclonal antibody (W3/25). Immunosuppressants were used as monotherapies and in combination. Results Isogeneic foetal rat pancreas transplantation resulted in the growth and development of mature insulin producing islets of Langerhans at the site of engraftment. Allogeneic foetal pancreatic transplantation without immunosuppression resulted in complete rejection of the grafts at 14 days post-transplantation. Histological assessment of allografts at 14 and 30 days post-transplantation showed that CsA was able to prevent acute rejection in our rat models although graft scores and survival were improved if CsA was combined with MMF. Intraperitoneal anti-CD4 monoclonal injections were well tolerated, and if given daily effectively prolonged graft survival up to 30 days. Combining DST with anti-CD4 and CsA induction therapy provided long-term graft survival without daily immunosuppression. This combination, together with allogeneic foetal rat pancreas transplantation, was effective in reversing the clinical signs of experimentally induced diabetes. To my knowledge these are the first published results in which reversal of streptozotocin induced diabetes was achieved by fully MHC mismatched foetal rat pancreatic transplantation. Conclusion Foetal rat pancreatic transplantation is a potential source of endocrine replacement, which, with effective immunosuppression allows for the development of functional islets able to reverse the clinical signs of experimentally induced diabetes in an allogeneic rat model. An unique immunosuppressive protocol, with potential clinical relevance in the human, combines anti-CD4 mAb, CsA and DST induction therapy, which alleviates the burden of daily immunosuppression and associated side effects. / AFRIKAANSE OPSOMMING: Inleiding Ten spyte van die vordering met modeme insulienterapie bly die newe-effekte, waarmee diabetes mellitus geassosieer is, steeds ‘n probleem vir diabete. Diabetiese pasiente met eindstadium nierversaking trek geweldig voordeel uit nier-pankreasoorplantings wat die diabetes omkeer en die progressie van diabetesverwantesiektes voorkom of selfs omkeer. Die newe-effekte van lewenslange immuunonderdrukking skakel pankreasoorplanting uit as ‘n lewensvatbare behandelingsopsie vir tipe I of II diabete. In ‘n streptozotosien-gei'nduseerde diabetiese rotmodel kan isogenei'ese fetale pankreasoorplanting die kliniese tekens van diabetes omkeer. Die omkering van streptozotosien-gei'nduseerde diabetes deur allogeneiese fetale pankreasoorplanting behoort moontlik te wees indien verwerping en die diabetogeniese newe-effekte van konvensionele immuunonderdrukkers oorkom word. Doelstellings Een van die mikpunte, fokusse en oogmerke van hierdie laboratorium studie in knaagdiere, is om ‘n betekenisvolle bydrae tot nuwe kennis in die wetenskaplike literatuur, te maak. Die potensiaal om die diabetiese toestand deur allogeneiese fetale pankeasoorplanting om te keer, was die hoof stimulus vir die studie. Metodes Fetale rotpankreata van 16-18 dae gestasie was in ‘n chirurgies voorbereide spasie onder die nierkapsel oorgeplant. Immuunonderdrukkende protokolle, vir die voorkomming van verwerping van die allogeneiese fetale pankreasoorplantings, het donorspesifiekeoortappings (DST), siklosporien [‘n kalsineurien inhibitor (CsA)], mikofenolaat mofetiel [‘n purien sintase inhibitor (MMF)] en ‘n anti-rot CD4 monoklonale antiliggaam (W3/25) ingesluit. Die immuunonderdrukkers is as mono- of as kombinasieterapie gebruik. Resultate IsogeneTese fetale rotpankreasoorplanting het tot die ontwikkeling van volwasse insulienproduseerende eilande van Langerhans gelei, wat die kliniese tekens van streptozotosien-gei'nduseerde diabetes kon omkeer. Allogenei'ese fetale rotpankreasoorplanting sonder immuunonderdrukking het tot algehele verwerping van die oorplanting binnel4 dae na oorplanting gelei. Histologiese beoordeling van die oorplantings 14 en 30 dae na oorplanting het getoon dat CsA akute verwerping van fetale pankreasoorplantings in die rotmodelle voorkom. Indien CsA met MMF gekombineer word, word die oorplantings-telling en oorlewing verbeter. Intraperitoneale anti-CD4 monoklonale inspuitings was goed verdra, en indien daagliks toegedien, het dit die oorlewing van die pankreasoorplantings effektief tot 30 dae verleng. Die kombinasie van DST, anti-CD4 en CsA induksieterapie het tot langtermyn oorlewing van die pankreasoorplantings gelei sonder verdere daaglikse immuunonderdrukking. Die induksieterapie in kombinasie met allogenei'ese fetale pankreasoorplanting was effektief in die omkering van die kliniese tekens van streptozotosien-gei'nduseerde diabetes in die rot. Hierdie is, sover ek weet, die eerste keer dat omkering van streptozotosien-gei'nduseerde diabetes suksesvol met ‘n volledige MHC onverenigbare allogenei'ese fetale pankreasoorplanting behaal is. Gevolgtrekkings Fetale rotpankreasoorplanting is ‘n potensiele bron vir endokrien vervangingsterapie, wat met effektiewe immuunonderdrukking tot die ontwikkeling van funksionele eilande van Langerhans lei, wat die vermoe het om die kliniese tekens van experimenteel-ge'induseerde diabetes in ‘n allogeneiese rotmodel om te keer. ‘n Unieke immuunonderdrukkingsprotokol, met kliniese relevansie, kombineer DST met anti-CD4 mAb en CsA induksieterapie wat die las van daaglikse immuunonderdrukking en die geassosieerde newe-effekte van konvensionele immuunonderdrukking verlig.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: A high fat diet (HFD) reduces beta-cell mass, impairs glucose signalling and is involved in the development of Type 2 diabetes. Malnutrition during gestation is hypothesized to irreversibly damage beta-cell development. The transcription factors Pdx-1 and Pax 4 are involved in islet cell development. Pdx-1 is reported to regulate expression of GLUT-2, glucokinase (GK) and the insulin gene. Aims The aim of this study is to investigate, in the neonatal and weanling rat, the effect of exposure to a HFD in utero and/or lactation on weight, glucose and insulin concentrations, islet cell development, pancreatic transcription factors and glucose sensing genes. Methods Neonatal and weanling rats were exposed to a maternal HFD for defined periods of gestation and/or lactation. After termination, pups were weighed and glucose and insulin concentrations determined. mRNA expression of Pdx-1, Pax 4, GLUT-2 and GK was quantified by LightCycler PCR. Pancreatic sections were immunostained for insulin and glucagon (islet cell development), and for Pdx-1, GLUT-2 and GK (beta-cell function) followed by image analysis. Results: Exposure to an in utero HFD throughout gestation resulted in hyperglycaemic pups with reduced beta-cell volume and number, Pdx-1 and GK immunoreactivity. In contrast the alpha-cell volume, number and size were augmented in neonates exposed to a HFD throughout gestation. Most weanlings were hyperglycaemic and hypoinsulinaemic. In some weanlings, reduced beta-cell number and beta- and alpha-cell size was observed. Pdx-1 mRNA was overexpressed in weanlings exposed to a maternal HFD for the final week of gestation or throughout both gestation and lactation, but reduced in those only exposed throughout lactation. Pax 4 mRNA was reduced in weanlings exposed to a maternal HFD for the first or final week of gestation, throughout gestation or throughout lactation. In most of the weanlings, GLUT-2 mRNA expression was reduced whereas immunoreactivity for GLUT-2 was increased. Both GK mRNA expression and immunoreactivity were reduced in most of the weanlings. Conclusions • Exposure to an in utero HFD throughout gestation induced hyperglycaemia in neonates. The reduced Pdx-1 expression appears to play a role in the compromised beta-cell development, and concomitant with the reduced GK levels, contributes to the hyperglycaemia in these neonates and may make them susceptible to beta-cell failure. • In most weanlings exposed to a HFD in utero and/or during lactation the hyperglycaemia and hypoinsulinaemia suggest compromised beta-cell function. The GK mRNA expression and immunoreactivity were reduced thereby impairing glycolysis which would result in reduced insulin secretion contributing to the hyperglycaemia. Furthermore, beta-cell development is adversely affected by the HFD in some weanlings. This would contribute to reduced beta-cell function and may eventually result in beta-cell failure. GLUT-2 immunoreactivity was increased in some, suggesting a compensatory adaptative mechanism to restore glucose homeostasis. • A maternal HFD has adverse effects both in neonates and weanlings on beta-cell development, transcription factor and glucose sensing gene expression and induced hyperglycaemia and hypoinsulinaemia in some of the offspring. Ways to ameliorate the HFD-induced attenuation of key beta-cell genes to ensure normal beta-cell function are important for future research in Type 2 diabetes. / AFRIKAANSE OPSOMMING: ‘n Hoe vet diet (HVD) verminder beta-sel masse, glukose signale en speel ‘n rol in Tipe 2 diabetes. Die hipothese is dat wanvoeding gedurende swangerskap lei tot onomkeerbare betasel beskadiging. Die transkripsiefaktore Pdx-1 en Pax 4 speel rolle in eilandselontwikkeling. Daar is bewyse dat Pdx-1 die uitdrukking van die GLUT-2, glucokinase (GK) en insulin gene reguleer. Doelstelling: Die doel van hierdie studie is om, in die pasgebore en gespeende rot, die effek van ’n HVD in utero en/of laktasie op gewig, glukose en insulin konsentrasies, eilandselontwikkeling, pankreatiese transksripsiefaktore en op glukosewaarnemingsgene te ondersoek. Metodes: Pasgebore en gespeende rotte is vir bepaalde periodes van gestasie en/of laktasie blootgestel aan ’n HVD van die moeder. Na terminase, is kleinjies geweeg en die glukose- en insulienkinsentrasies bepaal. mRNA uitdrukking van Pdx-1, Pax 4, GLUT-2 en GK is geoes met LightCycler PCR. Snitte van die pankreas is gekleur met insulien en glukagon (eilandsontwikkeling) en vir Pdx-1, GLUT-2 en GK (betaselfunksie) gevolg deur beeldanalise. Resultate: Bloodstelling aan ’n in utero HVD regdeur gestasie het hiperglisemie versoorsaak in pasgebore rotte met verlaagde betasel volume en aantal, Pdx-1 en GK immunoreaktiwiteit. In teenstelling daarmee was die alfasel se volume, aantal en grootte verhoog in pasgebore rotte wat regdeur gestasie aan 'n HVD blootgestel was. Meeste van die gespeende rotte was hiperglisemies en hipoinsulinemies. In sommige gespeende rotte, was daar ’n verlaging van betasel hoeveelheid en grootte en in alfasel groote. Oormatige uitdrukking van Pdx-1 mRNA het plaasgevind in speenlinge wat aan ’n HVD van die moeder vir die laaste week van gestasie of regdeur gestasie en laktasie blootgestel was, maar dit was verlaag in die speenlinge wat net tydens laktasie blootgestel was. Pax 4 mRNA was verlaag in speenlinge wat aan ’n HVD van die moeder blootgestel was vir die eerste of laaste week van gestasie, regdeur gestasie of regdeur laktasie. In meeste van die speenlinge is onder-uitdrukking van GLUT-2 mRNA, maar ’n verhoging van GLUT-2 immunoreaktiwiteit gevind. Beide GK mRNA uitdrukking en immunoreaktiwiteit was laer in meeste van die speenlinge. Gevolgtrekkings: • Blootstelling aan ’n in utero HVD regdeur gestasie lei tot hiperglisemie in pasgebore rotte. Die verlaagde Pdx-1 immunoreaktiwiteit speel klaarblyklik ’n rol by die geaffekteerde betaselontwikkeling. Dit, saam met die verlaagde immunoreaktiwiteit vir GK, kan bydra tot die hiperglisemie in hierdie pasgebore rotte. In die meeste van die speenlinge wat aan ’n HVD blootgestel was, dui die hiperglisemie en hipoinsulinemie op geaffekteerde betaselfunksie. Die GK mRNA uitdrukking en immunoreaktiwiteit is verlaag, wat weer glikolise benadeel, en dit sal lei tot verminderde insulienafskeiding wat bydra tot die hiperglisemie. Betaselontwikkeling word voorts negatief beinvloed deur die HVD, wat blyk uit die verlaagde aantal en grootte van betaselle. Dit sal bydra tot verminderde betaselfunksie. Dit kan uiteindelik tot betaselversaking lei. GLUT-2 immunoreaktiwiteit was verhoog in hierdie speenlinge, wat dui op ’n kompenserende aanpassingsmeganisme om glukose homeostase te herstel. ’n HVD van die moeder het ’n negatiewe uitwerking op betaselontwikkeling, transkripsiefaktor en glukosewaarneming geenuitdrukking in beide die pasgebore en gespeende rotte, en geinduseerde hiperglisemie en hipoinsulinemie in sommige kleintjies. Dis belangrik vir toekomstige Tipe 2 diabetes navorsing dat daar na gekyk moet word om die HVD-geinduseerde verlaging van sleutel betaselgene te verbeter vir optimale betaselfunksie.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy