Deciphering the mechanisms that underlie circuit function in the hippocampal formation is a key challenge for neuroscience. This region, which includes the medial entorhinal cortex (MEC), is critical for spatial learning and episodic memory in humans. Spatially modulated cells in the MEC, the grid cells, provide a topographical representation of space, but we are yet to establish the neuronal properties that underlie this or the contribution that particular cells in different regions of the MEC and hippocampus make to circuit function. This is partially because the specific targeting of the network with genetic tools is complicated by a multitude of cell types with predominantly unknown molecular profiles. To address our limited understanding of the molecular organisation of the MEC, I have characterised how the expression of genes is distributed throughout different layers of the MEC, using a custom-designed resource that facilitates analysis of in situ hybridisation data from the Allen Brain Atlas. Through simultaneous extraction of gene expression data across thousands of 2D aligned images, I reveal striking differences between layers within MEC, demonstrating that layer II contains the highest proportion of genes enriched in a single layer, whereas gene expression is very rarely confined to layer III. Of particular interest, layer II of MEC is highly enriched for Alzheimer’s disease pathway genes, providing insight into its vulnerability as one of the first brain regions to show pathology. I also identify over 1000 genes that are expressed with a dorso-ventral gradient that maps onto the topographic organisation of MEC connectivity, grid cell spatial resolution and synaptic integrative properties of cells. An intriguing group of genes that closely relate circuit activity to gene expression, the plasticity-related activity-dependent genes, often show this pattern of expression. Focussing on the activity-dependent expression of one such activity-regulated, plasticity-related gene, Arc, I provide a novel view of MEC function. During simple novel exploration, Arc expression is up-regulated to a much greater extent in the deep layers of dorsal MEC than in the grid cell-rich superficial layers. By selectively disrupting the predominant hippocampal input to dorsal MEC, which terminates in the deep layers, I show that the significance of this up-regulation is independent of hippocampal inputs. Thus, although research addressing MEC function is particularly focussed on the superficial layers, during the exploratory behaviour that potentially primes the system for representing an environment, important plasticity may be occurring at the synapses onto deep layer neurons. In summary, my investigations of baseline and activity-dependent gene expression in MEC have revealed a molecular organisation both across different layers and along a functionally relevant gradient. This may be important for specifically targeting microcircuits in MEC and for characterising how laminar and regional differences contribute to the encoding of space in the hippocampal formation.

Sepsis-related multiple organ dysfunction syndrome is a leading cause of death in intensive care units. There is overwhelming evidence that oxidative stress plays a significant role in the pathogenesis of sepsis-associated multiple organ failure; however, reactive oxygen species (ROS)-associated biomarkers and/or diagnostics that define mortality or predict survival in sepsis are lacking. Lung or peripheral blood gene expression analysis has gained increasing recognition as a potential prognostic and/or diagnostic tool. The objective of this study was to identify ROS-associated biomarkers predictive of survival in patients with sepsis. In-silico analyses of expression profiles allowed the identification of a 21-gene ROS-associated molecular signature that predicts survival in sepsis patients. Importantly, this signature performed well in a validation cohort consisting of sepsis patients aggregated from distinct patient populations recruited from different sites. Our signature outperforms randomly generated signatures of the same signature gene size. Our findings further validate the critical role of ROSs in the pathogenesis of sepsis and provide a novel gene signature that predicts survival in sepsis patients. These results also highlight the utility of peripheral blood molecular signatures as biomarkers for predicting mortality risk in patients with sepsis, which could facilitate the development of personalized therapies.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The fission yeast Schizosaccharomyces pombe is able to effectively degrade extracellular L-malate by means of a permease for the active transport of L-malate and a malic enzyme that catalyses the intracellular oxidative decarboxylation of L-malate to pyruvate and CO2. Sequence analysis of the S. pombe NAD-dependent malic enzyme gene, mae2, revealed an open reading frame of 1695 nucleotides, encoding a polypeptide of 565 amino acids. Mutational analyses of the mae2 promoter region revealed several putative cis-acting elements. Two of these elements have homology with binding sites for eukaryotic cAMPdependent regulatory proteins. The UAS I showed homology with the invert of the ADRI binding site, an AP-2 binding site and the TGGCA element. The other putative cAMPdependent site, UAS2, showed homology with the binding site for ATF/CREB and proved to be a strong activator sequence that is required for expression of the mae2 gene. Three negative acting elements, DRS I, DRS2 and DRS3 seem to function co-operatively to repress transcription of the mae2 gene. In this study northern and western blot analyses, as well as malic enzyme assays, showed increased levels of mae2 transcription and enzyme activity when cells were grown under fermentative conditions. The levels of mae2 expression increased approximately 4-fold in 30% glucose and 3-fold under anaerobic conditions. These increased levels of malic enzyme may provide additional pyruvate for various metabolic processes when the mitochondria are not fully functional under fermentative conditions. The regulated expression of the mae2 gene was further investigated using mae2-1acZ fusion plasmids that carried mutations in the DASI, UAS2 or the triple mutated DRSI/URS2/URS3 elements. These plasmids were transformed into S. pombe strains with mutations in the cAMP-dependent or stress-activated signal transduction pathways to determine the signal for the increased expression of the mae2 gene. The cAMP-dependent (Pkal ) and general stress activated (Styl) pathways often act in parallel to regulate the activation of transcription factors necessary for the expression of several S. pombe genes under different physiological conditions. The results presented here suggest that regulatory proteins involved in the Pka l and Styl pathways play a role in the regulation of the mae2 gene under fermentative conditions. Furthermore, some of the regulatory cis-acting elements in the mae2 promoter may interact with these trans-acting factors to regulate the transcription of the gene under different growth conditions. The mechanism of this interaction is not yet known and further research is required to identify all the transcription factors involved in the regulation of the mae2 gene. / AFRIKAANSE OPSOMMING: Die splitsingsgis S. pombe is in staat om ekstrasellulêre L-malaat effektief af te breek danksy 'n permease vir die aktiewe opname van L-malaat en 'n malaatensiem wat die intrasellulêre oksidatiewe dekarboksilering van L-malaat na pirovaat en C02 kataliseer. DNA-geen opeenvolgings van die NAD-afhanklike malaatensiemgeen, mae2, het 'n oopleesraam van 1695 nukleotiede getoon wat vir 'n polipeptied van 565 aminosure kodeer. Mutasie-analise van die mae2-promoter gebied het verskeie moontlike cis-werkende elemente getoon. Twee van die elemente toon homologie met bindingsetels vir eukariotiese cAMP-afhanklike regulatoriese proteïene. Die DAS 1 toon homologie met die omgekeerde volgorde van die ADRI bindingsetel, 'n AP-2 bindingsetel en 'n TGGCA element. Die ander moonlike cAMP afhanklike setel, DAS2, toon homologie met die bindingsetel vir ATF/CREB en is 'n sterk aktiveringselement wat vir die uitdrukking van die mae2-geen benodig word. Drie onderdrukker-tipe elemente, DRSI, DRS2 en DRS3, funksioneer moontlik gesamentlik om die transkripsie van die mae2-geen te onderdruk. In hierdie studie het northern en western klad analise, sowel as malaatensiem aktiwiteitstoetse verhoogde vlakke van mae2-transkripsie en ensiemaktiwiteit getoon wanneer die kulture onder fermentatiewe toestande gegroei het. Die uitdrukking van die mae2-geen het ongeveer 4-voudig toegeneem in 30% glukose en 3-voudig onder anaërobiese toestande. Hierdie verhoogde uitdrukking van die malaatensiem mag addisionele pirovaat vir verskeie metaboliese behoeftes voorsien wanneer die mitochondria onder fermentatiewe toestande nie volkome funksioneer nie. Die uitdrukking van die mae2-geen is verder onder fermentatiewe toestande bestudeer deur gebruik te maak van mae2-lacZ-fusie plasmiede wat mutasies in die moontlike DASI, DAS2, of die drievoudig-gemuteerde DRS I/URS2/URS3 setels bevat. Hierdie plasmiede is in S. pombe rasse met mutasies in die cAMP-afhanklike of stres-geaktiveerde seintransduksie paaie getransformeer om die sein vir die verhoogde mae2-geen uitdrukking te bepaal. Die cAMP-afhanklike (Pkal) en algemene stres-aktiverings (Styl) pad werk soms in parallel om die aktivering van transkripsiefaktore betrokke in die uitdrukking van verskeie S. pombe gene onder verskillende fisiologiese toestande to bewerkstellig. Ons resultate dui daarop dat die regulatoriese proteïene van die Pkal en die Styl paaie 'n rol in die regulering van die mae2- geen onder fermentatiewe toestande speel. Daar is ook aanduidings dat sommige van die regulatoriese cis-werkende elemente in die mae2-promoter wisselwerking met die transwerkende faktore toon om die transkripsie van die geen onder verskillende groeitoestande te reguleer. Die meganisme van hierdie interaksie is nog nie bekend nie en verdere navorsing is nodig om al die transkripsiefaktore wat by die regulering van die mae2-geen betrokke is, te identifiseer.

Psychiatric illnesses are disorders that affect millions worldwide. Evidence from quantitative and molecular genetics analysis suggests a strong genetic component to these disorders. There is also evidence that embryonic neurodevelopment is a key period in the progression schizophrenia. The aim of the present study was to use post-mortem human hippocampus from subjects of a variety of psychiatric phenotypes to investigate neurodevelopmentally- relevant gene expression in this region of the adult human brain. Particular interest is paid to schizophrenia risk gene DISC1; it has been shown to exhibit linkage and association to schizophrenia and is highly involved in embryonic and post natal neurodevelopmental processes. The results reported in this study indicate that DISC1 binding partners, and genes used to mark neurogenesis, can be found aberrantly expressed in schizophrenia and bipolar disorder, relative to controls. The results also suggest that DISC1 genotype may predict expression patterns of DISC1 binding partners and neurogenesis markers, irrespective of diagnosis. This may provide clues to the timing and nature of abnormal brain development in this illness and aid in development of treatment strategies.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy