Abstract The triosephosphate isomerase (TIM) barrel superfamily is a broad family of proteins, most of which are enzymes. At the amino-acid-sequence level, many of the members of this family share little, if any, homology. Yet, they adopt the same three-dimensional (βα)8 fold. The TIM barrel fold seems to be a good framework for many different kinds of enzymes, providing unique possibilities for both natural and human-designed evolution, as the catalytic center and the stabilizing features are separated to different ends of the barrel. Indeed, in the light of most recent studies, it seems likely that at least most of the different TIM barrel enzymes, catalyzing a huge variety of reactions, have evolved from a common ancestor. TIM can be considered a real text-book enzyme — its catalytic properties and stucture-function relationships have been studied for decades. Still, at present, we are quite far from understanding the structural features that make TIM and other enzymes such superior catalysts in both efficiency and precision. TIM is a dimeric enzyme that consists of two identical subunits of 250 residues. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in glycolysis. The basics of this reaction are well known, but there is ongoing discussion about the details of the proton transfer steps, and three alternative pathways have been suggested. In addition, it is a fascinating question how the enzyme succeeds in abstracting a highly stable proton from a carbon atom of the substrate. This study was undertaken to shed light on some of the questions concerning the structure-function relationships in TIM. The most important findings are the elucidation of the role of Asn11 as a catalytic residue and the meaning of the flexibility of both the catalytic Glu167 side chain as well as the substrate during catalysis, and the presence of a low-barrier hydrogen bond between Glu167 and a transition-state analogue, 2-phosphoglycolate. Furthermore, significant results were obtained on the importance of a conserved salt bridge, 20 Å away from the active site and the dimer interface, for the stability and folding of TIM as well as on the factors influencing the opening of the flexible loop 6 upon product release.

Master of Science / Department of Geology / Saugata Datta / Elevated levels of dissolved arsenic (As), iron (Fe) and manganese (Mn) are seen in the shallow, anoxic groundwaters of southeast Bangladesh on the Ganges- Brahmaputra- Meghna River delta. Over the past decade the mechanisms of As release have been widely debated. It is understood that As can sorb onto Fe-bearing minerals and can be subsequently released when reactions, such as microbially driven processes, occur. This study takes a multi disciplinary approach to understand the extent of the natural reactive barrier along the Meghna River and to evaluate the role of the natural reactive barrier in As sequestration and release in groundwater aquifers. River water and groundwater interactions occur in the hyporheic zone, which is defined as the transient subsurface region where river water and groundwater mix. The natural reactive barrier can develop within the hyporheic zone, where Fe-bearing minerals accumulate with a potential for As sorption, along with reworking and re-deposition of sediments along the riverbank. Shallow sediment cores, and groundwater and river water samples were collected from the east and west banks of the Meghna River in Jan. 2016. Groundwater and river water samples were tested for total dissolved Fe, Mn, and As concentrations; δ₂H, δ₁₈O isotopic ratios. Fluorescence spectroscopic characterization of groundwater organic matter provided insight into the hydro-geochemical reactions active in the groundwater and the hyporheic zone. Eight sediment cores of ~1.5 m depth were collected ~10 m away from the edge of the river. Vertical solid-phase concentration profiles of Fe, Mn, and As were measured by four different methods (hand-held XRF, and ICP-OES analysis of 3 digestions: aquaregia (HNO₃: HCl 1:3), 1.2 M HCl, and 1 M NaH₂PO₄ + 1 M L-ascorbic acid extractions). Enrichment of solid phase Fe, Mn, and As and the presence of possible Fe and Mn oxides in the sediments illustrate the existence of an natural reactive barrier at this reach of the Meghna. HCl extractions of sediment revealed solid-phase As accumulation along the west riverbank reaching concentrations of ~1500 mg/kg. Aqueous geochemical results showed the highest dissolved As concentrations in shallow wells (<30 m depth), where organic matter was fresh, humic-like, and aromatic. Humic-like dissolved organic matter present in the groundwater may enhance Fe oxide dissolution. Microbial reduction of organic matter prompts the reduction of Fe³⁺ to Fe²⁺, causing As to mobilize into groundwater. This study quantified the extent of As accumulation in the sediments along a 1 km stretch of the Meghna River. These findings contribute to the understanding of geochemical processes involved in As release into groundwaters from sediments within a fluvial deltaic environment.

>Magister Scientiae - MSc / The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.The aim of this study was to investigate in vitro effects of pure and street MA "tik" on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo® luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P<0.05 was denoted as significant. Results of this study showed that: 1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P>0.05). 2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P≤0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P ≤.0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P≤0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P≤0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of the Rf extract was able to partially alleviate excess ROS-induced effects.

Obesity is an independent risk factor for ischaemic stroke and may worsen stroke outcome. Therefore, the objective of this thesis was to identify potential interactions between obesity and stroke, with particular focus on inflammatory mechanisms. Ischaemic stroke was surgically induced in obese (ob/ob) and control (ob/-) mice by middle cerebral artery occlusion (MCAO). Outcome was worse in ob/ob mice, which showed rapid and severe ischaemic and vascular damage, characterised by blood vessel haemorrhage as early as 30 minutes post-stroke. The early vascular damage is likely an important driver of subsequent ischaemic damage by exacerbating inflammation in the brain. Assessment of systemic inflammation in ob/ob mice found that stroke had triggered a deregulated inflammatory response in the plasma, spleen, liver and bone marrow, and increased adipose tissue inflammation. Increased vascular damage was hypothesised to mediate the worse outcome found in obese rodents. The structural and inflammatory state of the vasculature was therefore assessed in ob/ob mice prior to and after stroke. Prior to stroke, increased vascular inflammation was found in ob/ob mice, though no differences in tight junction expression or structural alterations were noted. Stroke resulted in vascular damage that was similar on structural and molecular levels between genotypes, though ob/ob mice showed an increase in transcytosis in endothelial cells which may mediate their enhanced blood-brain barrier breakdown. Some studies have found obese patients have better outcome after stroke, perhaps because they are protected from detrimental post-stroke weight loss. The metabolic response to MCAO was therefore assessed, with ob/ob mice showing altered lipid metabolism post-stroke, such as increased fatty acid release from adipose tissue into the plasma. The potential role of fatty acid release in triggering adipose inflammation was also assessed in adipose tissue explants, though limited evidence for an inflammatory response to lipolysis was found. Overall, these findings suggest rapid and enhanced vascular damage may drive worse stroke outcome in ob/ob mice, as may their altered immunological and metabolic states.

The permeation of drugs across the blood-brain barrier (BBB) is a prerequisite for central nervous system (CNS) drug penetration. The BBB, possessing efflux transporters and tight junctions, limits drug penetration to the brain. Consequently, the discovery of novel drugs to treat CNS diseases remains problematic and is lagging behind other therapeutic areas. In vitro assays have progressed understanding of the factors that govern brain penetration. Central nervous system drug penetration is now thought to be modulated by three main processes, namely BBB permeability, active transport at the BBB and drug binding in blood and brain tissue. A more integrated approach to CNS drug discovery programmes is emerging which encompasses these processes in order to examine the rate and extent of drug brain penetration across species and improve predictions in human.A primary porcine in vitro BBB model was developed and characterised for the prediction of CNS drug permeability in vivo. Characterisation confirmed that the model exhibited physiologically realistic cell architecture, the formation of tight junction protein complexes, transcellular electrical resistance consistently >2000 Ω.cm2, functional expression the P-gp efflux transporter and ?-glutamyl transpeptidase and alkaline phosphatase activities.Transport of 12 centrally acting test drugs was investigated across four in vitro BBB models in order make comparisons between models and to generate in vitro permeability and efflux measurements. Blood-brain barrier permeability and active efflux processes are two major influences on the rate of drug penetration across the BBB. Species differences in fublood and fubrain, two prime influences on the extent of drug penetration, were investigated using equilibrium dialysis. Fraction unbound in brain was shown to be comparable across species suggesting that species differences in brain penetration could be due to variation in fublood for drugs that cross the BBB by passive diffusion, and/or species differences in transporter characteristics for drugs that are subject to active transport processes at the BBB. An in-house hybrid-PBPK rat CNS model was used to predict calculated rat Kp,uu using in vitro permeability, efflux, fublood and fubrain parameters generated during this work. The predicted Kp,uu generated using the rat CNS hybrid-PBPK model were within 3-fold of calculated Kp,uu. The rat CNS hybrid-PBPK model has potential use, as a tool for drug discovery scientists to aid the prediction of the extent of drug penetration in the early stages of drug discovery.This work has demonstrated that in vitro permeability and unbound drug fraction can be used to predict CNS drug penetration.

The electrically active defects introduced in GaAs by electron beam deposition (EB) of Ta were characterised. The effect of electron beam deposition on the electrical properties of GaAs was evaluated by current-voltage (I-V), capacitance¬voltage (C- V) and deep level transient spectroscopy (DL TS). However, when electronic devices are formed by EB, defects may be introduced into the semiconductor material, depending on the properties of the metal being deposited. Depending on the application, these defects may have either advantages or detrimental effects on the performance of such a device. I-V measurements indicated that the EB induced damage results in an increase in ideality factor and decrease in the barrier height with increasing the applied substrate bias, while C- V measurements showed that EB deposition also caused a decrease in the barrier height. DL TS studies on the same material in the temperature range of 20 - 350 K showed that at least three electrically active defects are introduced during EB deposition, with energies (0.102 ± 0.004, 0.322 ± 0.014 and 0.637 ± 0.029 eV) within the band gap. DL TS data was used to construct concentration profiles of these defects as a function of depth below the surface. It was found that the defect concentration increases with increasing substrate bias during the deposition, irrespective of the direction of the applied bias. This may be related to the I-V characteristics of the SBDs. The SBDs investigated by IV measurements showed that GaAs yields SBDs with poorer characteristic. The influence of EB deposition on the device properties of SBDs fabricated on GaAs is presented. These device properties were monitored using a variable temperature I-V and C- V apparatus. In order to have an understanding of the change in electrical properties of these contacts after EB deposition, it is necessary to characterise the EB induced defects. DL TS was used to characterise the defects in terms of their D L TS signature and defect concentration. / Dissertation (MSc (Physics))--University of Pretoria, 2006. / Physics / unrestricted

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

The state of North Carolina is home to some of the most spectacular barrier islands in the world. These features are constantly shifting, impacted by waves, tides, and wind. Studies of the Outer Banks, North Carolina have resulted in varied results, but a detailed analysis of the barrier system as a whole is lacking. Using historic topographic surveys (T-sheets) from the 19th, the positions of various barrier segments were analyzed in relation to modern imagery. Changes in area, width, and center line locations were evaluated over the past 150 years. In total, 74 percent of modern transects have decreased in area. Total reductions in size were 130 km2 for the study period. Mean centerlines as a function of migration showed that 53 percent of segments were demonstrating directional movement away from the ocean. The average movement towards the bay between modern and historic centerlines was 8 meters. Thusly, barrier islands in North Carolina are demonstrating both decreases in total area and directional movement inland in response to sea level rise.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.