Proteins play an important role in the function of biological systems. Developing a thorough understanding of the dynamics, structure and function is a goal of many biological spectroscopists. One commonly used tool for probing different features and properties of proteins and polypeptides is infrared (IR) spectroscopy. The spectra obtained however lacks resolution due to broad, featureless peaks that are hard to interpret. Overlapping of the bands is also an issue, especially from the amide I region; an important region that is sensitive to secondary structure elements of proteins. Isotope labelling of residues of interest is a solution to disentangle the band profiles, however the technique lacks any meaningful insight regarding coupling between different local sites. For the past few decades, 2D IR (an analogue of its NMR counterpart) has been used in various experiments to improve the quality of 1D IR by spreading the signal to a second frequency domain, thus revealing coupling interactions. The technique has helped reveal different relationships between the secondary structural elements of proteins or between protein-ligand complexes. The main challenge so far has been the computational requirement needed to compute the 2D IR signals of large proteins. This is due to the nonlinear response function scaling with the fourth power of the number of residues involved in the calculations. Moreover, convolution of the bands is still an issue as the number of residues grows, albeit not so much compared with 1D IR. In this thesis, we utilize the exciton approach and molecular dynamics (MD) simulations to compute the 1D and 2D IR signals of small peptides, globular proteins and [Leu]-Enkephalin in the presence of three opioid receptors of interest. The work presented in this thesis complements previous experiments on globular proteins and the behaviour of [Leu]-enkephalin and aims to provide more insight into the behaviour of such systems under the conditions outlined.

Increasing skeletal muscle carnitine content enhances PDC flux during 30 minutes of continuous exercise at 80% Wmax, reducing reliance on non-mitochondrial ATP production and improving work output. These studies in healthy volunteers evaluated a carnitine feeding strategy that did not rely on the high carbohydrate load previously used, then investigated whether manipulating muscle carnitine could alter the adaptations to a period of submaximal high-intensity intermittent training (HIT). The rate of orally ingested 2H3-carnitine uptake into skeletal muscle was directly quantified for the first time in vivo and increased 5-fold following ingestion of an 80g carbohydrate formulation. This positive forearm carnitine balance was entirely blunted when the carbohydrate load was supplemented with 40g of whey protein, suggesting a novel antagonisation of insulin-stimulated muscle carnitine transport by amino acids. Skeletal muscle biopsy sampling demonstrated minimal acetylcarnitine accumulation and non-mitochondrial ATP production during single-leg knee extension at 85% Wmax, suggesting that PDC flux does not limit oxidative ATP production under these conditions. Conversely, PDC flux declined over repeated bouts of cycling at 100% Wmax, as evidenced by greater non-mitochondrial ATP production in the face of similar acetylcarnitine accumulation. This suggested that muscle carnitine availability could influence oxidative ATP delivery during submaximal HIT. Manipulation of muscle carnitine content by daily carnitine/carbohydrate feeding elevated free carnitine availability and maintained PDC flux during repeated bouts of intense exercise. However, profound improvements in oxidative ATP delivery in response to HIT eclipsed any effect of this carnitine-mediated increase in PDC flux on non-mitochondrial ATP production and indeed, carnitine supplementation did not potentiate any increases in exercise capacity above submaximal HIT alone. These novel data advance our understanding of muscle carnitine transport and the interplay between carnitine metabolism, PDC flux and non-mitochondrial ATP production during intense exercise, having important implications for the development of nutritional and exercise prescription strategies to enhance human performance and health.

Tissue regeneration approaches involve the recreation of biochemical and mechanical cues dictating tissue fate. Gradients of chemical cues are common in the natural microenvironment and are usually accompanied with gradual changes in cellular responses. Consequently, thorough understanding of biomolecule gradient development, their effective concentrations and the corresponding cellular responses as a function of time and space are essential for efficient design of scaffolds for biomedical applications. Here, we developed a compartmental diffusion model to study the development and measurement of biomolecule gradients. The model was validated to ensure effective spatiotemporal measurements of diffusing species within three-dimensional (3D) hydrogels. Results confirmed that the factors regulating the diffusing molecules’ behaviour in hydrogel matrices were dependant on the size of the diffusing species and the interaction with the matrix. The source compartment was subsequently replaced by polymeric particulate depots with tuneable characteristics to maintain structural protein stability and provide controlled temporal release of proteins and the diffusion through the hydrogel compartment was accordingly monitored. Glycosaminoglycan enhanced transduction (GET) technology was employed to study 3D gradient transduction of reporter protein in cell-laden hydrogels and to examine the effect of cells on the diffusion of biomolecules. Results demonstrated that cellular uptake of GET proteins altered the diffusion pattern as compared to acellular scaffolds and cells themselves acted as a sink that maintained steep GET protein gradients over the 5 mm wide scaffold. Furthermore, the synergistic combination of poly-arginine cell penetrating peptide (CPP) together with the cell membrane binding peptide using the GET technology demonstrated significant intracellular transduction in a gradient fashion in comparison to CPP alone. Employing GET technology and the compartmental diffusion model in the gradient delivery of the transcription factor MyoD to cell-laden hydrogels, resulted in directing the cells towards myogenic differentiation. However, the gradient pattern of differentiation was not clearly observed due to the limited number of genes examined. In conclusion, the model can be employed for the effective spatiotemporal gradient delivery of functional proteins to achieve the tissue complexity observed in the native tissues.

NAD metabolism is increasingly implicated in a variety of biological functions, including regulation of gene transcription, lifespan, cell death, circadian rhythm and glucose home-ostasis. The three mammalian isoforms of the central NAD biosynthetic enzyme, nicotin-amide mononucleotide adenylyltransferases (NMNATs), have recently emerged as crucial players in neuronal maintenance and protection, in ageing-processes and in many neurodegenerative diseases, such as Alzheimer’s disease. The biological basis of ageing and its related pathologies are not fully elucidated and there is an urgent need to develop valid therapeutic strategies to minimize the impact that these conditions have on the society. In the present study, I used two mouse models, heterozygous Nmnat1 knockout mice (Nmnat1+/-) and mice overexpressing Nmnat1 (Nmnat1 tg), in order to assess the role of the nuclear isoform NMNAT1 in ageing and age-related diseases. First, I asked whether modulating the expression of NMNAT1 influences ageing-related mechanisms. To this aim, Nmnat1 tg and Nmnat1+/- mice were subjected to metabolic analysis and behavioral tests for measuring locomotor activity. Furthermore, NMNAT enzyme activity, NAD levels, gene expression and protein levels were analyzed with bio-chemical techniques at 6 and 24 months of age. I found that Nmnat1 tg mice had a reduced body weight and increased locomotor activity during ageing, while Nmnat1+/- weighted more at 12 months than their wild type littermates. NMNAT enzyme activity, significantly higher in Nmnat1 tg mice at 6 months, did not change during ageing. In contrast, NMNAT enzyme activity in Nmnat1+/-, which is already low in young mice, showed a trend of decrease with ageing. Second, in order to investigate the role of the nuclear isoform NMNAT1 in age-related diseases, I analyzed the effect of modulating NMNAT1 levels on behavioral and neuro-pathological traits in a mouse line which expresses non-mutant human tau isoforms (htau mouse), representing a model of tauopathy relevant to Alzheimer’s disease. To this aim, htau were crossed to Nmnat1 tg and Nmnat1+/- mice to produce experimental mouse groups with four genotypes that were all heterozygous for murine tau (mtau+/-): Nmnat1 tg/htau, Nmnat1+/-/htau and wild type littermates. Mice were subjected to a bat-tery of specific tests to assess potential behavioral abnormalities that correlate with dys-functions characteristic of AD. Furthermore, image analysis was performed to assess the integrity of the brain areas mainly impaired in AD. Finally, biochemical studies were conducted in order to test whether modulating NMNAT1 levels caused changes in NMNAT activity and in NAD levels in htau mice. I found that htau mice have an early, selective deficit in food burrowing, a behavioral task used to assess activities of daily living which are impaired early in Alzheimer’s dis-ease, and that overexpression of Nmnat1 ameliorates this defect. Despite the behavioral abnormalities, htau mice did not show neurodegenerative impairments in cortex and hippocampus. Modulating NMNAT1 levels produced a corresponding effect on NMNAT enzymatic activity but it did not alter NAD levels in htau mice. My results suggest beneficial effects of NMNAT1 on the early behavioral deficits in this mouse model of tauopathy. Finally, I asked whether modulation of Nmnat1 can influence the ischemic cell death, which is at least in part caused by an overactivation of PARP1 and NAD depletion. I hypothesized that reducing NMNAT1 levels exacerbates the ischemic brain damage, whereas increasing these levels confers protection by respectively decreasing or increas-ing NAD availability within the nucleus. To address this question, I used a stable NSC-34 clone expressing Wlds/NMNAT1, as well as wild type NSC-34 cells. I measured NAD levels and then tested cell viability af-ter genotoxic stress induced by N-Methyl-N-nitro-N-nitrosoguanidine (MNNG), which causes DNA damage and activation of PARP1, mimicking some mechanistic aspects of ischemic cell death. I first confirmed that Wlds was expressed in all cellular fractions in Wlds/NMNAT1 cells, but not in wild type cells. Furthermore, both wild type and Wlds/NMNAT1 cells showed lower NAD levels in the nuclear fraction compared to the cytoplasmic fraction. Paradoxically, NAD levels tended to be even lower in all cellular fractions of Wlds/NMNAT1 cells respect to the wild type cells. Correspondingly, they show higher susceptibility to cell death induced by MNNG. In addition to the in vitro experiments, I also started to set-up an in vivo model of cerebral ischemia. Even though time constraints did not allow analyzing the effects of Nmnat1 modulation, my study will be useful for future investigations to test whether overexpression of Nmnat1 or its downregulation may protect or exacerbate the effect of cerebral ischemia in an in vivo model. Taken together, my results suggest that modulating Nmnat1 correspondingly influences ageing and neurodegeneration processes and underline the utility of the Nmnat1 tg and Nmnat1+/- mice as a tool for future research in this field.

The nonspecific binding of proteins to various biological and non-biological surfaces has limited the potential of several detection techniques such as surface plasmon resonance (SPR), Luminex and ELISA (among others). Plasma proteins have been shown to decrease the sensitivity of instruments when working with complex fluids such as blood samples. This study examines the binding properties of several plasma proteins to a range of surfaces and utilises reagents in the media to eliminate the nonspecific binding of the plasma proteins. This has created a set of conditions that can reduce the nonspecific binding interaction, without affecting the specific interactions which the various techniques measure. These mechanisms were then applied to the assembly/disassembly of membrane microdomains. Membrane microdomains have been shown to be affected by from several factors such as acyl-chain length and temperature. This study demonstrates how reagents in the media can affect the assembly of these domains. We proposed a novel mechanism for the regulation of the domains, in which the reagents alter the intermolecular interactions between lipid head groups by altering the water network around these domains to promote domain assembly. These results that could have significant ramifications for the functional characterisation of membrane microdomains and the proteins that are known to associate with them. Finally, membrane binding affinity and kinetics of different polypeptides with various lipid membrane composition were characterised and resultant microdomains were monitored. Demonstrating how the previously uncontrolled, soluble factors in a model system can control the intermolecular interactions that occur in the system that is being measured.

The aryl hydrocarbon receptor (AhR) binds a wide range of structurally diverse compounds such as halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls which are abundant in the environment. Activation of AhR leads to the regulation of a battery of xenobiotic enzymes including cytochrome P4501A1 (CYP1A1). The purely chlorinated compounds feature in the World Health Organisation’s (WHO) evaluation of dioxin-like compounds derived from a meta-analysis of previous potency data (toxic equivalency factors; TEFs), which is used to calculate the total toxic equivalence (TEQ). The first aim of this work was to fully characterise the three most environmentally abundant mono-ortho-substituted polychlorinated biphenyls (PCBs; PCB 105, 118 and 156) including a re-evaluation of their putative antagonistic effects on AhR. Secondly, the effects of mixed halogenated compounds, currently not included in the TEQ estimation, were investigated as AhR agonists based on their environmental exposure and potency. Quantitative real-time PCR (qRT-PCR) was used to measure the AhR mediated induction of CYP1A1 mRNA in rat H4IIE and human MCF-7 cells. The three mono-ortho-substituted PCBs were shown to be antagonists of rat and human AhRs, an effect which is not currently included in the TEQ calculation. 2-bromo-3,7,8-trichlorodibenzo-p-dioxin (2-B-3,7,8-TriCDD) was found to be an AhR agonist that was 2-fold more potent than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; considered one of the most potent in the environment). The majority of the other tested compounds were found to be within 10-fold less potent than TCDD and could therefore have a significant impact on the TEQ. A family of putative AhR agonists from AstraZencea were investigated and one of the compounds was shown to be a highly potent AhR agonist, 5-fold more potent than TCDD at inducing CYP1A1. The results indicate approximately a 15-fold higher sensitivity of the rat cell line to the AhR agonists compared with the human cell line. It is not currently understood what confers these differences whether it is a difference in the mechanism of activation or purely as a result of differences in the AhR sequence. The mechanism of action is thought to be the same in both species and the associated proteins are both comparable. The amino acid sequences of the AhR, in both human and rat are quite similar but may play a significant role in the differences observed between species. Therefore in order to directly compare the rat and human AhRs, two novel cell line models were created using an inducible expression system to infect an AhR-deficient mouse cell line with a replication-defective virus containing either the rat or human AhR. The AhRs were activated with various compounds to induce mouse CYP1A1. The CYP1A1 mRNA was measured using qRT-PCR but showed that the two AhR genes were not expressed enough to produce a response detectable above the background CYP1A1 induction by the low levels of mouse AhR. This research has shown that these dioxin-like compounds can have very different potencies at AhRs in different species so it is not always possible to predict the potency in humans from in vitro or rat in vivo toxicity data. Furthermore, it has identified compounds, such as 5F-203, which are significantly more potent in human compared to rat. This thesis provides information on the AhR species differences between human and rat that can be applied to risk assessment.

The detection and control of microorganisms such as bacteria is important in a wide range of industries and clinical settings. Detection, binding and removal of such pathogenic contaminants can be achieved through judicious consideration of the targets which are available at or in the bacterial cell. Polymers have the ability to present a number of binding ligands for cell targeting on one macromolecule and so avidity of interaction can be greatly increased. The goal of the project was to test whether polymers generated with bacteria in situ would have their composition significantly altered to determine if a templating process was occurring. It was also anticipated that the templated polymers would have better re-binding properties than those produced in the absence of bacteria. A series of chemical functionalities were analysed for their binding properties to bacteria. The functionalities were chosen with consideration to the cell surface characteristics. Further to identification of the most binding and least binding functionalities the polymers were tested for their cytotoxicity against bacteria and human epithelial cells. Concentration ranges were determined which could facilitate bacterial binding and templating yet minimise the lethality of the processes. Templated polymers of the bacteria were generated using a novel method of atom transfer radical polymerisation (ATRP) which we have termed bacterial activated atom transfer radical polymerisation (b-ATRP). This polymerisation method has maximised the potential for templating processes to occur during the polymerisation. Templated polymers differed in both their composition and their binding behaviour to non-templated polymers. The bacterial organic reduction process has also been demonstrated to have greater scope for use within the organic chemistry field as demonstrated by the use of this system to enable in "click-chemistry" via the reduction of copper.

Proteins are the functional machines of nature and play an essential role in life. Understanding the structure and function of proteins is central to numerous areas of science and technology, including biotechnological, pharmaceutical and chemical industries. Protein crystals are an important objective for many researchers, but even though there are many examples of protein crystals reported, there are still many uncertainties in controlling the crystallisation process. In addition, while protein crystals have mostly been obtained for characterisation purposes, there are other applications where defined assemblies of proteins, either crystals or self-assembled protein nanoparticles, are desirable. These include industrial biocatalysis, therapeutic protein formulations, and even energy-harvesting systems. Accordingly, there remains a high demand for well-defined protein crystals or nanoscale aggregates. A variety of techniques have been used to produce protein crystals, the typical strategy involves the addition of nucleants surface, or surface-active materials into the crystallisation solution. Solution ‘additives’ of diverse types have proven crucial in the control of protein crystallisation. These additives can act via multiple roles in a crystallisation process, such as by enhancing intermolecular contacts between protein macromolecules, or disrupting unfavourable intermolecular association, or diminishing interactions between protein and solvent. Synthetic polymers are important additives for protein crystallisation control as there are many possible chemistries which can be introduced into the backbone and side-chains, giving a very wide range of functional behavior. However, because there are so many factors which can affect crystallisation, many of which are still poorly understood, to date only a very few classes of polymers have been studied as additives for protein crystallisation. Moreover, the mechanisms governing their interactions with protein crystals or surrounding solvent are elusive. In the study presented here, we show that polymers designed with varying degrees of charge, molecular weight and backbone structure influenced the crystallisation of three target proteins: hen-egg white lysozyme (HEWL); concanavalin A (Con A); and bovine liver catalase (BLC). Polymers were prepared as ‘additives’ into protein solution to influence the proteins crystallisation process, leading to changes in size, habit, morphology and even polymorph of the final crystals. A simple model linking polymer structure and charge to protein isoelectric point (pI) and crystallisation rate is proposed.

Os eritrócitos dos mamíferos são anucleados e desprovidos de organelas citoplasmáticas, contando somente com o ciclo da glicólise, o ciclo das pentoses e algumas enzimas anexas, o que garante o fornecimento de energia calórica sob a forma de adenosina-5\'-trifosfato (ATP) e de energia redutora sob a forma de nicotinamida adenosina dinucleotídeo reduzida (NADH), nicotinamida adenosina dinucleotídeo fosfato reduzida (NADPH) e glutationa reduzida (GSH). A via glicolítica possui o desvio denominado de ciclo de Rapaport-Luebering, onde há a síntese de 2,3- difosfoglicerato (2,3-DPG), que é um importante metabólito e atua como modulador da afinidade da hemoglobina ao O2. Havendo poucos estudos comparativos sobre o metabolismo eritrócitário dos mamíferos propôs-se investigar as atividades das enzimas glicolíticas, anexas (2,3-difosfogliceratomutase, glicose-6-fosfato desidrogenase e 6-fosfogliconato desidrogenase) e a concentração dos compostos intermediários adenosina-5\' -trifosfato e 2,3-difosfoglicerato. Mamíferos das ordens Primates, Rodentia, Camivora, Lagomorpha, Artiodactyla, Didelphimorphia e Xenarthra oriundos da Fundação Parque Zoológico de São Paulo e Centro de Bioterismo da Faculdade de Medicina da USP foram investigados. O sangue foi colhido em ACD, os eritrócitos foram lavados em solução fisiológica a 4°C e hemolisados em solução hemolisante 1:20 por congelamento e descongelamento e as atividades das seguintes enzimas foram determinadas de acordo com Beutler (Red Cell Metabolism, a Manual of Biochemical Methods, Ed. Grune & Stratton, 3rd ed, 1984): hexoquinase, glicose fosfato isomerase , fosfofrutoquinase, aldolase, triose fosfato isomerase, gliceraldeído 3-fosfato desidrogenase, fosfoglicerato quinase, monofosfogliceromutase, enolase, piruvato quinase, lactato desidrogenase, bem como a 2,3-difosfoglicerato mutase, glicose-6-fosfato desidrogenase, 6-fosfogluconato desidrogenase, os metabólitos intermediários 2,3-difosfoglicerato e adenosina-5\'-trifosfato. As enzimas e os compostos intermediários estudados apresentaram grande variabilidade entre as espécies de mamíferos estudadas. Foi observada correlação positiva entre a atividade da triose fosfato isomerase e a 2,3-difosfoglicerato mutase e os teores de adenosina-5\'-trifosfato das espécies, bem como correlação positiva entre a 2,3-difosfoglicerato mutase em relação ao 2,3-difosfoglicerato. Os teores de adenosina-5\'-trifosfato mantiveram-se dentro de um patamar estável, ao redor de 4.000 a 6.000 nmoles / gHb, com as exceções das espécies das ordens Carnivora (Panthera leo, Leopardus pardalis, Canis lupus and Chrysocyon brachyurus) e Artiodactyla (Cervus elaphus), que exibiram teores ao redor de 2.000 a 3.000 nmoles / g Hb. Já os valores da concentração de 2,3-difosfoglicerato apresentaram variação considerável entre as espécies e ordens estudadas. / Mammalia red cells are non-nucleated and do not have cytoplasm organeles as well, and present the glycolytc pathway, the pentose shunt to attend the requirements in caloric energy as adenosine-5-triphosphate (ATP) and reducing power as reduced nicotinamide adenosine dinucleotide (NADH) and reduced nicotinamide adenosine dinucleotide phosphate (NADPH) and reduced glutathione (GSH). The glycolytic pathway exhibit besides the Luebering-Rappaport shunt, in which the 2,3-diphosphoglycerate is formed, which regulates the hemoglobin affinity to the molecular oxygen. As there are not so many studies on comparative about mammalian red cell metabolism, it was decided to study the glycolytic enzyme activities, the glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 2,3-diphosphoglycerate mutase and the metabolites adenosine-5-triphosphate and 2,3-diphosphoglycerate (2,3-DPG). Mammalia representatives from Primates, Rodentia, Carnívora, Lagomorpha, Artyodactyla, Didelphimorphia and Xenarthra orders, obtained from Fundação Parque Zoológico de São Paulo and Centro de Bioterismo da Faculdade de Medicina da USP, were studied. The blood was collected in ACD, the red cells were washed in saline at 4° C, lysed 1:20 in hemolysing solution by freeze-and-thaw, and the following enzymes were assayed according to Beutler (Red Cell Metabolism, a Manual of Biochemical Methods, Ed. Grune & Stratton, 3rd ed, 1984): hexokinase, glucose-6-phosphate isomerase, phosphofructo kinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, monophosphoglycerate mutase, enolase, pyruvate kinase, lactate dehydrogenase activities, as well as 2,3-diphosphoglycerate mutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities, and adenosine-5-triphosphate, 2,3-diphophoglycerate concentrations. A remarkable variation among the studied species was observed. However, it was detected a significant positive correlation between the adenosine-5-triphosphate concentrations and triosephosphate isomerase and 2,3-diphosphoglycerate mutase activities, as well as significant positive correlation between 2,3-diphosphoglycerate concentration and 2,3-diphosphoglycerate mutase activity in all studied species as a whole. Most of studied species exhibited a steady ATP concentration range between 4,000 and 6,000 nmoles.g Hb -1 but the Artiodactyla (Cervus elaphus) and Carnivora (Panthera leo, Leopardus pardalis, Canis Lupus and Chrysocyon brachyurus,) which presented values between 2,000 and 3,000 nmoles Hb - 1. However, the 2,3-DPG concentration showed remarkable variation among the studied species and orders.

Thesis (PH.D. (Medical Sciences)) --University of Limpopo, 2010 / Refere to document