nÃo hà / No mundo, existem cerca de 3.000 espÃcies de serpentes das quais 10 a 14% sÃo peÃonhentas. Dentre os paÃses sul-americanos, o Brasil à o que apresenta maior nÃmero de acidenÂtes/ano com cerca de 20.000 acidentes ofÃdicos por ano. De acordo com o MinistÃrio da SaÃde, as serpentes do gÃnero Bothrops sÃo as principais envolvidas nos acidentes ofÃdicos no paÃs e a insuficiÃncia renal aguda (IRA) à uma complicaÃÃo grave dos envenenamentos produzidos por estas serpentes. Tendo em vista que a fraÃÃo L-aminoÃcido oxidase (LAAO) constitui grande parte da composiÃÃo total do veneno de serpentes, em algumas serpentes chegando a constituir mais de 30% do total de proteÃnas do veneno, neste trabalho, foram investigados os efeitos renais da fraÃÃo L-aminoÃcido oxidase isolada do veneno da serpente Bothrops marajoensis (LAAOBM) em sistema de perfusÃo de rim isolado e em cultura de cÃlulas tubulares renais da linhagem MDCK (Madin-Darby Canine Kidney). Para perfusÃo de rim isolado foram utilizados ratos Wistar pesando entre 250 e 300g, cujos rins foram excisados cirurgicamente e perfundidos com soluÃÃo de Krebs-Hanseleit contendo 6%p/v de albumina bovina previamente dialisada. Foram investigados os efeitos da LAAOBM (10 Âg/mL; n=4) sobre a PressÃo de PerfusÃo (PP), ResistÃncia Vascular Renal (RVR), Fluxo UrinÃrio (FU), Ritmo de FiltraÃÃo Glomerular (RFG), Percentual de Transporte Tubular Proximal de SÃdio (%pTNa+),Percentual de Transporte Tubular de SÃdio (%TNa+), de PotÃssio (%TK+) e de Cloreto (%TCl-). As cÃlulas MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% v/v de Soro Bovino Fetal e entÃo incubadas com a LAAOBM nas concentraÃÃes de 50; 25; 12,5; 6,25; 3,125 e 1,652 Âg/mL. ApÃs 24 horas de incubaÃÃo, foram realizados os ensaios de viabilidade e proliferaÃÃo celular utilizando-se o mÃtodo do MTT. A LAAOBM promoveu uma reduÃÃo da pressÃo de perfusÃo aos 90 minutos de experimento e esta reduÃÃo foi ainda discretamente maior aos 120 minutos. Observou-se tambÃm queda da resistÃncia vascular renal aos 120 minutos. Houve uma queda abrupta e acentuada do fluxo urinÃrio aos 90 minutos que, apesar da tendÃncia à recuperaÃÃo observada aos 120 minutos, ainda mostrou-se bastante reduzido quando comparado ao grupo controle. A infusÃo da LAAOBM tambÃm promoveu uma reduÃÃo do ritmo de filtraÃÃo glomerular aos 90 minutos quando comparada ao grupo controle e este parÃmetro manteve-se ainda no mesmo patamar de reduÃÃo aos 120 minutos. A LAAOBM reduziu gradativamente o percentual de transporte tubular de sÃdio aos 90 e 120 minutos e o percentual de transporte tubular de cloreto nos tempos de 60, 90 e 120 minutos. A anÃlise histolÃgica dos rins perfundidos com LAAOBM mostrou a presenÃa de alteraÃÃes morfolÃgicas significativas, como acÃmulo de proteÃnas nos espaÃos tubulares e glomerulares. Na cultura de cÃlulas MDCK a LAAOBM promoveu uma reduÃÃo da viabilidade celular a partir da concentraÃÃo de 3,25Âg/mL atà a concentraÃÃo de 50Âg/mL, com valor da CI50 de 2,43Âg/mL. Foram observadas tambÃm, atravÃs de microscÃpio Ãptico invertido, alteraÃÃes morfolÃgicas destas cÃlulas, tais vacuolizaÃÃo citoplasmÃtica, alteraÃÃo do estado de confluÃncia e desprendimento das mesmas do substrato de cultura. Estes resultados demonstram que a LAAOBM alterou todos os parÃmetros vasculares e renais avaliados na perfusÃo de rim isolado e possui aÃÃo citotÃxica sobre as cÃlulas MDCK apÃs 24 horas de incubaÃÃo. / There are about 3.000 species of snakes worldwide, but only 10 to 14% are venomous. Among South American countries, Brazil is the one with the largest number of accidents, with approximately 20.000 snakebites each year. According to the Department of Health of Brazil, the genus Bothrops are the main involved in snakebites in the country. Acute renal failure (ARF) is a serious complication of snake poisoning. The fraction L-amino acid oxidase (LAAO) constitutes a main part of the total composition of snake venoms. In some cases this amount can reach 30% of total venom proteins. The renal effects of fraction L-amino acid oxidase isolated from the venom of Bothrops marajoensis (LAAOBM) was investigated in this study. Isolated perfused rat kidney and the cultured renal tubular cells line MDCK (Madin-Darby Canine Kidney) were used here. For the isolated perfused rat kidney method, we used Wistar rats weighing between 250 and 300. Their right kidneys were surgically excised and perfused with Krebs-Hanseleit containing 6% w/v bovine albumin previously dialyzed. The effects of LAAOBM (10 mg/mL, n=4) were analyzed on the Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR) Percentage of Sodium Proximal Tubular Transport (%pTNa+), Percentage of Sodium (%TNa+), Potassium (%TK+) and Chloride (%TCl-) Tubular Transport. MDCK cells were cultured in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum and incubated with LAAOBM at concentrations of 50, 25, 12.5, 6.25, 3.125 and 1.652 mg/mL. After 24 hours of incubation, assays were performed on cell proliferation and viability using the MTT method. The LAAOBM promoted a reduction of perfusion pressure at 90 minutes of the experiment and this reduction was even slightly higher at 120 minutes. It was also observed decrease in renal vascular resistance at 120 minutes. There was a sharp and sudden drop in urine flow at 90 minutes, despite the tendency of recovery observed at 120 minutes, still proved to be quite small when compared to the control group. The infusion of LAAOBM also promoted a reduction in glomerular filtration rate at 90 minutes compared to the control group and this parameter still remained at the same level of reduction at 120 minutes. The LAAOBM gradually reduced the percentage of sodium tubular transport at 90 and 120 minutes and the percentage of chloride tubular transport in the periods of 60, 90 and 120 minutes. Histological analysis of kidneys perfused with LAAOBM showed the presence of significant morphological changes such as accumulation of proteins in tubular and glomerular spaces. In the MDCK cell culture LAAOBM promoted a reduction in cell viability from concentrations of 3.25 mg / mL until 50μg/mL, with IC50 value of 2.43 mg/mL. Inverted light microscopy showed morphological changes of these cells, such as vacuolation, alteration of the state of confluence and detachment of the substrate culture. These results demonstrated that LAAOBM changed all the parameters evaluated in renal and vascular perfusion of isolated kidney and had cytotoxic activity on MDCK cells after 24 hours of incubation.

To establish comprehensive pharmacology of P2X₇ receptors, membrane current recording, intracellular calcium transient recording and ethidium bromide uptake were carried out to examine several selective (A-740003, A- 438079) and non-selective (suramin) P2X₇ antagonists across mammalian P2X₇ receptors (human, mouse and rat). These P2X₇ receptors demonstrated species-dependent sensitivities to antagonists. In each species, A-740003 revealed variant IC50 values with different assays, indicating the assay- dependent pharmacology of P2X₇ receptors. Conventionally, pharmacology can be used to define a native current but not in the case of the human breast cancer cell line, Hs578T. It is found that P2X₇ was expressed at both mRNA and protein level. The ATP-evoked currents recorded from Hs578T cells were P2X₇-like with distinctive electrophysiological features. But the pharmacology profile of the currents did not fit with P2X₇ receptor. Further experiments are needed to either include or exclude the existence of functional P2X₇ receptors in Hs578T. Transmembrane domain 2 (TM2) is known as the pore-forming region for P2X receptors. TM2 of P2X₇ receptor was investigated with cysteine substitution scanning. The predicted α-helix structure of the TM2 segment was in good agreement with the results from the substituted cysteine accessibility method (SCAM). Thr336, Ser339, Tyr343, Phe344 and Thr348 were found important for both channel dilation and aqueous pore formation. Ser339 was further studied. Various substitutions at Ser339 were explored. The results suggest that the polarity of the side chain at Ser339 is essential for the channel dilation. Furthermore, disulfide bond formation was identified between S339C in the trimeric receptor, implying that the side chains of Ser339 might turn very close to each other during the channel opening and dilation.

Life is anti-entropic and highly organized phenomenon with two characteristics reinforcing each other: homochirality and the stereospecific catalysis of chemical reactions. The exclusive presence of L-amino acids and R-sugars in living world well depict this. Hypothetically, the amino acids and sugars of reverse chirality could form a parallel kingdom which is highly orthogonal to the present world. The components from this mirror kingdom, such as protein or nucleic acid, will be much more resistant to the defensive mechanism of present living system, which could be of great value. Therefore, by gradually rewiring the present bio-machineries, we look to build a bridge leading us to the space of mirror-imaged biomolecules. We begin by investigating protein synthesis with mirror amino acid since most amino acids contain one chiral center to be inversed comparing to sugars. In this work, we analyzed three stages critical for the incorporation of D-amino acid into ribosomal protein synthesis: amino acylation, EF-Tu binding of amino acyl-tRNA and delivery bias, and ribosome catalyzed peptidyl transfer. We have demonstrated that the affinity between EF-Tu and amino acyl-tRNA plays critical role on D-amino acid incorporation, and built a platform aimed to select for ribosome tolerating D-amino acid better. / Chemistry and Chemical Biology

The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.

We have developed a novel computational approach to functional de novo protein design using gradient based continuous optimization techniques. Motivated by many engineering applications in which a cost function is optimized subject to a set of constraints, we pose a functional protein design task as a continuous optimization problem to search sequence and conformation spaces simultaneously. The methods used in sequence-space search are analogous to the material-design formulations in the topology optimization of structures, whereas the conformation search techniques are similar to mechanical-link like models and modal analysis of structures. Computationally efficient techniques such as the nonlinear conjugate gradient and interior point optimization are used to solve optimization problems. Both the sequence and conformation search techniques are individually validated with real proteins. Coarse-grained as well as atomistic potentials are used to model the energy. Finally, we combine the sequence and conformation search methods and propose a new strategy for a simultaneous search in sequence and conformation spaces for designing functional de novo proteins. In view of the lack of experimental resources, the proposed computational scheme is validated by re-designing an existing protein, the hen-egg white lysozyme. Since the thrust of this method is on developing computationally efficient models, we developed an amino acid grouping scheme based on metric multi-dimensional scaling. Some structure-prediction problems are also solved using Graphics Processing Unit (GPU) based Compute Unified Device Architecture (CUDA) programming.

Proteins are the basic building blocks of biological organisms, and are responsible for a variety of functions within them. Proteins are composed of unique amino acid sequences. Some has only one sequence, while others contain several sequences that are combined together. These combined amino acid sequences fold to form a unique three-dimensional (3D) shape. Although the sequences may fold proteins into different 3D shapes in diverse environments, proteins with similar amino acid sequences typically have similar 3D shapes and functions. Knowledge of the 3D shape of a protein is important in both protein function analysis and drug design, for example when assessing the toxicity reduction associated with a given drug. Due to the complexity of protein 3D shapes and the close relationship between shapes and functions, the prediction of protein 3D shapes has become an important topic in bioinformatics. This research introduces a new approach to predict proteins’ 3D shapes, utilizing a multilayer artificial neural network. Our novel solution allows one to learn and predict the representations of the 3D shape associated with a protein by starting directly from its amino acid sequence descriptors. The input of the artificial neural network is a set of amino acid sequence descriptors we created based on a set of probability density functions. In our algorithm, the probability density functions are calculated by the correlation between the constituent amino acids, according to the substitution matrix. The output layer of the network is formed by 3D shape descriptors provided by an information retrieval system, called CAPRI. This system contains the pose invariant 3D shape descriptors, and retrieves proteins having the closest structures. The network is trained by proteins with known amino acid sequences and 3D shapes. Once the network has been trained, it is able to predict the 3D shape descriptors of the query protein. Based on the predicted 3D shape descriptors, the CAPRI system allows the retrieval of known proteins with 3D shapes closest to the query protein. These retrieved proteins may be verified as to whether they are in the same family as the query protein, since proteins in the same family generally have similar 3D shapes. The search for similar 3D shapes is done against a database of more than 45,000 known proteins. We present the results when evaluating our approach against a number of protein families of various sizes. Further, we consider a number of different neural network architectures and optimization algorithms. When the neural network is trained with proteins that are from large families where the proteins in the same family have similar amino acid sequences, the accuracy for finding proteins from the same family is 100%. When we employ proteins whose family members have dissimilar amino acid sequences, or those from a small protein family, in which case, neural networks with one hidden layer produce more promising results than networks with two hidden layers, and the performance may be improved by increasing the number of hidden nodes when the networks have one hidden layer.

Hepatitis C virus (HCV) infects an estimated 170 million people worldwide and is a major cause of chronic hepatitis and hepatocellular carcinoma. As there are limited treatment options, the elucidation of novel host-viral interactions during HCV pathogenesis will be critical for the development of new therapeutics. My thesis work has identified cell death-inducing DFF45-like effector B (CIDEB) as a host factor that is disregulated during HCV infection, and has delineated the relevance of CIDEB’s dual roles in apoptosis and lipid metabolism in the context of the HCV lifecycle. Moreover, additional host factors necessary for the HCV lifecycle were investigated using unnatural amino acid (UAA) technology. With this technique, the photo-cross-linking UAA p-azido-phenlyalanine (AZF) and 3’-azibutyl-N-carbamoyl-lysine (Abk) were incorporated into viral proteins by expanding the genetic code of the host organism. This conferred diverse physicochemical and biological properties to these proteins that were exploited to investigate protein structure and function in vitro and in vivo. In summary, gaining insight into the numerous host-viral interactions that take place during HCV infection will both advance our understanding of HCV pathogenesis and uncover potential therapeutic targets.

The necessity of reprocessing spent nuclear fuel has arisen from increasing awareness and concern for the environment, in addition to the potential of minimising proliferation. A number of different reprocessing techniques are currently being developed around the world to allow useful spent nuclear fuel (SNF) to be recycled and reused and the remaining waste to be treated. One such technique, currently being developed in the USA is the TALSPEAK process, an advanced reprocessing method for the separation of trivalent lanthanide (Ln3+) and minor actinide (MA3+) components. This process, developed in the 1960s at Oak Ridge National Laboratory, uses DTPA to act as a holdback reagent for MA3+, in a lactate buffered aqueous phase at pH 3.6, allowing Ln3+ to be selectively extracted by organophosphate HDEHP into an organic phase of DIPB or dodecane. TALSPEAK is one of the most promising techniques being researched due to its numerous advantages, particularly its relative resistance to radiolysis and its ability to be carried out without the need for high reagent concentrations. Additionally it gives high separation factors, in the region of ~50-100, comparable to other advanced reprocessing methods under development. The chemistry of the process is very complex and not particularly well understood so it would be advantageous to simplify the process by removing the need for a separate holdback reagent and buffer. In collaboration with colleagues at the Idaho National Lab, the use of amino acids as a potential combined buffer and soft donor was investigated. Although it was found that amino acids do not act as holdback reagents in their own right, optimisation of an L-alanine buffered TALSPEAK system with DTPA was found to allow the process to be carried out effectively at a lower pH of 2, which is more preferable for industrial application. As an extension of this, separation studies were carried out using the tripeptide L-glutathione (GSH) to determine its potential for use as a combined buffer and soft-donor. As with the studies with amino acids, it was found that GSH also does not act as a holdback reagent in its own right, however it does interact with Ln-DTPA complexes at pH 4. When optimised at this pH, separation factors of up to 1200 were achieved for Eu3+/Am3+, whilst still maintaining low MA3+ partitioning. However, further studies by ICP-MS and luminescence spectroscopy showed that a GSH buffered system was not effective for extraction of heavier lanthanides, although the results show the potential for further investigation into other short and longer chain peptide buffered systems and possibly lanthanide-lanthanide separations. Further studies were carried on amino acid appended DTPA ligands which were synthesised in a one step reaction in order to create a combined buffer and soft donor. The ligands were found to self-buffer at around pH 2 and allow successful separation of Eu3+/Am3+ (SF ~ 100). The results from initial investigations by luminescence spectroscopy and solvent extraction are promising and are presented here. Further work is needed on these systems in order to optimise their extraction capability and minimise Am3+ partitioning. In the future this work could promote studies for better understanding of TALSPEAK chemistry that could be used in industrial partitioning processes.

Magister Scientiae - MSC (Biochemistry) / Proteins play an important role in cells, as the morphology, function and activities of the cell depend on the proteins they express. The key to understanding how different proteins function lies in an understanding of the molecular structure. The overall aim of this thesis was the determination of the structure of DWNN domains. This thesis described the preparation of samples of human DWNN suitable for structural analysis by nuclear magnetic resonance spectroscopy (NMR), as well as NMR analysis. / South Africa

This thesis focuses on the two research projects which have applied machine learning techniques to the protein-related topics. The first project is to use protein sequences and the interaction graph to address the protein-protein interaction prediction problem. The second project is to leverage the sequences of protein loops within and beyond homologs to predict the protein loop structures. In the protein-protein interaction prediction project, we applied the pretrained language models, which were trained on large sets of protein sequences, as one of the protein feature extraction methods. Another feature extraction method is the graph learning on the protein interaction graph. The graph learning embeddings and the language model embeddings were fed into classification models to predict if two proteins are interacting or not. We trained and tested our methods on the S. cerevisiae dataset and the human dataset. Our results are comparable to or better than other state-of-art methods, with the advantages that our method is faster at the sample preparation step and has a larger application scope for requiring only protein sequences. We also did experiments with datasets from different similarity cutoffs between the train and test set of the human dataset, and our method has shown an effective prediction ability even with a strict similarity cutoff. In the protein loop prediction project, we utilized the attention-based encoder-decoder language models to predict the protein loop inter-residue distances from the protein loop sequences. We fed the model with the loop sequences and received arrays of numbers representing the distances between each C_α pair in the loops. We utilized two different strategies to reconstruct the loops from the predicted distances. One was firstly to calculate the C_α coordinates from the predicted distances, and then apply a fast full-atom reconstruction method starting from C_α coordinates to build the local loop structures. Our local loop structure prediction results of this method are very competitive with low local RMSDs, especially with the lowest standard deviations. The second method was to integrate the predicted inter-residue distances as constraints to the de novo loop prediction method PLOP (Jacobson et al. 2004). We tested the loop reconstruction process on the 8-res and 12-res loop benchmark sets. This method has the best performance compared to other state-of-art methods, and the incorporation of such machine learning step decreased the computing time of the standalone PLOP program.