Hepatitis C Virus (HCV) is a positive-strand RNA virus that has infected more than 3% of the world population. Chronic infections by the virus lead to cirrhosis and hepatocellular carcinoma. HCV is currently the leading cause for liver transplantation in the US. The nonstructural protein NS5B of HCV is the RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA on host derived membranous structures. Structurally NS5B has the characteristic fingers, thumb and palm domains seen in all polymerase proteins. However, extensive interactions between the fingers and thumb domains completely encircle the active site of NS5B as seen in solved X-ray diffraction crystal structures. These interactions are primarily mediated by a short (35 residues) flexible loop called the Delta 1 loop. NS5B produced from heterologous systems can initiate RNA synthesis by a de novo initiation mechanism from 3?ends of RNA templates or can also extend from 3'ends of primers that are annealed stably to a template RNA in biochemical assays. The closed conformation of NS5B as per X-ray crystal structures can only accommodate a ssRNA but not a dsRNA, hence necessitating a conformational change between de novo initiation and elongation. The details of these conformational changes are not known and will prove to be important to design potent polymerase inhibitors. The study performed for this dissertation focused on the conformational requirements of NS5B during de novo initiation and primer extension (or elongation). Biochemical assays utilizing template RNAs that can lead to both de novo initiation and primer extension products were utilized, and a systematic mutational analysis of the template channel of the RdRp was performed. Mutants W397A and H428A were identified that showed only primer extension but no de novo initiation. Structural analysis of NS5B suggested that these residues were important contact points in the Delta 1 loop and thumb domain interactions. A deletion mutant, m26-30 with a five amino acid deletion at the apex of the Delta 1 loop also failed in de novo initiation but not primer extension reactions. Biophysical and gel shift assays showed that m26-30 was in a more open conformation than the WT enzyme. Furthermore, oligomerization of NS5B was demonstrated and its role in RNA synthesis was examined. It was found that the de novo initiation competent conformation of NS5B is maintained by oligomeric contacts between individual subunits, likely by stabilizing the Delta 1 loop and thumb domain interactions. Mutations disrupting the Delta 1 loop and thumb domain interactions as well as those in the allosteric GTP binding site induced conformational changes in the protein partially explaining the defect in de novo initiation activity in enzymes carrying those mutations. These results not only contribute to the overall mechanism of RNA synthesis in viral RdRps but also open new avenues for developing HCV polymerase inhibitors.

Thèse de doctorat : Chimie : Strasbourg 1 : 2008. Thèse de doctorat : Chimie : Université de Barcelone : 2008. / Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Notes bibliogr.

No description available.

Helical oligomers made up of the achiral amino acid Aib (2-aminoisobutyric acid) have great potential in relaying stereochemical information over nanometre distances by control of their macromolecular conformation. In a synthetic signalling pathway, the helical domain acts as a transducer, mediating the information flow between an input and an output. This thesis describes the advancement of these helical systems into functioning, dynamic transduction pathways capable of nanoscale information processing by a variety of different means. Through studying the effect of C-terminal chiral amino acids, alanine was found to give maximal control over the helical screw-sense. It was also discovered that the hydrogen bonding preference of the terminal group (ester, amide etc.) determined the screw-sense direction. By modifying the terminus, this directional preference was inverted using light or pH as secondary stimuli. Non-covalent interactions with solvent were found to be important in controlling the flow of information through the helical domain. In less polar solvents (THF, chloroform) a single screw sense can prevail for up to 200 monomers, leading to very low signal loss. A zinc binding site was developed to allow a chiral ligand to act as the input in the signalling process. Both amino and phosphoric acids led to remarkable levels of screw-sense induction. Using the reversible ligand binding interaction a pH switch was set up to moderate the binding of these two ligands, creating a ternary information switch. Significant progress was also made towards a light switch using a spiropyran as a competing ligand. Finally, a novel light-responsive switch capable of releasing a signalling molecule in solution was conceived and developed.

La lécithine:rétinol acyltransferase (LRAT) est une enzyme de l'épithélium pigmentaire rétinien (EPR) qui joue un rôle essentiel dans le cycle visuel des rétinoïdes au niveau de l'estérification du rétinol tout-trans en rétinyl ester tout-trans. Plusieurs travaux de caractérisation de cette protéine ont été publiés depuis les 20 dernières années, mais ce n'est que depuis 2003 qu'il est possible d'utiliser une forme tronquée et purifiée de la LRAT qui correspond à sa portion cytosolique (tLRAT) (Bok et al. 2003). Bien que plusieurs mécanismes biochimiques aient été approfondis avec la tLRAT purifiée, très peu d'investigations à propos de sa structure et de son interaction membranaire ont été publiées jusqu'à maintenant. De plus, même si les paramètres enzymatiques de la tLRAT ont été étudiés par différents groupes de recherche, ces travaux ont été effectués avec un protocole expérimental indadéquat conduisant à des niveaux d'activité non-reproductibles d'un article à l'autre. Nous avons donc produit la tLRAT afin d'étudier en profondeur sa structure secondaire, son interaction membranaire et son activité enzymatique. En élaborant un nouveau protocole de recherche pour étudier l'activité enzymatique de la tLRAT, nous avons pu obtenir des niveaux d'activité très élevés et reproductibles. Par surcroît, les méthodes de spectroscopie infrarouge (IR) dichroïsme circulaire électronique et vibrationnel ont permis de déterminer que la tLRAT était constituée d'une structure secondaire majoritairement en hélice alpha. Ensuite, la spectroscopie de réflexion-absorption par modulation de polarisation en IR (PM-IRRAS) a permis de déterminer que la tLRAT interagissait fortement et hydrolysait des monocouches de phospholipides. L'interaction entre la tLRAT et différents types de lipides retrouvés dans les membranes de l'EPR a également été caractérisée avec la méthode des monocouches de Langmuir par la détermination de la pression d'insertion maximale (PIM). Cette méthode a permis de déterminer que la tLRAT s'adsorbait spontanément aux monocouches de lipides à des pressions de surface au-delà la pression latérale des membranes biologiques. Les deux segments hydrophobes en N- et C-terminal de la LRAT qui n'ont pas été surexprimés dans la tLRAT ont été synthétisés afin de les étudier en PM-IRRAS. Ces expériences ont permis de démontrer qu'ils interagissaient fortement avec les monocouches de lipide et qu'ils adoptaient une structure en hélice alpha. Enfin, le mutant S175R responsable de la rétinite pigmentaire a été produit afin de comprendre les impacts moléculaires de cette mutation. La méthode des monocouches de Langmuir et le dichroïsme circulaire ont permis de démontrer que son interaction membranaire et sa structure secondaire n'étaient pas modifiés en comparaison à la forme sauvage de la tLRAT. Cependant, comme son activité enzymatique est absente, il a été conclu que l'entrée du substrat dans le site actif devait être compromise par la mutation de la serine en arginine.

Equines have been used for utilized for manual labor, recreation, and companionship amongst many other valuable conveniences since their domestication. As the modern horse progressed from livestock to athlete, attention was paid to the body conformation to be used as an indicator of biomechanics and can dictate equine performance. Poor conformation can put physical limitations on the body and predispose the horse to injury and chronic disease. When not managed properly, these flaws can lead to injury, lameness, and premature retirement in sport horses. The distal limb is composed of tendons and ligaments that are all susceptible to tear or rupture. Protective wraps or boots are typically applied to the distal limb prior to exercise to prevent superficial injury from the environment or interference. However, these preventatives can trap heat against the skin which can have detrimental effects on the fibroblasts which can lead to failure. It was not until the early twentieth century that the idea of equine physiotherapy was adopted, and practices changed to meet remedial needs and create a sustainable, healthy equine athlete. Equine physiotherapy is a broad-spectrum term used to describe the therapeutic efforts made to keep the body in good health by means of prevention of injury to improve or maintain performance. Traditionally, therapeutics are administered by a veterinarian or trained professional in the event of an existing injury. In recent years therapeutics have been commercialized and are readily available for everyday preventative use. The most common readily available treatments being variations of pulsating electromagnetic fields (PEMF), vibration therapy, cryotherapy, and thermotherapy. When used prior to or after exercise, the therapeutics are designed to prepare the body for exercise and improve recovery by increasing circulation and down regulating the inflammatory response. The studies performed evaluate the efficacy of Rambo®Ionic (Horseware, Dundalk,Ireland), Lux Ceramic Therapy® (Schneider Saddlery Co., Inc., Ohio, USA), and Ice-Vibe® (Horseware, Dundalk,Ireland) therapeutic boots when applied to the distal limb as per manufacturer recommendation. The first study evaluated the therapeutic boots ability to alter performance performing gait analysis using the ALOGO™ MovePro (Alogo Technologies, Switzerland) stride sensor, blood analysis measuring serum concentrations of C reactive protein (CRP), basic fibroblast growth factor (bFGF) and tenascin-C (TN-C), and capturing thermal images of the distal limb using an HT-19 thermal imaging camera (HTI, La Vergne, TN). In this study, eight healthy horses were exercised for approximately ten minutes per day for five consecutive days. There was a ten-day washout period where the horse received no treatment between each period; there was a total of four periods. The second study only evaluated Rambo®Ionic (Horseware, Dundalk,Ireland) and Ice-Vibe® (Horseware, Dundalk,Ireland) therapeutic boots on seventeen healthy horses in the Virginia Tech equitation lesson program. There were three periods with five days of consecutive data collection and a ten-day washout period in between where the horses received no treatment. Gait analysis was measured using the ALOGO™ MovePro (Alogo Technologies, Switzerland) stride sensor and a blind behavioral analysis was performed to analyze behavioral changes under saddle in response to a rider. / Master of Science / Equines have been used for utilized for manual labor, recreation, and companionship amongst many other valuable conveniences since their domestication. As the modern horse progressed from livestock to athlete, attention was paid to the structure of the horse, otherwise known as conformation. Conformation is an indicator of physical movement and can dictate what uses the horse is best suited for. In undesirable cases, poor conformation can put physical limitations on the body and predispose the horse to injury and chronic disease. When not managed properly, these flaws can lead to injury, lameness, and premature retirement in sport horses. The distal limb is a particularly vulnerable structure. It is free of muscle and is comprised of tendons, ligaments, and mobile joints. A protective wrap or boot is typically applied to the distal limb prior to exercise which can have detrimental effects on the cellular components of the associated structures which can lead to failure. It was not until the early twentieth century that the idea of equine physiotherapy was adopted, and practices changed to meet remedial needs and create a sustainable, healthy equine athlete. Equine physiotherapy is a broad-spectrum term used to describe the therapeutic efforts made to keep the body in good health by means of prevention of injury to improve or maintain performance. Traditionally, therapeutics are administered by a veterinarian or trained professional in the event of an existing injury. In recent years therapeutics have been commercialized and are readily available for everyday preventative use. The most common readily available treatments being variations of pulsating electromagnetic fields (PEMF), vibration therapy, cryotherapy, and thermotherapy. When used prior to or after exercise, the therapeutics are designed to prepare the body for exercise and improve recovery by increasing circulation and down regulating the inflammatory response. The studies performed evaluate the efficacy of Rambo®Ionic (Horseware, Dundalk,Ireland), Lux Ceramic Therapy® (Schneider Saddlery Co., Inc., Ohio, USA), and Ice-Vibe® (Horseware, Dundalk,Ireland) therapeutic boots when applied to the distal limb as per manufacturer recommendation. The first study evaluated the therapeutic boots ability to alter performance performing gait analysis using the ALOGO™ MovePro (Alogo Technologies, Switzerland) stride sensor, blood analysis measuring serum concentrations of C reactive protein (CRP), basic fibroblast growth factor (bFGF) and tenascin-C (TN-C), and capturing thermal images of the distal limb using an HT-19 thermal imaging camera (HTI, La Vergne, TN). In this study, eight healthy horses were exercised for approximately ten minutes per day for five consecutive days. There was a ten-day washout period where the horse received no treatment between each period; there was a total of four periods. The second study only evaluated Rambo®Ionic (Horseware, Dundalk,Ireland) and Ice-Vibe® (Horseware, Dundalk,Ireland) therapeutic boots on seventeen healthy horses in the Virginia Tech equitation lesson program. There were three periods with five days of consecutive data collection and a ten-day washout period in between where the horses received no treatment. Gait analysis was measured using the ALOGO™ MovePro (Alogo Technologies, Switzerland) stride sensor and a blind behavioral analysis was performed to analyze behavioral changes under saddle in response to a rider.

Foal scores from the International Sporthorse Registry and Oldenburg Registry North America were used for statistical and genetic analysis. Scored traits include type and conformation (TC), athletic ability of movement (AM), overall development as related to age (OD), and total score (TS) calculated as a weighted average of TC, AM, and OD. Premium status (PS) was analyzed as a binary trait. Preliminary statistical analysis determined significant fixed effects of sex, year of birth, dam breed, and inspection period. Offspring of stallions with only one offspring in the dataset and non-warmblood sires were deleted. Non-warmblood or non-Thoroughbred dams were also removed. Variance components were estimated using ASReml methodology to obtain genetic parameters. Traits were moderately to highly heritable with heritabilities of 0.45, 0.47, 0.49, and 0.55 for TC, AM, OD, and TS, respectively. PS had a heritability of 0.32 on a binary scale and 0.51 when transformed to the normal scale. Genetic correlations between TC, AM, OD, and TS were all high and favorable, ranging from 0.80 to 0.99. Genetic correlations with PS were inestimable. Foal inspection scores are heritable and should respond to selection. Selection for improvement in one trait should result in improvement in all traits. If genetic parameters can be correlated to data obtained in older horses, incorporating foal scores in selection decisions could improve warmblood breeding programs. Utilizing foal inspection scores should be beneficial to breeding objectives of the International Sporthorse Registry and Oldenburg Registry North America. / Master of Science

Submitted in fulfillment of the requirements of the Degree of Doctor of Technology: Chemistry, Durban University of Technology, 2011. / Understanding the dynamics and mechanism of protein folding continues to be one of the central problems in molecular biology. Peptide folding experiments characterize the dynamics and molecular mechanisms of the early events of protein folding. However, generally the highly flexible nature of peptides makes their bioactive conformation assessment reasonably difficult as peptides fold at very fast rates experimentally, requiring probing on the nanosecond time resolution. On the other hand, determining the bioactive conformation of biological peptides is a requirement for the design of peptidomimetics in computer-aided drug design. Peptides offer a unique opportunity to bridge the gap between theoretical and experimental understanding of protein folding. Therefore, the present work focuses on the exploration of the conformational space of biologically active neuropeptides with the aim of characterizing their conformational profile. Specifically, bombesin, neuromedin B (NMB) and neuromedin C (NMC), have been chosen for the current investigations. These peptides are widely distributed in the gastrointestinal tract, spinal cord and brain, and are known to elicit various physiological effects, including inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. These peptides act as a growth factor in a wide range of tumours including carcinomas of the pancreas, stomach, breast, prostate, and colon. This work is intended to get some insight into the performance of different procedures used to explore the configurational space to provide an adequate atomic description of these systems. Different methodological studies involving utilization of molecular dynamics (MD), multicanonical replica exchange molecular dynamics (REMD) and simulate annealing (SA) are undertaken to explore the folding characteristics and thermodynamics of these neuropeptides. MD and REMD calculations on bombesin peptide have revealed its dual conformational behaviour never discovered before and is described in chapter 3. These results explain the known structure-activity studies and open the door to the understanding of the affinity of this peptide to two different receptors: BB1 and BB2. In the case of NMC, REMD calculations are carried out in explicit and implicit solvents, using the Generalized Born (GB) surface area, and are then complemented with two additional MD simulations performed using Langevin and Berendsen thermostats. The results obtained clearly reveal that REMD, performed under explicit solvent conditions, is more efficient and samples preferentially folded conformations with a higher content of  and γ turns. Moreover, these results show good agreement with the experimental results supporting the role of two -turns for its biological action, as reported in the literature. Finally, the results obtained from MD, REMD and SA calculations on NMB reveal that the peptide has a tendency to adopt both turns and helices suggesting its two different receptor recognizing and binding conformations during its biological action. Hence, the present work provides comprehensive information about the conformational preferences of neuropeptides which could lead to a better understanding of their native conformations for future investigations and point the way towards developing their new antagonists.

The main focus of this thesis is on the ring conformations of carbohydrate molecules; how the conformational equilibria and the rates of the associated interconversions are affected by the molecular constitution and their surroundings. The conformational equilibria of a group of amine linked pseudodisaccharides, designed as potential glycosidase inhibitors, comprising α-D-altrosides are described in Chapter 3. The OS2 conformation was largely populated, and the ring conformation was found to depend on the charge of the amine functionality. The conformations of β-D-xylopyranoside derivatives with naphthyl-based aglycones, which are potential anti-cancer agents, are described in chapter 4. Solvent dependent flexibility was observed. Intramolecular hydrogen bonds were concluded to be involved in the stabilization of 1C4 conformers in non-hydrogen bonding solvents of low polarity. Chapter 5 describes the first measurements of the conformational exchange rates of mannuronic acid ester derivatives between the 4C1 and 1C4 conformations, through DNMR measurements. The relative reactivity of glycosyl triflates as electrophiles in glycosylation reactions were investigated with NMR-based competition experiments. In Chapter 6, investigations of ruthenium-catalyzed epimerizations of the allylic alcohols of glycal derivatives, and stereoselective synthesis of esters through a DYKAT protocol, are described. The kinetics of the epimerizations were elaborated through different NMR-spectroscopic methods. Chapter 7 describes additions of NMR chemical shift data of mono- and oligosaccharides to database of the computer program CASPER, and applications thereof. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript.</p>

Recent technological advances underpinned by high throughput sequencing have given new insights into the three-dimensional structure of mammalian genomes. Chromatin conformation assays have been the critical development in this area, particularly the Hi-C method which ascertains genome-wide patterns of intra and inter-chromosomal contacts. However many open questions remain concerning the functional relevance of such higher order structure, the extent to which it varies, and how it relates to other features of the genomic and epigenomic landscape. Current knowledge of nuclear architecture describes a hierarchical organisation ranging from small loops between individual loci, to megabase-sized self-interacting topological domains (TADs), encompassed within large multimegabase chromosome compartments. In parallel with the discovery of these strata, the ENCODE project has generated vast amounts of data through ChIP-seq, RNA-seq and other assays applied to a wide variety of cell types, forming a comprehensive bioinformatics resource. In this work we combine Hi-C datasets describing physical genomic contacts with a large and diverse array of chromatin features derived at a much finer scale in the same mammalian cell types. These features include levels of bound transcription factors, histone modifications and expression data. These data are then integrated in a statistically rigorous way, through a predictive modelling framework from the machine learning field. These studies were extended, within a collaborative project, to encompass a dataset of matched Hi-C and expression data collected over a murine neural differentiation timecourse. We compare higher order chromatin organisation across a variety of human cell types and find pervasive conservation of chromatin organisation at multiple scales. We also identify structurally variable regions between cell types, that are rich in active enhancers and contain loci of known cell-type specific function. We show that broad aspects of higher order chromatin organisation, such as nuclear compartment domains, can be accurately predicted in a variety of human cell types, using models based upon underlying chromatin features. We dissect these quantitative models and find them to be generalisable to novel cell types, presumably reflecting fundamental biological rules linking compartments with key activating and repressive signals. These models describe the strong interconnectedness between locus-level patterns of local histone modifications and bound factors, on the order of hundreds or thousands of basepairs, with much broader compartmentalisation of large, multi-megabase chromosomal regions. Finally, boundary regions are investigated in terms of chromatin features and co-localisation with other known nuclear structures, such as association with the nuclear lamina. We find boundary complexity to vary between cell types and link TAD aggregations to previously described lamina-associated domains, as well as exploring the concept of meta-boundaries that span multiple levels of organisation. Together these analyses lend quantitative evidence to a model of higher order genome organisation that is largely stable between cell types, but can selectively vary locally, based on the activation or repression of key loci.