Molecular subtyping of feline immunodeficiency virus from domestic cats in Australia

Objective To determine the prevalent subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population of Australia. Method Blood samples were collected from 41 FIV antibody positive cats from four cities across Australia. Following DNA extraction, polymerase chain reaction (PCR) was performed to amplify the variable V3-V5 region of the envelope (env) gene. Genotypes were assessed by direct sequencing of PCR products and comparison with previously reported FIV sequences. Phylogenetic analysis allowed classification of the Australian sequences into the appropriate subtype. Results Of the 41 FIV samples, 40 were found to cluster with previously reported subtype A isolates, whilst the remaining sample grouped within subtype B. Conclusions Subtype A was found to be the predominant FIV subtype present in Australia, although subtype B was also found. These results broaden our knowledge of the genetic diversity of FIV and the associated implications for preventative, diagnostic and therapeutic approaches.

Different routes to pure food allergens: extraction, recombinant techniques and total chemical synthesis for obtaining the plant-food pan-allergen LTP

La richiesta di allergeni puri è in continuo aumento per scopi diagnostici, come standard per metodi di rilevamento e di quantificazione, per l'immunoterapia e per lo studio a livello molecolare dei meccanismi delle reazioni allergiche, al fine di facilitare lo sviluppo di possibili cure. In questa tesi di dottorato sono descritte diverse strategie per l’ottenimento di forme pure di non-specific Lipid Transfer Proteins (nsLTPs), le quali sono state riconosciute essere rilevanti allergeni alimentari in molti frutti e verdure comunemente consumati e sono state definite come modello di veri allergeni alimentari. Una LTP potenzialmente allergenica, non nota in precedenza, è stata isolata dalle mandorle, mentre una LTP dall’allergenicità nota contenuta nelle noci è stata prodotta mediante tecniche di DNA ricombinante. Oltre a questi approcci classici, metodi per la sintesi chimica totale di proteine sono stati applicati per la prima volta alla produzione di un allergene, utilizzando Pru p 3, la LTP prototipica e principale allergene della pesca nell'area mediterranea, come modello. La sintesi chimica totale di proteinepermette di controllarne completamente la sequenza e di studiare la loro funzione a livello atomico. La sua applicazione alla produzione di allergeni costituisce perciò un importante passo avanti nel campo della ricerca sulle allergie alimentari. La proteina Pru p 3 è stata prodotta nella sua intera lunghezza e sono necessari solo due passaggi finali di deprotezione per ottenere il target nella sua forma nativa. Le condizioni sperimentali per tali deprotezioni sono state messe a punto durante la produzione dei peptidi sPru p 3 (1-37) e sPru p 3 (38-91), componenti insieme l'intera proteina. Tecniche avanzate di spettrometria di massa sono state usate per caratterizzare tutti i composti ottenuti, mentre la loro allergenicità è stata studiata attraverso test immunologici o approcci in silico.

Molecular phase space transport in water:non-stationary random walk model

Molecular transport in phase space is crucial for chemical reactions because it defines how pre-reactive molecular configurations are found during the time evolution of the system. Using Molecular Dynamics (MD) simulated atomistic trajectories we test the assumption of the normal diffusion in the phase space for bulk water at ambient conditions by checking the equivalence of the transport to the random walk model. Contrary to common expectations we have found that some statistical features of the transport in the phase space differ from those of the normal diffusion models. This implies a non-random character of the path search process by the reacting complexes in water solutions. Our further numerical experiments show that a significant long period of non-stationarity in the transition probabilities of the segments of molecular trajectories can account for the observed non-uniform filling of the phase space. Surprisingly, the characteristic periods in the model non-stationarity constitute hundreds of nanoseconds, that is much longer time scales compared to typical lifetime of known liquid water molecular structures (several picoseconds).

HLA-DP2 binding prediction by molecular dynamics simulations

Major histocompatibility complex (MHC) II proteins bind peptide fragments derived from pathogen antigens and present them at the cell surface for recognition by T cells. MHC proteins are divided into Class I and Class II. Human MHC Class II alleles are grouped into three loci: HLA-DP, HLA-DQ, and HLA-DR. They are involved in many autoimmune diseases. In contrast to HLA-DR and HLA-DQ proteins, the X-ray structure of the HLA-DP2 protein has been solved quite recently. In this study, we have used structure-based molecular dynamics simulation to derive a tool for rapid and accurate virtual screening for the prediction of HLA-DP2-peptide binding. A combinatorial library of 247 peptides was built using the "single amino acid substitution" approach and docked into the HLA-DP2 binding site. The complexes were simulated for 1 ns and the short range interaction energies (Lennard-Jones and Coulumb) were used as binding scores after normalization. The normalized values were collected into quantitative matrices (QMs) and their predictive abilities were validated on a large external test set. The validation shows that the best performing QM consisted of Lennard-Jones energies normalized over all positions for anchor residues only plus cross terms between anchor-residues.

The molecular design of a new solvent for the absorption of carbon dioxide

Gas absorption, the removal of one or more constitutents from a gas mixture, is widely used in chemical processes. In many gas absorption processes, the gas mixture is already at high pressure and in recent years organic solvents have been developed for the process of physical absorption at high pressure followed by low pressure regeneration of the solvent and recovery of the absorbed gases. Until now the discovery of new solvents has usually been by expensive and time consuming trial and error laboratory tests. This work describes a new approach, whereby a solvent is selected from considerations of its molecular structure by applying recently published methods of predicting gas solubility from the molecular groups which make up the solvent molecule. The removal of the acid gases of carbon dioxide and hydrogen sulfide from methane or hydrogen was used as a commercially important example. After a preliminary assessment to identify promising moecular groups, more than eighty new solvent molecules were designed and evaluated by predicting gas solubility. The other important physical properties were also predicted by appropriate theoretical procedures, and a commercially promising new solvent was chosen to have a high solubility for acid gases, a low solubility for methane and hydrogen, a low vapour pressure, and a low viscosity. The solvent chosen, of molecular structure Ch3-COCH2-CH2-CO-CH3, was tested in the laboratory and shown to have physical properties, except for vapour pressures, close to those predicted. That is gas solubilities were within 10% but lower than predicted. Viscosity within 10% but higher than predicted and a vapour pressure significantly lower than predicted. A computer program was written to predict gas solubility in the new solvent at the high pressures (25 bar) used in practice. This is based on the group contribution method of Skold Jorgensen (1984). Before using this with the new solvent, Acetonyl acetone, the method was show to be sufficiently accurate by comparing predicted values of gas solubility with experimental solubilities from the literature for 14 systems up to 50 bar. A test of the commercial potential of the new solvent was made by means of two design studies which compared the size of plant and approximate relative costs of absorbing acid gases by means of the new solvent with other commonly used solvents. These were refrigerated methanol(Rectisol process) and Dimethyl Ether or Polyethylene Glycol(Selexol process). Both studies showed in terms of capital and operating cost some significant advantage for plant designed for the new solvent process.