Chirality is the property of objects, including molecules, which are not superimposable on their materialized mirror image. Chiral molecules are omnipresent in living organisms and the constituents of biological macromolecules (proteins and nucleic acids) are chiral. Amino-acids (constituting proteins), ribose and 2-deoxy-ribose (the only chiral constituent of RNA and DNA nucleotides respectively) are furthermore generally present in living organisms only under one of their enantiomeric forms. This is referred to as the homochirality of the living world. The origin of this homochirality is still unexplained, even if many partial scenarios have been proposed in the literature. All scenarios involve the creation of a small enantiomeric excess for certain molecules, amplification of this excess and chirality transfer to other chiral molecules. The origin of homochirality on Earth is closely related to the origin of life, and is currently supposed to have preceded life. As no-one will ever be able to directly observe the phenomena which lead to homochirality, and life, on our planet, the only scientific approach to try and help explain how this occurred is to build scenarios, and test them taking into account all available information on the physical and chemical conditions on the primitive Earth (Earth before life appeared). In our work, we investigated three scenarios related to the origin of homochirality on Earth. One of these scenarios also relates to a very precise step of the origin of life: the selection of beta-d-ribofuranose as component of RNA nucleotides. Enantiomeric excesses (up to 15 %) of alpha-methylated alpha-amino-acids have been detected in meteorites which fell on Earth during the 20th century. No enantiomeric excess is detected for the corresponding alpha-hydroxy-acids in the same meteoritic samples and small (2% at most) or no enantiomeric excesses have been measured for non-methylated alpha-amino-acids. In the first part of our work, we investigated if photolysis by circularly polarized light (CPL) in space could be at the origin of the presence (or absence) of an enantiomeric excess for these compounds. Experiments to reproduce UV-CPL photolysis are difficult to undertake: they require high-energy circularly polarized photons, hence the use of a synchrotron. In our work, we used quantum mechanical calculations to obtain the electronic circular dichroïsm (ECD) spectra of two -methylated -amino-acids, their corresponding alpha-hydroxy-acids and one non-methylated alpha-amino-acid. Differences are observed between these spectra, and we propose a scenario to explain the experimental measurements reported here above: the enantioselective photolysis, in the gas phase at low temperatures (20K at most), of the alpha-amino-acids by UV-CPL with lambda>210 nm. Under these conditions no photolysis of the alpha-hydroxy-acids would occur. This scenario concerns the first step in the origin of homochirality on Earth: the creation of a small enantiomeric excess for some chiral molecules. The second scenario that we investigated relates to the enantiomeric amplification step of the origin of homochirality on Earth, for which the role of the alpha-amino-acid serine has been suggested in the literature. Serine clusters have been observed in the gas phase by mass spectrometry. Among these clusters the octamer has been shown to be a magic number cluster and to have a preference for homochirality. An enantiomeric amplification via cycles of formation and dissociation of the octamer has been suggested. No complete scenario has however been proposed in the literature to explain how this could have occurred on the primitive Earth, but any scenario would most probably include an aqueous phase. We aimed at determining if the homochiral preference of serine octamers also exists in solution and therefore we first investigated if serine octamers exist in solution. For this study, we used nuclear magnetic resonance and infrared spectroscopies, which are well-adapted to the study of molecular assemblies in solution. We were able to demonstrate that most probably serine clusters are not present in solution, and if they are it could only be in extremely low concentration. The scenario suggested in the literature is discussed in the light of our results and of literature data on serine clusters. As last hypothesis, we investigated a possible scenario for the selection of beta-d-ribofuranose as component of RNA nucleotides. The currently known prebiotic synthesis pathways to ribose also lead to the formation of many other carbohydrates, and ribose is only a minor product of these syntheses. Our hypothesis is that beta-d-ribofuranose could have been selected through favorable interactions with -amino-acids already present on the primitive Earth under one enantiomeric form. Indeed, it is plausible that a peptidic world emerged before the presence of RNA and that homochiral -amino-acids were present on Earth when RNA was synthesized. Under this hypothesis, we investigated the role that alpha-l-amino-acids could have played in the selection of alpha-d-ribofuranose as component of RNA nucleotides. This work is related to the last step of the origin of homochirality: chirality transfer. Our scenario was investigated via nuclear magnetic resonance studies of the interaction between alpha-amino-acids and carbohydrates. We were able to show that, in the systems that we studied, when an interaction occurs it is very weak (affinity constant less than 1M−1) and non enantioselective. Our results most probably discard the role that alpha-amino-acids alone could have played in the selection of beta-d-ribofuranose as component of RNA nucleotides, but does not discard the role that peptides could have played in this selection.

Mathematical models are now gaining in importance for descriptions of biological processes. In this thesis, such models have been used to identify and analyze principles that govern bacterial protein synthesis under amino acid limitation. New techniques, that are generally applicable for analysis of intrinsic fluctuations in systems of chemical reactions, are also presented. It is shown how multi-substrate reactions, such as protein synthesis, may display zero order kinetics below saturation, because an increase in one substrate pool is compensated by a decrease in another, so that the overall flow is unchanged. Under those conditions, metabolite pools display hyper sensitivity and large fluctuations, unless metabolite synthesis is carefully regulated. It is demonstrated that flow coupling in protein synthesis has consequences for transcriptional control of amino acid biosynthetic operons, accuracy of mRNA translation and the stringent response. Flow coupling also determines the choices of synonymous codons in a number of cases. The reason is that tRNA isoacceptors, cognate to the same amino acid, often read different codons and become deacylated to very different degrees when their amino acid is limiting for protein synthesis. This was demonstrated theoretically and used to successfully predict the choices of control codons in ribosome mediated transcriptional attenuation and codon bias in stress response genes. New tools for the analysis of internal fluctuations have been forged, most importantly, an efficient Monte Carlo algorithm for simulation of the Markov-process corresponding to the reaction-diffusion master equation. The algorithm makes it feasible to analyze stochastic kinetics in spatially extended systems. It was used to demonstrate that bi-stable chemical systems can display spontaneous domain separation also in three spatial dimensions. This analysis reveals geometrical constraints on biochemical memory circuits built from bistable systems. Further, biochemical applications of the Fokker-Planck equation and the Linear Noise Approximation have been explored.

Graduation date: 1992

The following study investigates the intramolecular and intermolecular interactions responsible for invoking stereoselectivity and stereospecificity in the synthesis of a chiral original species and amino acid receptor. The former commences with a brief overview of the nature, scope and applications of helical chirality, and culminates in the formation of a permanent helix via the synthesis of a novel chiral lactone. The latter is discussed as an extension of a naturally occurring cofactor whose identity is modulated to furnish a tailored receptor selective to the binding of amino-acid enantiomers. The study and analysis is executed via both synthetic and computational methods.

The following study investigates the intramolecular and intermolecular interactions responsible for invoking stereoselectivity and stereospecificity in the synthesis of a chiral original species and amino acid receptor. The former commences with a brief overview of the nature, scope and applications of helical chirality, and culminates in the formation of a permanent helix via the synthesis of a novel chiral lactone. The latter is discussed as an extension of a naturally occurring cofactor whose identity is modulated to furnish a tailored receptor selective to the binding of amino-acid enantiomers. The study and analysis is executed via both synthetic and computational methods.

To examine the response of wheat to different temperatures and photoperiods at the proteomic level, a series of experiments was performed at the University of Saskatchewan, College of Agriculture and Bioresources, Department of Plant Science. Tandem-mass spectrometry (MS/MS) was used for protein identification. The iTRAQ approach was used to generate raw data for protein quantification. The Pro Group protein identification software was used for protein identification and quantification of differentially expressed proteins. Despite the input samples being from a plant,the software reported non-plant proteins. The traditional approach used by scientists to deal with this problem is to use sequence alignment software to find close green-plant homologs of the non-plant proteins from a plant-only database. Such a technique is problematic since homology-based sequence similarity does not generally equate to similarity of mass spectra. In this work a more radical approach was investigated and implemented. A bioinformatics pipeline was designed and implemented to report plant proteins misidentified by the Pro Group software. The approach drew its idea from the fact that MS/MS-based protein identification uses peptide fragments/ions bearing unique m/z values in the mass spectra. From the reported non-plant proteins and associated peptides, putative m/z values of the peptides are generated and then used to find alternate hits from a green plant-only database. The pipeline uses three different heuristics, each generating a list of candidate proteins. The proteins reported consistently across the three reported lists have the highest likelihood to be present in the original sample. To evaluate the performance of the pipeline, three separate experiments were performed. A set of known plant peptides, a combination of known plant and non-plant peptides and a set of known non-plant peptides were used as input to the pipeline. For each experiment a stringency value (threshold value) was set by the user. Better results were observed by specifying a tighter stringency; that is, more plant proteins were reported consistently across the three reported lists. The research presented in this thesis shows that m/z values, consideration of unique peptides and accounting for proteins with shorter sequences can be used to identify proteins. These characteristics can be used to identify proteins when limited information is available, in this case a list of non-plant proteins reported as being present in a plant-derived sample. The information available was limited because the original input data was already processed by the Pro Group software. The approach presented here is an alternative to a wet lab scientist using sequence alignment tools, sequence databases, and homology-based search. The pipeline can be enhanced by adding various other modules. The results presented here could be used as a foundation for a further study.

A series of experiments were conducted to examine protein digestion in the young bird and the physiological changes that affect protein digestion as the bird matures. Trial one determined the effect of age on pH of the gastrointestinal tract. The results showed that the pH of the proventriculus and gizzard decreases with age suggesting that gastric acid secretion increases proportionally with age and this may impact protein digestion. Experiment two examined the effect of age on ileal amino acid (AA) digestibility of feed ingredients. AA digestibility increased from 5 to 21 d and the degree of improvement differed among ingredients and specific AA. This finding coupled with the low AA content of some ingredients demonstrates the importance of using appropriate nutrient digestibility values for young birds when formulating pre-starter and starter diets. Experiment three determined the acid binding capacity (ABC) of feed ingredients and the effect of formulating diets based on ABC on diet ABC, gastrointestinal pH and performance. The research confirmed dramatic differences in ABC among ingredients and that diets could be formulated on the basis of ingredient ABC. However, the range in diet ABC was less than predicted suggesting interactive effects among ingredients. Intestinal pH was reduced however broiler performance was not improved when diets low in ABC were fed. Experiment four examined the effect of diet acidification with HCl on diet ABC, gastrointestinal pH, ileal amino acid digestibility and broiler performance. Improvements in performance and reductions in mortality were observed when broilers were fed acidified diets. Adding acid to diets did not improve AA digestibility and therefore was not the reason for improved performance indicating an alternative mechanism of action. It is concluded that acid production by the proventriculus of young birds is low and increases with age however this does not impact protein digestion. Methods of improving performance of broiler chickens may include diet acidification however this is not the effect of remedying the low acid production by young birds. Therefore, alternative mechanisms are positively affecting broiler performance.

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies. / February 2007

Protein restriction during rodent pregnancy is a well-established model of developmental programming. Although the Southampton low-protein diet model of developmental programming has been accepted to produce hypertensive offspring, the mechanism of this programming remains unclear. Currently the effects of protein restriction in the Southampton low-protein diet are confounded by a relative elevation of the amino acid methionine. The aim of this project was thus to clarify the roles of protein reduction and methionine elevation within this model, especially within the context of amino acid and one-carbon metabolism. Pregnant Wistar rats were fed casein-based diets ad libitum varying in casein (18% or 9%) and methionine content (0.5% or 1.0%) from day 0 through 20 of pregnancy. Two diets exactly replicated the Southampton control and low-protein diets (Con: 18% casein, 0.5% methionine; LP: 9% casein, 0.5% methionine), while a third low-protein high-methionine diet (LP-MET: 9% casein, 1.0% methionine) was employed as a positive control for methionine stress. On day 20 of gestation dams were sacrificed and the feto-placental unit was excised; maternal and fetal blood was collected for HPLC analysis of free amino acids. Maternal plasma was also analyzed for homocysteine content using a spectrophotometric-based enzyme assay. Diet did not affect maternal weight gain, food consumption, litter size or fetal weight. In dams and fetuses, methionine was significantly elevated in both low-protein groups. Maternal homocysteine was significantly elevated only in dams fed the low-protein high-methionine diet. Reductions in maternal serine, proline and glycine levels also occurred in dams fed the low-protein high-methionine diet; fetuses of these dams had significantly reduced levels of all three branch chain amino acids (leucine, isoleucine and valine). Both low-protein diets resulted in drastic reductions in circulating threonine levels in dams and fetuses. Thus, ingestion of low-protein diets with a relative (0.5%) or overt excess (1.0%) of methionine appears to disrupt one-carbon metabolism at the level of homocysteine remethylation to methionine. This may place strain on the folate cycle, as may be indicated by the reduced levels of threonine, serine and glycine. Further testing is necessary to clarify the extent to which folate stores are being utilized for homocysteine remethylation. Increased competition for placental amino acid transport may explain the alternations in circulating free fetal amino acids. Further investigations into levels of other one-carbon metabolites in dams and fetuses are necessary to fully characterize the effect of low-protein high-methionine diets, particularly within the context of the Southampton model of developmental programming.

A series of experiments were conducted to examine protein digestion in the young bird and the physiological changes that affect protein digestion as the bird matures. Trial one determined the effect of age on pH of the gastrointestinal tract. The results showed that the pH of the proventriculus and gizzard decreases with age suggesting that gastric acid secretion increases proportionally with age and this may impact protein digestion. Experiment two examined the effect of age on ileal amino acid (AA) digestibility of feed ingredients. AA digestibility increased from 5 to 21 d and the degree of improvement differed among ingredients and specific AA. This finding coupled with the low AA content of some ingredients demonstrates the importance of using appropriate nutrient digestibility values for young birds when formulating pre-starter and starter diets. Experiment three determined the acid binding capacity (ABC) of feed ingredients and the effect of formulating diets based on ABC on diet ABC, gastrointestinal pH and performance. The research confirmed dramatic differences in ABC among ingredients and that diets could be formulated on the basis of ingredient ABC. However, the range in diet ABC was less than predicted suggesting interactive effects among ingredients. Intestinal pH was reduced however broiler performance was not improved when diets low in ABC were fed. Experiment four examined the effect of diet acidification with HCl on diet ABC, gastrointestinal pH, ileal amino acid digestibility and broiler performance. Improvements in performance and reductions in mortality were observed when broilers were fed acidified diets. Adding acid to diets did not improve AA digestibility and therefore was not the reason for improved performance indicating an alternative mechanism of action. It is concluded that acid production by the proventriculus of young birds is low and increases with age however this does not impact protein digestion. Methods of improving performance of broiler chickens may include diet acidification however this is not the effect of remedying the low acid production by young birds. Therefore, alternative mechanisms are positively affecting broiler performance.