Systems formed by the supramolecular assemblages of organic molecules known as organogelators and hydrogelators are currently, and only recently, a subject of great attention and promise. In this context, low molecular weight gelators (LMWGs) are of particular interest because they provide a bottom-up approach to the formation of supramolecular architectures through self-assembly. Gelator molecules do so via the initial formation of a one-dimensional array of individual molecules bound non-covalently through forces such as: hydrogen bonds, electrostatic forces, Van der Waals interactions, and other weak forces such as π-π interactions. These interactions then lead to secondary structure formation through a similar assembly mechanism. Understanding the gelation process through characterization techniques is critical to the development of a design rationale for gelator molecules. Past and current research performed by the Wang group indicates that analogues of various 4,6-benzylidene acetals form stable gels in organic, aqueous, and organic/aqueous solvents at varying concentrations. The basis of varying the 4,6-protecting groups on glucose and glucosamine derivatives is to discern the relative structure activity relationships of these systems, and as well to fabricate functional systems which respond to external stimulus. Stimuli responsive or trigger release gel systems formed by sugar based low molecular weight gelators (LMWGs) have applications as smart biocompatible materials, and such responsiveness in various media was explored and developed to determine the feasibility of such applications using monosaccharide derivatives.

Low molecular weight gelators (LMWGs) have gained great attention over the past two decades. These compounds form self-assembled fibrous networks like micelles, cylindrical, sheets, fibers, layers and so on. The fibrous network entraps the solvent and form gel. LMWGs are interesting compounds with many potential applications in material and biomedical sciences. Many different structures have been found to be good LMWGs. Our interests focus on the carbohydrate based LMWGs. Previously, we have found that several ester derivatives of methyl 4, 6-O-benzylidene-α-D-glucopyranoside are good gelators for organic solvents and aqueous solutions. In this study, in order to understand the structure requirement, we systematically investigated the influence of sugar head groups and the attached hydrophobic tails towards gelation. The design, synthesis and gel properties of esters, amides, ureas, carbamates which derived from sugar head groups show above will be discussed in chapter II, III, IV.

Os modelos moleculares são construídos com base na caracterização da composição do petróleo, que consiste em conhecer informações moleculares detalhadas com o objetivo de identificar os compostos presentes. Este trabalho tem como objetivo construir um banco de dados para a classificação de componentes constituintes de asfaltenos em grupos moleculares. A classificação é feita através da semelhança entre suas massas moleculares em grupos de séries homólogas. Tal classificação é feita por meio da utilização dos dados provenientes da análise de espectrometria de massa e de um algoritmo de busca em linguagem VBA (Visual Basic for Applications), com o objetivo de identificar uma molécula específica em um conjunto de moléculas conhecidas (banco de dados). O método proposto nesse trabalho é baseado na classificação dos compostos através de suas massas moleculares, isto é, os compostos presentes em uma mistura são separados em classes por uma diferença de massa tão pequena quanto se queira. Pode-se, assim, observar a que grupo de moléculas um composto de petróleo pertence por meio de sua massa molecular. Como o banco de dados foi gerado por compostos cuja composição é conhecida, sabe-se que composição um dado composto terá entre as possíveis composições. Para gerar moléculas baseado no conhecimento da composição de petróleo, usam-se as fórmulas gerais dos compostos orgânicos que são formados por séries homólogas. Séries homólogas são séries de compostos orgânicos com propriedades químicas similares cujos componentes diferem entre si por uma massa molecular relativa constante (CH2). Tais séries são geradas e seus pesos moleculares são calculados e armazenados juntamente com suas fórmulas moleculares, em ordem crescente de massa, podendo assim, gerar os grupos moleculares com o auxílio de uma rotina computacional em linguagem VBA. / Molecular models are built based on the characterization of the composition of oil, which consists of detailed molecular information to know in order to identify the compounds present. This work aims to build a database for classifying constituent components of asphaltenes in molecular groups. The classification is done by the similarity of their molecular weights with the use of groups of homologous series. This classification is made by using data from the analysis of mass spectrometry and a search algorithm in language VBA (Visual Basic for Applications) with the aim of identifying a molecule in a specific set of molecules known (database data). The method proposed in that work is based on the classification of the compounds through their molecular weights, i.e., the compounds in a mixture are separated into classes by a mass difference as small as one likes. One can therefore see that the group of molecules from petroleum belongs through its molecular weight. Because the database is generated from compounds whose composition is known, it is known that a given compound composition will have all the possible compositions. To generate molecules based on knowledge of the oil composition, it is used the general formulas of organic compounds which are formed by homologous series. Homologous series are series of organic compounds with similar chemical properties whose components differ by a constant relative molecular mass (CH2). Such series are generated and their molecular weights are calculated and stored together with their molecular formulas, in ascending order of mass, being able to generate the molecular groups with the help of a computing routine in VBA language.

O propósito deste trabalho é avaliar as características e classificação de compostos orgânicos, presentes em frações de petróleo, em saturados, aromáticos, resinas e asfaltenos (SARA) e identificar a classe homóloga (compostos de enxofre, nitrogênio, oxigênio, enxofre-oxigênio, nitrogênio-oxigênio ou apenas hidrogênio e carbono), construindo um modelo, com base no peso molecular, e utilizando um banco de dados em Excel. No modelo, os compostos orgânicos são organizados em uma matriz atômica de séries homólogas. Essas são séries de compostos com propriedades químicas similares, que diferem por um peso molecular constante (CH2). Esses compostos são classificados por relações heurísticas de Hidrogênio/ Carbono (H/C) e Ligação Dupla Equivalente (DBE). DBE é o número de anéis ou ligações ? envolvendo o átomo de carbono, porque cada anel ou ligações ? resultam na perda de um átomo de hidrogênio. O banco de dados foi desenvolvido em Excel, usando programação em VBA (Visual Basic for Applications). Os dados experimentais foram obtidos utilizando a técnica analítica de espectrometria de massa por MALDI-TOF. Os resultados obtidos mostram que o algoritmo em VBA é capaz de identificar os compostos de uma amostra, dentro da faixa de erro definida e de acordo com a calibração do espectrômetro de massa. / The purpose of this study is to evaluate the characteristics and classification of organic compounds in petroleum fractions as saturates, aromatics, resins and asphaltenes (SARA) and identify the possible homologous class (compounds of sulfur, nitrogen, oxygen, sulfur-oxygen, nitrogen-oxygen or only hydrogen and carbon), building a model and using a database in Excel, based on molecular weight. In the modeling, organic compounds are organized in an atomic matrix of homologous series. These are series of similar chemical properties, which differ by a constant molecular weight (CH2). Then, these compounds are classified through heuristics of hydrogen/ carbon ratio (H/C) and DBE (Double Bond Equivalent) relations. DBE is the number of rings or ? bonds involving carbon, because each ring or ? bonds results in a hydrogen atoms loss. The database was developed in Excel using VBA (Visual Basic for Applications) programming. The experimental data were obtained by analytical technique of MALDI-TOF mass spectrometry. The results show that the VBA algorithm is able to identify compounds from a given sample, within an error marge defined according to the mass spectrometer calibration.

An efficient and simple purification procedure for cow brain glutaminase (L- glutamine amidohydrolase, EC 3.5.1.2) is described. The main steps consisted of acetone extraction and French press treatment in the presence of phosphate ions, precipitation of nucleic acids and lipids by 0.08 % protamine·S04 and high speed centrifugation (300,000xg), ammonium sulfate fractionation, and gel filtration on Sepharose 4B first as the low molecular weight and then as the aggregated form. The yield was 22% and the final preparation had a specific activity of 142 μmoles/min/mg. The purification was more than 8000-fold over crude brain homogenate. The enzyme showed one strong and one diffuse band on SDS gel electrophoresis, suggesting that the enzyme was highly purified. Phosphate activated cow brain glutaminase with a sigmoidal concentration dependence. The activation was time dependent, a function of the pretreatment, and was enhanced by high concentrations of protein. The molecular weight of cow brain glutaminase also depended on the nature of the buffer in which it is dissolved. Gel filtration on Sepharose 4B was used to determine the molecular weight. Three forms of glutaminase were observed, one each in tris/acetate, phosphate, and borate/ phosphate buffer. These were interconvertible and have molecular weights of 170,000, 300,000, and >10^6 respectively. It appeared that activation of cow brain glutaminase by phosphate was due to formation of dimers or higher molecular weight polymers.

The focus of this study was to look at relationship between polydispersity caused by high pressure homogenization and molecular weight dependent antimicrobial activity of chitosan. It has been shown that chitosan has antimicrobial activity against bacteria, fungi, and viruses. Chitosan is obtained by partial de-N-acetylation of chitin which, consists of a ß 1-4 copolymer of glucosamine and N-acetylglucosamine residues. In this experiment we compared chitosan of sixteen different molecular weights after being processed through a high pressure homogenizer. Processed chitosan (420 kDa average molecular weight, 30% of acetylation) was dissolved in a 1% (v/v) acetic acid in water to a final concentration of 1% (w/v) and apparent viscosity of 183 MPa. The chitosan solution was passed through a high pressure homogenizer with 0-5 passes at pressure levels 0, 100, 200, and 300 MPa. After processing, the chitosan acetate was investigated to determine the effect on polydispersity in terms of molecular weight and the antimicrobial properties of chitosan at different molecular weights. All compounds were tested against Escherichia coli K-12 to determine antimicrobial activity. There is growing interest in the application of chitosan in food industry due to its wide range of desirable properties including being non-toxic and biodegradability. However, as a hydrophobic material, it is very challenging to work with. Though chitosan is a challenge to work my findings indicated a strong antimicrobial relationship with chitosan at 1% concentration with a molecular weight of 200 kDa and lower against E. coli. In conclusion chitosan has viable application with a variety of foods and can be used as a preservative that decrease bacterial activity below detection level’s and helps to prolong shelf-stable products.

Statement of the problem The primary objective of this thesis was to determine the best study design to evaluate the safety and effectiveness of an intermediate dose of low molecular weight heparin for secondary prevention of pregnancy associated VTE (PAVTE). An RCT was deemed unfeasible,so the use of a single arm study with prior evaluation of feasibility with a pilot study is proposed. // Methods - A systematic review was conducted to evaluate the efficacy of current strategies used for secondary prevention of PAVTE.A survey was used to elicit the non-inferiority margin. // Results - The pooled proportion of recurrent VTE in patients treated with full dose LMWH was 0.012(95% CI 0.006 to 0.02) and the rate of major bleeding was 0.025(95% CI=0.01 to 0.041). The non-inferiority margin was elicited at 2.5%. // Conclusions - Although a randomized controlled trial should be conducted whenever possible, in certain scenarios they are unfeasible. Therefore, an alternative study design should perhaps be used to evaluate the safety and efficacy of therapeutic strategies.

Molecular weight and concentration are two most important characteristics of polymers synthesized through chemical or microbial processes. However, current methods for characterizing polymer molecular weight such as Multi-Angle Laser Light Scattering (MALLS) or Gel Permeation Chromatography (GPC) require precise information on concentration as well as extensive sample preparation. Additionally, these current methods are also generally expensive, low throughput, and require large sample titers. These limitations prevent dynamic time-point studies of changes in molecular weight, which would be very useful for monitoring synthesis progress in microbes or in chemical synthesis. In this thesis, we designed, fabricated, and tested a rapid, low cost, high throughput, modular microfluidic system for determining polymer molecular weight in samples of unknown concentrations. To assess the accuracy of this system, we first constructed theoretical predictions for its accuracy, and then compared these to the experimental results from our microfluidic system. The system evaluated molecular weight by correlating the behavior of polymers in various solvent conditions to their molecular weights. The system consists of two modules for measuring fluid viscosity, and for controlling solvent conditions. Results of this study will show that this system is able to evaluate the differences in polymer viscosity for varying molecular weights and solvent conditions. For the solvent control module, we show that salt concentrations in small titers of polymer solutions can be rapidly added or subtracted and evaluated compared with current methods. Next, we will show the efficacy of the viscosity module at rapidly and accurately assessing fluid viscosity over a wide range of molecular weights. Finally, we will show the effects of solvent changes on molecular weight viscosity, and thus the efficacy of the system in determining molecular weight from fluid viscosity. This system will be applied to the evaluation of both the biologically produced polymer Hyaluronic Acid (HA) as well as the synthetically produced polymer Poly-ethylene Oxide (PEO).

Condensed tannins (CT) are polyphenolic compounds that have demonstrated biological activities in ruminants including suppression of enteric methane (CH4) production, protein binding and suppression of gastrointestinal nematode (GIN) infections. Some forage CT have been reported to be biologically active, whereas others have demonstrated no biological activity at all. While the chemical structure of CT has been postulated to be a key contributing factor affecting biological activity, the specific factors that determine whether or not CT from a specific forage have bioactive properties remain unknown. Results from previous studies have shown that as molecular weight of CT increases, CT biological activity also increases. Others have reported no effect of CT molecular weight on biological activity. The relationship between molecular weight of CT and CT biological activity remains inconclusive. The effect of molecular weight of CT from a variety of warm-season perennial legumes commonly consumed by ruminants on biological activity has not been adequately explored. The objectives of this study were to determine if molecular weight of CT from warm-season perennial legumes could predict the biological activity of CT relative to suppression of enteric CH4 production, protein-binding ability (PB) and anthelmintic activity, and to compare the biological activity of CT from native warm-season perennial legumes to that of the introduced species Lespedeza cuneata, a plant that has gained attention in recent years due its anthelmintic properties. All or a combination of the following warm-season perennial legume species were evaluated for in vitro gas production, protein-precipitable phenolics (PPP) and PB, and percent larval migration inhibition (LMI). Eight North American native warm-season perennial legumes: Leucaena retusa Benth. (littleleaf leadtree), Desmanthus illinoensis (Michx.) MacMill. Ex B.L. Rob. & Fernald (Illinois bundleflower), Lespedeza stuevei Nutt. (tall lespedeza), Mimosa strigillosa Torr. & A. Gray (powderpuff), Neptunia lutea (Leavenworth) Benth. (yellow puff), two ecotypes of Acacia angustissima var. hirta (Nutt.) B.L. Rob (prairie acacia), Desmodium paniculatum (L.) DC. var. paniculatum (panicledleaf ticktrefoil), and two introduced legumes: Arachis glabrata Benth. (rhizoma peanut) and Lespedeza cuneata (Dum. Cours.) G. Don (sericea lespedeza) were included. In vitro CH4 production regressed on CT MW resulted in a R2 of 0.0009 (P = 0.80). There was no correlation between PPP or PB and MW of CT (R^2 0.11; P = 0.17 and R^2 0.02; P = 0.54, respectively). There was a weak correlation between CT MW and percent LMI (R^2 0.34; P = 0.05). The results of our study strongly suggested that CT MW does not explain the biological activities of enteric methane suppression or protein-binding ability. Condensed tannin MW may be involved in anthelmintic activity of CT from the forage legumes surveyed. North American native legumes containing biologically active CT, as compared to introduced species, were identified as having promise for use in ruminant diets.

Hyaluronic acid (HA) is a high value biopolymer that has numerous biomedical and cosmetic applications. It is currently derived from two sources, namely animal tissues and bacterial fermentation (Fong Chong et al. 2005). The molecular weight (Mw) of HA can vary from several hundred thousand dalton (Da) to approximately 8 MDa (Widner et al. 2005). High Mw HA has surgical applications, and therefore constitutes a major component of the lucrative HA market. The current need is largely met by extraction from animal tissues, e.g., rooster comb and bovine vitreous humor (Shiedlin et al. 2004). However, the potential of contamination with adventitious agents (e.g., viruses) have raised regulatory concerns regarding the use of animal extracts in pharmaceutical products. Moreover, with recent reports of zoonotic diseases (e.g., bovine spongiform encephalitis and avian influenza virus), pharmaceutical companies are moving towards microbial HA sources. Although HA obtained from bacterial fermentation does not have the problem of viral contamination, this approach has not yet resulted in a process where HA of sufficiently high Mw for surgical applications can be derived. While attempts have been made to produce higher Mw HA through cross-linking, cross-linked HA is undesirable for certain medical procedures (e.g., ophthalmic applications) which requires a natural polymer with a short half-life. Nevertheless, due to its availability and the relative ease of purification, bacterial fermentation has the potential of replacing extraction from animal tissues as a preferred commercial source of HA. This thesis presents a good example of a metabolic engineering study where modern techniques (e.g., molecular biology, fermentation and omics technologies) are used to explain complex cell metabolism. The hypothesis for this study was that the precursors to HA, i.e., UDP-glucuronic acid and UDP-N-acetylglucosamine and consequently the genes involved in precursor generation, are important for HA Mw. However, environmental manipulation, e.g., anaerobic versus aerobic or glucose versus maltose, often results in large global changes in metabolite concentration and enzyme activities. This makes it impossible to resolve issues related to Mw control. Classical statistical methods do not provide a meaningful inference as the number of explanatory variables always exceeds the number of independent observations. Hence, it is difficult to distinguish between causative and accidental correlation. This work first examined the influence of manipulation of metabolite concentrations in the hyaluronan pathway to find an explanation for the mechanism of Mw control. To achieve this, the five essential genes of the hyaluronan synthesis (has) operon in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) were first examined. These genes are involved in two pathways which lead to the production of either UDP-glucuronic acid or UDP-N-acetylglucosamine. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased HA Mw, while overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased HA Mw. The Mw variation generated provided a stepping stone for further understanding of Mw control of HA. The highest Mw observed was achieved with combined overexpression of pgi and glmU. This study proved that there is a positive correlation between UDP-N-acetylglucosamine and Mw. The first model for HA Mw control based on the concentration of activated sugar precursors is described in this study (Chapter 3). This correlation observed led to the hypothesis that high Mw HA can be achieved when an appropriate balance of the two HA precursor is maintained. Three genes in the two precursor pathways are not found in the has operon of S. zooepidemicus. To obtain a complete overview of all genes in the HA pathway, these genes were also examined using overexpression studies. Individual overexpression of these genes had negligible effects on HA Mw and production. Despite the positive correlation previously observed between UDP-N-acetylglucosamine and Mw, sequential overexpression of genes involved in the UDP-N-acetylglucosamine precursor pathway did not increase Mw of HA produced. This is surprising since the highest pool of UDP-N-acetylglucosamine was achieved in this case. This suggests that a threshold effect is present in the correlation between UDP-N-acetylglucosamine and Mw. This threshold effect may be defined by a balance between the two precursors. To investigate this phenomenon further, the precursor ratio was also manipulated by co-metabolising glucose and N-acetylglucosamine. Similar to the previous experiment, a significant increase in UDP-N-acetylglucosamine levels was observed despite only a marginal increase in Mw (Chapter 4). Surprisingly, an increase in Mw was observed with the introduction of a plasmid in S. zooepidemicus. This plasmid effect was studied on a global scale using transcriptome and proteome analysis to understand the changes occurring in the system. The increase in Mw due to the plasmid effect is independent of the functions, i.e., nisin promoter or antibiotic resistance, encoded in the plasmid. A gene involved in UDP-N-acetylglucosamine production, UDP-N-acetylglucosamide 1-carboxivinyltransferase (murA), was significantly down-regulated in both the plasmid bearing strain and the high Mw strain (pgi). In addition, overexpression of murA decreased both the concentration of activated sugar precursors and HA Mw. There was however no evidence of down-regulation of murA in the plasmid containing strain from transcriptomics data. This suggests that control is exerted either at the translation level or by protein degradation (Chapter 5). This thesis contributes and represents an ongoing effort to understand the elusive mechanism of Mw control of HA.