The surface and structural change of human hair fibre have been analysed to determine the oxidation effects for bleached hairs. Three types of bleached hairs (6% H2O2 bleach, 9% H2O2 commercial bleach and commercial persulphate bleach (contains 9% H2O2)) as well as virgin hair were evaluated with the increasing treatment time using Scanning Electron Microscopy (SEM), Reflective Spectrophotometry, Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared (FTIR) Spectroscopy. It is obvious that longer treatment times result in the greater surface and structural damage. However, commercial persulphate bleach causes less surface damage for the cuticle. 6% H202 bleach has overall moderate damage effects on both cuticle and cortex over the treatment time. 9% H2O2 commercial bleach indicates two different damage stages. The first 1.5h bleached hairs show mild oxidation to the surface, whereas the damage becomes heavy after 2h. This phenomenon results in that 9% H2O2 commercial bleach has a more intensive oxidation damage in the cortex than the commercial persulphate bleach. This is in line with DSC investigation which shows that the intermediate filament of 9% H2O2 commercial bleach is heavily damaged after the extensive oxidation time (greater than or equal to2h). Although commercial persulphate bleach contains the stronger oxidising agent, it has a less surface damage than 9% H2O2 commercial bleached hair in FTIR-ATR measurement, and a similar oxidation effect on the matrix as 6% H202 bleached hair in FTIR transmission investigation. In addition, it has been verified by colour measurements that bleached hairs have an overall lighter, yellowish and reddish colour. Consequently, commercial persulphate bleached hair is much lighter and more yellow than 9% H2O2 commercial bleached hair and 6% H202 bleached hair. DSC investigations reveal that the three bleaches have a homogenous oxidation effect on IFs and IFAPs. The deconvolution results using three Gaussian distributions confirm this observation. The stronger bleach results in a homogenous structural damage on both para- and ortho-cortex with increasing bleaching time. Commercial persulphate bleach and 9% H2O2 commercial bleach have a progressive damage effect on the ortho- and para- cortex than 6% H202 bleach. Kinetics analysis is conducted for the virgin and bleached hairs by using various heating rates according to ASTM-E698. The activation energies of 260 kJ/mol for the virgin hair and 295 kJ/mol for the commercial persulphate bleached hair (2h) are determined from the slope of the regression line of peak temperature, TD (as 1/TD) and heating rate, β (as lnβ) on the basis of the Arrhenius-equation. The predominant structural damage for various heating rates only occurs in the IF. It is shown that a linear increase in DeltaHD occurs for lower heating rates, while it is constant for higher heating rates. This can be ascribed to the hypothesis that a lower heating rate favours a crystal transformation change (alpha-β transformation), while a higher rate favours a crystalline-amorphous transformation. SEM examines the morphological changes of hair samples after DSC. The cortex has been dissolved at the lower heating rate. The commercial persulphate bleached hairs (2h) show an overall shrunk cuticle surface and fewer and smaller hydrolysed protein granules, due to the previous damage of the alpha-helix in the cortical cell.

Ce travail se propose de montrer l’articulation de la nature et de l’artifice au sein de la pensée de Rousseau. Nous verrons que la connaissance de la nature est bornée par une origine qui nous échappe et une puissance de transformation qui ne semble pas avoir de fin. Cela conduit notre auteur à remettre en cause l’existence d’un ordre de la nature sur le plan spéculatif et sur le plan moral.. Dans l’homme la nature va se révéler comme vide. La perfectibilité est l’effet de l’inachèvement de la nature de l’homme. Le devenir social de l’homme sera pensé comme l’effet de la découverte érotique de l’autre. Cette découverte altère le moi absolu de l’homme à l’état de nature et le reconfigure en un moi relatif exposé au manque de l’autre. De cette exposition résultent toutes les formes de la socialité, l’aliénation et la liberté. L’étiologie amoureuse de la dénaturation nous a permis de reconnaitre l’importance de la réflexion sur le goût dans la constitution de la sociabilité. La différence sexuelle apparait comme le résultat d’un processus de différentiation dont le ressort est le désir de plaire. Le désir de plaire sera pour Rousseau un obstacle à l’uniformité de la société. Il préserve les différences qui sont à la fois les conditions du bon goût et de la volonté générale. La dépendance de l’homme devenu social est paradoxalement ce qui fait de la liberté une propriété en puissance de la vie en société. Dans ce cadre, le peuple n’est ni un sujet, ni une instance juridique, il est la forme du lien sociale sous la détermination de la liberté. Il y a peuple lorsque le lien unissant chacun à l’État, et unissant les hommes entre eux sous les conditions de l’égalité et de la liberté, devient un intérêt particulier. / This thesis shows the articulation between nature and artifice in Rousseau's thought. Rousseau argues that the knowledge of nature is limited by an origin that escapes from us and by a transformational power that seems immeasurable. This leads him to interrogate the existence of a natural order, both at speculative and moral levels. The man of nature is interpreted as unfinished with an unlimited changing potency. Rousseau argues that man´s becoming is the result of the erotic discovery of the other. This discovery distorts the absolute I of the man in the natural state and reshapes them in a relative I - exposed to the other's absence. From this recognition stems all forms of sociality, alienation and liberty. “Denaturation”'s amorous aetiology allows us to recognize the centrality of taste in the constitution of sociability. Sexual difference appears as the consequence of a differentiation process guided by the desire to please. For Rousseau, the desire to please challenges society´s uniformity. He argues that both the conditions for the good taste and general shape/foster/configure difference. The dependency of the human – as social being- is what, paradoxically, makes freedom a potential property in social life. In this scenario, “the people” is neither a subject nor a juridical element. It represents the social tie shaped by the conditions of freedom and equality. “The people” emerges under the conditions of equality and liberty and when every individual is tied to the state and to each other reflecting the union of everybody´s particular interests / Naturaleza y artificio en la constitución de la sociabilidad en el pensamiento de Rousseau.Este trabajo se propone mostrar la articulación de la naturaleza y el artificio en el pensamiento de Rousseau. El conocimiento de la naturaleza está para Rousseau limitado por un origen inalcanzable y una potencia de transformación que parece infinita. Lo cual lleva al autor a cuestionar la existencia de un orden de la naturaleza tanto en el plano especulativo como en el plano moral. El hombre de la naturaleza se presenta como un ser inacabado, con una potencia de transformación ilimitada. El devenir social del hombre resulta del descubrimiento erótico del otro. Este descubrimiento altera el yo absoluto del hombre natural y lo reconfigura en un yo relativo expuesto a la falta del otro. De esta exposición resultan todas las formas de sociabilidad, la alienación y la libertad. La etiología amorosa de la desnaturación permite reconocer la importancia del gusto en la constitución de la sociabilidad. La diferencia sexual es el resultado de un proceso de diferenciación cuyo móvil es el deseo de gustar. El deseo de gustar es un obstáculo a la uniformidad de la sociedad. Preserva las diferencias, estas son la condición del buen gusto y de la voluntad general. La dependencia del hombre devenido social es paradójicamente lo que hace que la libertad sea una propiedad en potencia de la vida en sociedad. En este marco, el pueblo no es ni un sujeto, ni una instancia jurídica, es la forma del lazo social determinado por las condiciones de la igualdad y la libertad. Hay pueblo cuando el lazo que une cada uno al Estado, y a los hombres entre sí, bajo la condición de la igualdad y de la libertad, se transforma en el punto de unión de todos los intereses particulares.

The objective of this project was to analyze the thermodynamics (thermal conductivity and diffusivity as well as protein denaturation) and physical properties (percent expressible moisture, cooking loss, change in steak thickness, shear force, texture profile analysis and rheological behavior) of beef steaks of different USDA quality grades (Upper 2/3 Choice and Select), thicknesses (thick and thin), and grill surface temperatures (high and low) cooked to the same internal degree of doneness to determine if a specific set of cooking parameters would create a profound difference in the eating characteristics, described by the tenderness and juiciness of cooked beef strip steaks. The elastic behavior of the surface and centers of beef steaks were analyzed to determine how the microstructure of the beef responded to applied stress. The elastic behavior of steak centers was influenced in a three-way interaction between USDA Quality Grade, steak thickness, and grill surface temperature while the elastic behavior of the surface of steaks was influenced only by USDA Quality Grade and steak thickness. These interactions along with the differences in the thermal characteristic of proteins suggest that the microstructure of beef steaks is significantly affected by each cooking treatment group. The physical properties in the beef steaks further support through more tangible applications that the composition, thickness, and cooking regiments impact the microstructure and thermal properties of beef and thus final tenderness and texture. This project identified cooking preparation should take into consideration that quality grade, thickness and cooking temperature will affect the textural eating qualities of beef steaks. Choice steaks were shown to be ideally sliced thick and cooked on a low grill surface temperature supported by the springiness, hardness, expressible moisture, and rheological data. Select steaks were not always effected by grill surface temperature and had similar results among the different measurements but the hardness, resilience and chewiness values along with viscosity suggest a thick steak cooked at a high grill surface temperature. Therefore, cooking parameters may be utilized as a mechanism to enhance beef steak palatability.

It is well known that folded proteins in water are destabilized by the addition of urea. When a protein loses its ability to perform its biological activity due to a change in its structure, it is said to denaturate. The mechanism by which urea denatures proteins has been thoroughly studied in the past but no proposed mechanism has yet been widely accepted. The topic of this thesis is the study of the mechanism of urea-induced protein denaturation, by means of Molecular Dynamics (MD) computer simulations and Nuclear Magnetic Resonance (NMR) spectroscopy. Paper I takes a thermodynamic approach to the analysis of protein – urea solution MD simulations. It is shown that the protein – solvent interaction energies decrease significantly upon the addition of urea. This is the result of a decrease in the Lennard-Jones energies, which is the MD simulation equivalent to van der Waals interactions. This effect will favor the unfolded protein state due to its higher number of protein - solvent contacts. In Paper II, we show that a combination of NMR spin relaxation experiments and MD simulations can successfully be used to study urea in the protein solvation shell. The urea molecule was found to be dynamic, which indicates that no specific binding sites exist. In contrast to the thermodynamic approach in Paper I, in Paper III we utilize MD simulations to analyze the affect of urea on the kinetics of local processes in proteins. Urea is found to passively unfold proteins by decreasing the refolding rate of local parts of the protein that have unfolded by thermal fluctuations. Based upon the results of Paper I – III and previous studies in the field, I propose a mechanism in which urea denatures proteins mainly by an enthalpic driving force due to attractive van der Waals interactions. Urea interacts favorably with all the different parts of the protein. The greater solvent accessibility of the unfolded protein is ultimately the factor that causes unfolded protein structures to be favored in concentrated urea solutions.

Heparin binding (HB) proteins mediates a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins could bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to the currently available methods. One of the most important classes of heparin binding protein is the fibroblast growth factors (FGFs) and its receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which exhibit several disadvantages, including time-consuming experimental procedures and regeneration and results in high cost for production of recombinant proteins. Authenticity of the purified proteins was verified by SDS-PAGE and MALDI mass spectrum analysis. Results of the heparin binding chromatography and steady state fluorescence experiments showed that the FGF-1 and the D2 are in a native biologically active conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

In Streptococcus pyogenes, the protein SiaA (HtsA) is part of a heme uptake pathway system and involved in heme transfer from Shp to the ABC transporter. SiaA mutants, in which alanine replaces the axial histidine (H229) and methionine (M79) ligands, as well as a lysine (K61) and cysteine (C58) located near the heme propionates, are reported. Studies on a mutant of a cysteine expected to be at a distance from the propionates (C47A) are also reported. The coordination state and spin state of the selected mutants were determined via Resonance Raman studies. The pKa values of mutants ranged from 9.0 to 9.4, which were close to the pKa of the WT SiaA (9.7). The midpoint reduction potential of lysine (K61A) mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the WT protein (68 ± 3 mV). The addition of guanidinium hydrochloride resulted in protein denaturation that could show more than one process and occurred over days. The ease of protein unfolding was directly related to the extent of interaction of the residues with the heme: changes in the axial ligands resulted in far greater changes in heme protein stability than changes in the residues near the heme propionates. The causative agent of diphtheriae, Corynebacterium diphtheriae, imports heme via an ABC uptake transporter. In this research, two of the five proteins in the heme uptake pathway of C. diphtheriae were studied. These proteins were HmuT, lipoprotein component of the ABC transporter, and HtaA, the heme receptor. UV-visible spectroscopy and fluorescence spectroscopy showed that HmuT protein as isolated bound a porphyrin, rather than heme. Electrospray ionization mass spectrometry (ESI-MS) studies suggested that two tetrapyrroles were bound. To assess stability of this protein towards heme release, thermal denaturation studies were performed. For HtaA, UV-visible and fluorescence spectroscopy also showed the protein as isolated was also bound a porphyrin, rather than heme. Homology studies showed that HtaA protein is quiet distant from homologous heme uptake proteins and could be a member of novel heme binding domain family.

The possible existence of extraterrestrial life forms has been of interest to humans for many millennia. In the past few decades space travel has provided an opportunity to search life outside of Earth. Chiral molecules are critical molecules in Earth-based life and are among the first chemical molecules sought after as proof of potential extraterrestrial life; however, identification of these chiral molecules is difficult due the lack of sensitive instruments. The objective of this work is to develop a benchmark reaction to be used as a guide in the development of instrumentation, such as a polarimeter, to be used in the search for extraterrestrial life. To achieve this objective, to investigate the enzyme mandelate racemase (MR), which catalyzes the racemization between the enantiomers of mandelate. MR is a member of the enolase superfamily, which contains a (alpha/beta)7-b barrel domain, the fold most frequently found among all known protein structures. Activity of the enzyme was measured at low temperatures and in non-aqueous media, as these are the conditions that represent extraterrestrial terrain. We find that mandelate racemase (MR) is active in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between 30C to 70C; however, the enzyme is not active in several organic cryosolvents. The stability of the structure of MR was also explored. Using differential scanning calorimetry (DSC) we observe the unfolding of the enzyme was irreversible and therefore kinetically controlled. We also found proof for divergent evolution of the enolase superfamily, providing evidence for divergent evolution across the MR and muconate lactonizing enzyme (MLE) subfamilies has been demonstrated. However, we also conclude that reactions yielding a polarimetric signal, such as racemizations employed in this work, are suitable as a tool to find signs of life.

This dissertation focuses on the spectroscopic and thermodynamic characterization of the unusual metal-RNA cluster found in the P5abc subdomain of the Tetrahymena group I intron. The P5abc subdomain is a part of the P4-P6 domain found in the Tetrahymena thermophila group I intron selfsplicing RNA. From both X-ray crystal structures of the P4-P6 domain, a remarkable cluster of Mg2+ or Mn2+ ions was found in the P5abc subdomain (Cate et al. 1996; Juneau et al. 2001). It is believed that the metal ion core in the P5abc subdomain stabilizes the active conformation of the RNA (Cate et al. 1996). An understanding of the role of these metal ions in facilitating the correct structure of the P5abc subdomain provides insight into how metal ions help overcome the folding barriers of complex RNA structures. Under solution conditions, the properties of this uncommon metal ion core and its influence on the truncated P5abc subdomain structure have been investigated. Both EPR spectroscopy and thermal denaturation experiments have been employed to search for a spectroscopic signature of metal ion core formation and also determine the thermodynamic contribution of the metal ion core on the stability of the folded P5abc structure. A spectroscopic signature of metal ion core formation was assigned for the P5abc subdomain by EPR microwave power saturation studies. Power saturation studies of the P5abc subdomain, P4-P6 domain and corresponding mutants reveal that the addition of 5 equivalents of Mn2+ are required for the wild type P5abc subdomain to form the metal ion core under solution conditions in 0.1 M NaCl. Results from both domain and subdomain microwave power saturation studies suggest that this technique can be applied for detecting clustering of Mn2+ ions in other RNA structures. The thermodynamic consequence of this metal ion core was probed by thermal denaturation techniques including UV-Vis spectroscopy and differential scanning calorimetry (DSC). DSC experiments were utilized to directly determine the thermodynamic contribution of the metal ion core. This value was determined to be an average of ∆∆G of -5.3 kcal/mol and is consistent with ∆∆G values obtained for other RNA tertiary structures.

In this thesis, we have advanced a set of distinct bioinformatic and computational tools to address the structure and function of proteins. Using data mining of the protein data bank (PDB), we have collected statistics connecting the propensity between the protein sequence and the secondary structure. This new tool has enabled us to evaluate new structures as well as a family of structures. A comparison of the wild type staphylococcal nuclease to various mutants using the proposed tool has indicated long-range conformational deviations spatially distant from the mutation point. The energetics of protein unfolding has been studied in terms of the forces observed in molecular dynamics simulations. An adaptive integration of the steered molecular dynamics is proposed to reduce ground state dominance by the rare low energy trajectories on the estimated free energy profile. The proposed adaptive algorithm is utilized to reproduce the potential of mean force of the stretching of decaalanine in vacuum at lower computational cost. It is then used to construct the potential of mean force of this transition in solvent for the first time as to observe the hydration effect on the helix-coil transformation. Adaptive steered molecular dynamics is also implemented to obtain the free energy change during the unfolding of neuropeptide Y and to confirm that the monomeric form of neuropeptide Y adopts halical-hairpin like pancreatic-polypeptide fold.

The physical understanding of biological processes such as transcription requires the knowledge of double-stranded DNA (dsDNA) physics. A notable thermo- dynamic property of dsDNA is its denaturation, at the melting temperature, in which it unwinds into two single-stranded DNAs via the formation of denat- uration bubbles (segment of consecutive unpaired base-pairs). The dynamics of denaturation has been studied so far at the base-pair (bp) scale, ignoring conformational chain degrees of freedom. These studies do not explain the very long closure times of 20 to 100 s, measured by Altan-Bonnet et al., of 18 bps long bubbles at room temperature. In this thesis, we study the closure of pre-equilibrated large bubbles, by using Brownian dynamics simulations of two simple DNA coarse- grained models. We show that the closure occurs via two steps: rst, a fast zipping of the initial bubble occurs until a meta-stable state is reached, due to the large bending and twisting energies stored in the bubble. Then, the meta-stable bubble closes either via rotational di usion of the sti side arms until their alignment, or bubble di usion until it reaches the chain end, or locally by thermal activation, depending on the DNA length and elastic moduli. We show that the physical mechanism behind these long timescales is therefore the dynamical coupling between base-pair and chain degrees of freedom.