Turkish red pine is coastal tree and is a drought resistant pine that withstands more aridity and poor soils than most other timber species growing in the same climatic conditions. In Turkey, this species grows in southern and western Anatolia and is also found in the Marmara region. Drought results in a water deficit in plant tissues, which, in turn, can lead to an imbalance in the redox poise of plant cells, and thus inducing oxidative stress in plants. Resistance to conditions associated with oxidative-stress must, in part, rely on endogenous antioxidative defense mechanisms required to maintain cellular homeostasis. Glutathione is one of the major endogenous antioxidants in plants known to play an important role in plant defense mechanisms. Glutathione S-transferase (GST, EC 2.5.1.18) is a GSH dependent detoxifying enzyme in plants, which catalyzes the conjugation of GSH. In this study, we investigated the changes in cytosolic glutathione S-transferase enzyme activity using CDNB as substrate and total thiol amount in Pinus brutia Ten., related to the drought stress during four months, June to September. The osmotic pressure in the needles was also determined as an indirect measure of drought condition. Together with the increase in the temperature values from June to July, GST enzyme activity increased from 15,78 &plusmn / 1,36 &micro / moles min-1 mg protein-1 to 22,91 &plusmn / 1,99 &micro / moles min-1 mg protein-1 which was statistically significant. However in August, GST activity had fallen to 16,54 &plusmn / 1,61 &micro / moles/min/mg protein, which may be because of a local rainfall at the beginning of the August in the sampling area. In September, GST activity significantly increased with respect to June, in accordance with high temperatures. The total thiol amount was not changed significantly during the sampling period. Although there were statistically significant changes in osmotic pressure in the needdles collected during the same sampling period, it did not exactly correlated to the changes in GST activity.

Knowledge about selenium content of foods containing selenium species is very important in terms of both nutrition and toxicity. Bioavailability of selenium species for human body is different from each other. Hence, speciation of selenium is more important than total selenium determination. In the selenium speciation study, chicken breast samples, selenium supplement tablets and egg samples were analyzed for their selenium contents. In chicken breast study, chickens were randomly categorized into three groups including the control group (25 chickens), inorganic selenium fed group (25 chickens) and organic selenium fed group (25 chickens). After the optimization of all the analytical parameters used throughout the study, selenomethionine, selenocystine, Se(IV) and Se(VI) were determined using Cation Exchange-HPLC-ICP-MS system. In selenium supplement tablet study, anion and cation exchange chromatographies were used to determine selenium species. Arsenic is known as toxic element, and toxicity of inorganic arsenic species, As(III) and As(V), is much higher than organic arsenic species like arsenobetaine and arsenosugars. Hence, speciation of arsenic species in any matrix related with human health is very important. In the arsenic speciation study, Cation Exchange-HPLC-ICP-MS and Cation Exchange-HPLC-ES-MS systems were used to determine arsenobetaine content of DORM-2, DORM-3 and DOLT-4 as CRMs. All of the parameters in extraction, separation and detection steps were optimized. Standard addition method was applied to samples to eliminate or minimize the matrix interference. Thiols play an important role in metabolism and cellular homeostasis. Hence, determination of thiol compounds in biological matrices has been of interest by scientists. In the thiol study, Reverse Phase-HPLC-ICP-MS and Reverse Phase-HPLC-ES-MS systems were used for the separation and detection of thiols. For the thiol determination, thiols containing &ndash / S-S- bond were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be around 100%. Reduced and free thiols were derivatized before introduction on the column by p-hydroxymercuribenzoate (PHMB) and then separated from each other by using a C8 column. In the real sample measurement, yeast samples were analyzed using HPLC-ES-MS system.

Work in the Turos group over the past five years has focused on the development of N-alkylthio beta-lactams, which show antibacterial activity against Staphylococcus (including MRSA), Bacillus, and others. These compounds do not function in the manner of the traditional beta-lactam antibiotics, but were thought to undergo an intracellular thiol-transfer to coenzyme A. In expanding the SAR of these novel compounds, it was found that N-alkylthio-2-oxazolidinones also exhibit antibacterial activity. Although CoA acts as the thiol-redox buffer in the genera most susceptible to the N-alkylthio beta-lactams, studies on Coenzyme A disulfide reductase (CoADR) show that the redox buffer is not affected by these compounds. However, the recent finding that fatty acid synthesis is affected by the N-alkylthio beta-lactams led to the discovery that these compounds act as prodrugs, and that the asymmetric CoA disulfides produced by in vivo thiol transfer are potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH) through a novel thiol-disulfide exchange with the active site cysteine. Lactams 2a and 2g were also found to be potent inhibitors of this enzyme. In an effort to produce a CoA mixed-disulfide mimic which could cross the cell membrane, a series of simple aryl-alkyl disulfides were synthesized and tested against E. coli, S. aureus, and B. subtilis. Several of these compounds were found to be very potent antibacterials both in vitro and in vivo, with MICs less than 0.125 micrograms/mL. Comparison of the activities of these disulfides with those of acyl-CoA analogs and CoA mixed disulfides support the assertion that FabH is indeed the cellular target of these potent new compounds.

No description available.

The research described herein is aimed at exploring the thermo-mechanical properties of thiol-ene polymers in bulk form, investigating the ability of thiol-ene polymers to behave desirably as photolithographic media, and providing the first characterization of the mechanical properties of two-photon stereolithography-produced polymer structures. The thiol-ene polymerization reaction itself is well-characterized and described in the literature, but the thermomechanical properties of thiol-ene and thiol-ene/acrylate polymers still require more rigorous study. Understanding the behavior of thiol-ene networks is a crucial step towards their expanded use in bulk form, and particularly in specialized applications such as shape memory devices. Additionally, the thiol-ene polymerization reaction mechanism exhibits unique properties which make these polymers well suited to photolithography, overcoming the typical dichotomy of current materials which either exhibit excellent photolithographic behavior or have controllable properties. Finally, before two-photon stereolithography can create mechanisms and devices which can serve any mechanically functional role, the mechanical properties of the polymers they produce must be quantitatively characterized, which is complicated by the extremely small scale at which these structures are produced. As such, mechanical characterization to date has been strictly qualitative. Fourier transfer infrared spectroscopy revealed functional group conversion information and sol-fraction testing revealed the presence of unconverted monomer and impurities, while dynamic mechanical analysis and tensile testing revealed the thermomechanical responses of the systems. Nanoindentation was employed to characterize the mechanical properties of polymers produced by two-photon stereolithography. Optical and electron microscopy were exploited to provide quantitative and qualitative evaluations of thiol-ene/acrylate performance in small-scale polymerization regimes. The broad objective of the research was to explore thiol-ene polymer behavior both in bulk and at the small scale in an effort to supplement the material library currently used in these fields and to expand the design envelope available to researchers. The significance of the research is the advancement of a more complete and fundamental understanding of thiol-ene polymerization from kinetics to final properties, the quantitative establishment of the mechanical properties of materials created with two-photon stereolithography, and the comprehensive characterization of a supplementary class of photopatternable polymers with greater property tunability than is possible with currently used materials.

Η συνεισφορά των πρωτεϊνικών θειολών στη διαμόρφωση της θειολικής οξειδοαναγωγικής κατάστασης είναι πολύ σημαντική και αλλαγές της τελευταίας μπορεί να επιφέρουν καθοριστικές μεταβολές στη κυτταρική λειτουργία. Στόχος της παρούσας μελέτης είναι η βελτίωση προηγούμενων μεθόδων ποσοτικοποίησης των πρωτεϊνικών θειολών με τη χρήση νέων αντιδραστηρίων αναγωγής δισουλφιδικών δεσμών και ανίχνευσης -SH ομάδων. Σχεδιάστηκε μια νέα φωτομετρική μέθοδος προς την κατεύθυνση της ακριβούς ποσοτικοποίησης των πρωτεϊνικών θειολών PSH, PSSP και PSSNP. Ο νέος αναγωγικός πάραγοντας που αναδείχθηκε είναι το tributylphosphine (TBP) και το νέο αντιδραστήριο- ανιχνευτής ελεύθερων -SH ομάδων, το DPS ή 4- Aldrithiol. / The protein thiols contribution in the regulation of the thiol redox status is very important and changes in the last can produce serious cell disfunction. The purpose of this study was the improvement of precedent protein thiol quantification methods using new reagents of disulfide bonds reductants and –SH group tracers. A new photometric method has been planned leading to the exact quantification of protein thiol compounds such as PSH, PSSP and PSSNP. The new reductant used is tributylphosphine (TBP) and the new free -SH tracer reagent is DPS or 4- Aldrithiol.

Orientador: Roberto Carlos Burini / Resumo: A prevalência mundial de Síndrome Metabólica (SM) está aumentando drasticamente nos últimos anos. Esta desordem consiste de um conjunto de condições metabólicas, contemplando hiperadiposidade abdominal, resistência insulínica (RI), dislipidemia e hipertensão, e é fator de risco para o desenvolvimento de Diabetes Mellitus tipo 2 (DM2) e doenças cardiovasculares (DCVs). Muitos estudos indicam a participação do estresse oxidativo (EO) junto com o estado estresse inflamatório (EI) como parte do desenvolvimento e agravamento da SM e surgimento das doenças crônicas não-transmissíveis (DCNTs). O EO é caracterizado como estado de desequilíbrio entre os sistemas oxidativo e antioxidante, resultando no excesso de espécies reativas de oxigênio (EROs), menor capacidade antioxidante e efeitos deletérios a tecidos e células. A regulação das reações de oxidação e redução são importantes para o controle de funções fisiológicas e combate ao estresse. Diante disso, os aminotióis ou compostos tio-redox (homocisteína, cisteína e glutationa) exercem importante papel no equilíbrio do estado redox celular. Neste artigo de revisão, analisamos os aspectos fisiopatológicos de compostos tio-redox no curso da SM. / Abstract: The worldwide prevalence of Metabolic Syndrome (MetS) has been increasing dramatically in recent years. MetS is a set of metabolic conditions, including abdominal hyperadiposity, insulin resistance, dyslipidemia and hypertension, and is a risk factor for the development of type 2 diabetes mellitus and cardiovascular diseases. Many studies indicate the participation of oxidative stress along with the pro-inflammatory state as part of the development and aggravation of MetS and emergence of chronic noncommunicable diseases. Oxidative stress is characterized as a state of imbalance between oxidative and antioxidant systems, resulting in excess of reactive oxygen species, lower antioxidant capacity and deleterious effects on tissues and cells. The regulation of the oxidation and reduction reactions are important for the control of physiological functions and the fight against stress. In view of this, the aminothiols or thiol-redox compounds (homocysteine, cysteine and glutathione) play an important role in the equilibrium of the cellular redox state. In this review article, we analyze the pathophysiological aspects of thiol-redox compounds in the course of MetS. / Mestre

Submitted by Hugo Tadashi Kano null (tada_tk@aluno.ibb.unesp.br) on 2018-03-15T12:00:25Z No. of bitstreams: 1 Dissertação Mestrado - Hugo Tadashi Kano.pdf: 1976397 bytes, checksum: 22cf9f00e7caf878327b4634bb6d02ad (MD5) / Approved for entry into archive by Luciana Pizzani null (luciana@btu.unesp.br) on 2018-03-19T12:53:29Z (GMT) No. of bitstreams: 1 kano_ht_me-bot.pdf: 1976397 bytes, checksum: 22cf9f00e7caf878327b4634bb6d02ad (MD5) / Made available in DSpace on 2018-03-19T12:53:29Z (GMT). No. of bitstreams: 1 kano_ht_me-bot.pdf: 1976397 bytes, checksum: 22cf9f00e7caf878327b4634bb6d02ad (MD5) Previous issue date: 2018-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A prevalência mundial de Síndrome Metabólica (SM) está aumentando drasticamente nos últimos anos. Esta desordem consiste de um conjunto de condições metabólicas, contemplando hiperadiposidade abdominal, resistência insulínica (RI), dislipidemia e hipertensão, e é fator de risco para o desenvolvimento de Diabetes Mellitus tipo 2 (DM2) e doenças cardiovasculares (DCVs). Muitos estudos indicam a participação do estresse oxidativo (EO) junto com o estado estresse inflamatório (EI) como parte do desenvolvimento e agravamento da SM e surgimento das doenças crônicas não-transmissíveis (DCNTs). O EO é caracterizado como estado de desequilíbrio entre os sistemas oxidativo e antioxidante, resultando no excesso de espécies reativas de oxigênio (EROs), menor capacidade antioxidante e efeitos deletérios a tecidos e células. A regulação das reações de oxidação e redução são importantes para o controle de funções fisiológicas e combate ao estresse. Diante disso, os aminotióis ou compostos tio-redox (homocisteína, cisteína e glutationa) exercem importante papel no equilíbrio do estado redox celular. Neste artigo de revisão, analisamos os aspectos fisiopatológicos de compostos tio-redox no curso da SM. / The worldwide prevalence of Metabolic Syndrome (MetS) has been increasing dramatically in recent years. MetS is a set of metabolic conditions, including abdominal hyperadiposity, insulin resistance, dyslipidemia and hypertension, and is a risk factor for the development of type 2 diabetes mellitus and cardiovascular diseases. Many studies indicate the participation of oxidative stress along with the pro-inflammatory state as part of the development and aggravation of MetS and emergence of chronic noncommunicable diseases. Oxidative stress is characterized as a state of imbalance between oxidative and antioxidant systems, resulting in excess of reactive oxygen species, lower antioxidant capacity and deleterious effects on tissues and cells. The regulation of the oxidation and reduction reactions are important for the control of physiological functions and the fight against stress. In view of this, the aminothiols or thiol-redox compounds (homocysteine, cysteine and glutathione) play an important role in the equilibrium of the cellular redox state. In this review article, we analyze the pathophysiological aspects of thiol-redox compounds in the course of MetS.

Dans cette thèse, nous décrivons sommairement les techniques utilisées qui sont l’imagerie parrésonance plasmon de surface (SPRi) et la diffusion Raman exaltée de surface (SERS). Le butprincipal du projet Piranex dans lequel la thèse s’inscrit consiste au développement d’une biopucenanostructurée bimodale permettant le couplage des deux techniques SPRi et SERS. Cettebiopuce est constituée d’un film d’or par-dessus lequel nous avons déposé un réseau carré denanocylindres en or. Un ensemble d’études ont été effectuées pour caractériser ses propriétésplasmoniques du biocapteur afin d’en optimiser le signal SERS. Nous avons ainsi constaté quel’émission du signal était fortement anisotrope, dus à l’excitation du Mode de Bragg et que lechamp proche était principalement exalté sur les bords de la nanostructure. Les propriétés furentégalement comparées avec celles de réseaux identiques déposés directement sur un substrat diélectrique.Par la suite un ensemble d’études plasmoniques et SERS ont été effectuées pourl’aluminium, autre matériaux plasmonique d’intérêt. Enfin, un protocole de détection par SERSde l’ochratoxine basé sur un aptamère fut développé et a permis la détection de l’ochratoxine dès10 pM, bien en dessous de la limite autorisée par les organismes de régulation en agroalimentaire. / In this thesis, we briefly describe the techniques used, which are surface plasmon resonanceimaging (SPRi) and surface enhanced Raman scattering (SERS). The main goal of the Piranexproject in which the thesis is based is the development of a bimodal nanostructured biochipallowing the coupling of the two techniques SPRi and SERS. This bio-chip consists of a goldfilm over which we have deposited a square array of gold nanocylinders. A set of studies hasbeen carried out to characterize plasmonic properties of the biosensor in order to optimize theSERS signal. We have thus found that the emission of the signal was strongly anisotropic, due tothe excitation of the Bragg Mode and that the near field was mainly enhanced on the edges of thenanostructure. The properties were also compared with those of identical gratings depositeddirectly on a dielectric substrate. Subsequently a set of plasmonic and SERS studies were carriedout for aluminum, other plasmonic materials of interest. Finally, a detection protocol by SERS ofochratoxin based on an aptamer was developed and allowed the detection of ochratoxin with adetection threshold of 10 pM, well below the limit allowed by food regulatory agencies

Aqueous Phase Partitioning has a long history of applications to the analytical characterisation of biomolecules. However process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The main aim of this work was to explore the proposition that partition in Aqueous Two Phase Systems (ATPS) can be used as an analytical tool to detect protein isoforms and to assess the applicability of the method in clinical assays and for quality control in bioprocessing through examination of several analytical problems. The work also examined the development of automated methods of system preparation and sampling techniques to determine the partition coefficient in ATPS. The study demonstrated that the geometrical form of the phase diagram co-existence curve was of crucial importance since this directly affected the accuracy with which systems of defined Tie Line Length and Mass Ratio could be constructed. The TLL %Bias (accuracy) of a theoretical system range in the PEG1000-(NH4)2SO4 system at shorter TLL (12.2) was in the range +80.6% to -100% while at a longer TLL (53.1) the %Bias (accuracy) was reduced to +0.1% to -1.9%. At the same time the MR %Bias (accuracy) at shorter TLL (12.2) was in the range +59.5% to -21.3% while at the longer TLL (53.1) this was reduced to +2.7% to -2.6%. By contrast in the PEG8000-Dextran500 system the TLL %Bias (accuracy) at shorter TLL (13.1) was in the range +3.7% to -4.12%, while at a longer TLL (31.1) the range was +0.74% to -0.67%. The MR %Bias (accuracy) at the shorter TLL (13.1) was in the range +3.6% to -3% while at the longer TLL (31.1) the range was +1.1% to -1.4%. This illustrated that it is more difficult to work with a high degree of accuracy (e.g. %Bias <5%) close to the critical point in PEG-salt systems than in PEG-dextran systems. Two different approaches were taken to examine analytical phase partitioning. In the first approach the structure of the isoforms of a model protein (ovalbumin) were altered enzymatically. Analytical methods involving Strong Anion-Exchange chromatography were developed and applied to the separation of the ovalbumin isoforms. Removal of the phosphorylated groups (dephosphorylation of ovalbumin) was undertaken using alkaline phosphatase and de-glycosylation was attempted using neuraminidase and Endo-glycosidase F. However, both enzymatic approaches to deglycosylation were unsuccessful. Dephosphorylated isoforms were successfully produced and characterised. After partitioning in ATPS a clear difference was demonstrated between the behaviour of the native and dephosphorylated forms of ovalbumin. The mean % recovery in a PEG-salt ATPS was 99.8% (± 3.59) for the naive protein and 75.6% (± 4.03) for the dephosphorylated form. On the other hand, in a PEG3350-Dextran500 system, where solubility was maintained, a significant difference in the partition coefficient (K) of native and dephosphorylated ovalbumin was found. K for native ovalbumin was 0.85 while the partition coefficient of the dephosphorylated ovalbumin was 0.61. Analysis of covariance (ANCOVA) indicated that the regression coefficients of the respective partition isotherms were significantly different (p value < 0.05). In a second approach to examine analytical phase partitioning, chemical modification of a specific target surface amino acid of another model protein (serum albumin) was used to determine the degree of conjugation of the protein and also to determine its oxidative state. The method examined the reactivity of a free surface thiol to a wide range of labels ( (a) 2-methylsulfonyl-5-phenyl -1,3,4 oxidiazole reagent, (b) N-Ethylmaleimide (NEM) reagent, (c) 5, 5’-dithiobis (2-nitrobenzoate)(DTNB) (Ellman’s reagent), (d) N-pyrenylmaleimide (NPM) reagent, (e) Fluorescein-5-maleimide (F-5-M) Reagent). Only DTNB was found to modify the surface free thiol of serum albumin in a highly specific and quantitative manner. In the course of the development of a partitioning assay for surface free thiols of serum albumin significant oxidative properties were found to be associated with poly(ethylene glycol) PEG solutions and several attempts were made to find an oxidatively safe partitioning system by including antioxidants and by removal of contaminants by freeze drying. PEG3350-Dextran500 was found to provide an oxidatively safe environment for the development of a partitioning assay for the determination of albumin free thiols. A phase partitioning assay system capable of quantitatively resolving protein associated free thiols and low molecular weight thiols from a mixture of the two was developed. Correlation coefficients (R2) for the regression of experimentally determined protein free thiols in the presence of different levels of added LMW free thiol on the known addition of protein ranged from 0.77 to 0.83. The results demonstrated that the assay could quantify and distinguish both types of thiol in a simple two-step procedure.