Η ολοκληρωμένη εκτίμηση της θειολικής οξειδοαναγωγικής κατάστασης (ΘΟΚ) ενός οργανισμού ιστού ή κυττάρου είναι πολύ σημαντική καθώς οξειδοαναγωγικές αλλαγές των διαφόρων θειολικών μορίων συνδέονται με το οξειδωτικό στρες και με αρκετές ασθένειες. Η γενική ΘΟΚ (ΓΘΟΚ) χαρακτηρίζεται από τις συγκεντρώσεις ορισμένων συνόλων θειολικών μορίων στην αναγμένη και την οξειδωμένη μορφή τους (θειολικά οξειδοαναγωγικά ζεύγη). Αυτά τα ζεύγη μπορεί να είναι μη πρωτεϊνικά (non-protein ή NP) (όπως NPSH και NPSSNP με το NP να συμβολίζει οποιαδήποτε άλλη μη πρωτεϊνική θειόλη) ή πρωτεϊνικά (protein ή P) (όπως PSH, PSSP και PSSNP). Ειδικότερα, οι κυριότερες μη πρωτεϊνικές θειόλες γλουταθειόνη (GSH) και κυστεΐνη (CSH) μαζί με τα συμμετρικά, μεικτά δισουλφίδιά τους και τις οξειδωμένες τους μορφές (GSSG, PSSG, PSSC, NPGSHox, NPCSHox,) είναι τα οξειδοαναγωγικά ζεύγη τα οποία χαρακτηρίζουν την ειδική ΘΟΚ (ΕΘΟΚ), καθώς είναι εκείνα που απαντώνται σε υψηλότερη συγκέντρωση στους οργανισμούς. Στη διεθνή βιβλιογραφία δεν υπάρχει μεθοδολογία για την ταυτόχρονη ποσοτικοποίηση των θειολικών μορίων που χαρακτηρίζουν τη ΘΟΚ των οργανισμών. Συνεπώς, στόχος της παρούσας μελέτης είναι η ανάπτυξη μιας νέας μεθόδου ποσοτικοποίησης τόσο της ΓΘΟΚ όσο και της ΕΘΟΚ, που να είναι εφαρμόσιμη σε όλους τους οργανισμούς. Για το διαχωρισμό πρωτεϊνικών και μη πρωτεϊνικών μορίων χρησιμοποιήθηκε το τριχλωροακετικό οξύ που σε ορισμένη συγκέντρωση (>5%) καταβυθίζει αποτελεσματικά όλες τις πρωτεΐνες. Ο ποσοτικός προσδιορισμός των δισουλφιδικών μορίων και οξειδωμένων μορφών (NPSSNP, PSSP, PSSNP, GSSG, NPGSHox, NPCSHox, PSSG και PSSC) πραγματοποιήθηκε μετά από αναγωγή τους (με το αντιδραστήριο tributyl phosphine), ενώ ο ποσοτικός προσδιορισμός των ελεύθερων θειολών (PSH, NPSH, GSH και CSH) πραγματοποιήθηκε χωρίς την αναγωγή τους. Ειδικότερα, η ποσοτικοποίηση των αναγμένων διθειολικών ομάδων (δισουλφιδίων) και των ελεύθερων θειολών έγιναν με τα αντιδραστήρια 4,4-dithiodipyridine (για τις -SH ομάδες των αναγμένων δισουλφιδίων, καθώς και για τις ελεύθερες NPSH και PSH), o-phthalaldehyde (για την GSH, GSSG και NPGSHox) και νινυδρίνη (για την CSH και την NPCSHox), σε συνδυασμό με κατάλληλη μαθηματική επεξεργασία βασισμένη στη στοιχειομετρία των αντιδράσεων αναγωγής. Η υψηλή ευαισθησία της μεθόδου (στο επίπεδο του nmol) την καθιστά εφαρμόσιμη ακόμη και σε βιολογικά δείγματα χαμηλής περιεκτικότητας σε θειόλες (όπως πχ. το οφθαλμικό και το εγκεφαλονωτιαίο υγρό). / The thiol redox state (TRS) is an essential condition of prokaryotic and eukaryotic cells associated with all major biological processes. The general TRS (GTRS) part of it, is characterized by the levels of all thiol compounds of protein or non-protein origin in their reduced or oxidized form (thiol redox couples), while the specific TRS (STRS) by the levels of certain thiols, reduced and oxidized, free or membrane bound. The GTRS redox couples are composed of non-protein (NP) (such as NPSH and NPSSNP) or protein (P) (such as PSH, PSSP and PSSNP) thiols. On the other hand, the STRS redox couples are composed of the main non-protein thiol glutathione (GSH) and cysteine (CSH) together with their symmetric, mixed disulfides and oxidized forms (GSSG, PSSG, PSSC, NPGSHox, NPCSHox). In light of the fact that there is not available any appropriate method in literature for the simultaneous determination of the main thiol components that characterize TRS, a new method is developed for the purpose of this study for the quantification of GTRS and STRS, applicable to any organism. For the separation of protein from non protein thiols, trichloroacetic acid was chosen (at 5%) as the most effective protein precipitant. The determination of disulfides and oxidized forms (NPSSNP, PSSP, PSSNP, GSSG, PSSG, PSSC, NPGSHox and NPCSHox) was accomplished after their reduction with the tributyl phosphine (which, because of its hydrophobicity effectively reduces protein thiols as well), whereas the quantification of free thiols (PSH, NPSH, GSH and CSH) was accomplished without reduction. Reduced disulfides and free thiols were quantified by the more effective than DTNB 4,4-dithiodipyridine (for the determination of -SH groups of reduced disulfides as well as of free NPSH and PSH), o-phthalaldehyde (for the specific determination of GSH, GSSG and NPGSHox) and ninhydrin (for the specific determination of CSH and NPCSHox). The high sensitivity of the method (in the level of nmoles) makes it applicable even in biological samples of very low thiol concentration (such as ophthalmic or cerebrospinal fluid).

Macrophage death is likely to contribute to the transformation of fatty streaks into advanced atherosclerotic lesions. Previous work in the laboratory showed that OxLDL promotes cell death in human macrophages by a mechanism involving intracellular peroxide formation. Here we show that glutathione depletion induced by OxLDL occurs independent of peroxyl radical formation. Our data suggest that the depletion of glutathione is the fundamental defect that renders macrophages susceptible to OxLDL-induced cell injury, but alone is not sufficient to kill macrophages. We indicate that increased protein-Sglutathionylation is involved in OxLDL-induced macrophage death. A potentiation of OxLDL toxicity was observed in macrophages transfected with siRNA directed against either glutathione reductase or glutaredoxin. Our data suggests that OxLDL-induced cell injury in human macrophage is mediated by the depletion of GSH, a decreased in the GSH/GSSG ratio and peroxyl radical formation. All three signals are required for OxLDL-induced macrophage death. Our results also show that the glutathione reductase/glutaredoxin system protects macrophages from OxLDL-induced cell death.

Compartmentalization of redox reactions is essential to all life forms. Protein activity can respond to changes in the local redox environment through the reversible oxidation of cysteine thiols. For the majority of cysteines in the proteome, this interaction takes place through equilibration with the glutathione pool; this raises the question whether this redox pool acts as a buffer, or instead as a sensitive media, transducing information from a local physiological state into protein function.

The objective of the research was to investigate proton-coupled electron transfer (PCET) reactions involving antioxidants to gain insight into the detailed mechanisms of glutathione (GSH), Trolox, and α-tocopherol (α-TOH). PCET reactions are complex redox reactions that transfer electrons and protons sequentially or in concert. These reactions are ubiquitous in natural or artificial processes that produce electrochemical energy that is extractable as electricity or as chemical fuels of high energy content. Examples of processes based on PCET are photosynthesis, respiration, nitrogen fixation, carbon dioxide reduction, redox fuel cells, and artificial photosynthesis. Antioxidants were selected as a PCET model to understand the coupling between proton transfer (PT) and electron transfer (ET) in order to elucidate structure-reactivity relationships under different experimental conditions. PCET reactions were studied with a set of electrochemical techniques to propose a preliminary mechanism that could be validated with digital simulations matching the electrochemical response. In some cases, other analytical techniques were used to aid in the system characterization. This thesis presents the results and discussion of the effects of oxidant-base pairs on the mediated oxidation of GSH, the -2e-/-H+ process of Trolox in aqueous and nonaqueous solvents with various pH values, and the particle collision electrolysis of α-tocopherol in oil-in-water emulsion droplets on an ultramicroelectrode. Ultimately our goal was to determine the kinetic and thermodynamic factors that control PCET reactions so that they can be applied in designing artificial systems for the production of energy using more abundant reagents with lower cost and better yields.

Autism is a lifelong neurodevelopmental disorder. The etiology of autism still remains unclear due to the heterogeneous and complex nature of the disorder, however synergistic actions between genetic components and environmental factors have been suggested. Acetaminophen (APAP) is one of the most popular over-the-counter drugs that possess antipyretic and analgesic effects. It is considered a relatively safe and effective within therapeutic doses. Recently, early exposure to APAP has been suggested to be one of the underlying cause of autism. Children are often prescribed APAP to lessen fever or irritability after vaccination during the first year, and APAP may adversely affect the normal brain development. In order to better understand the association with APAP and autism, we used an inbred mouse strain BTBR T+tf/J (BTBR). BTBR exhibits behavioral deficits that mimic the core behavioral deficits of human autism. In the study, investigated 1) if BTBR mice showed differences in thiol biochemistry and EAAT3 levels in brain compared with C57BL/6J (C57) mice, 2) if early exposure to APAP induced behavioral changes worsening the autistic phenotypes of BTBR in adolescence, and 3) if APAP exposure in neonatal mice induced possible toxicity at various doses. As a result, we observed that BTBR mice have significantly lower plasma sulfate levels and EAAT expression levels in the frontal cortex compared to C57 mice. Surprisingly, neonatal therapeutic dose of APAP administration did not induce behavioral changes in both C57 and BTBR in adolescence. However, we showed that a supratheraputic dose of APAP significantly elevated levels of oxidative stress marker in the brain. Overall, the results suggested that BTBR mice would be a useful mouse model to investigate effects of various environmental factors that have been associated with autism. In addition, early exposure to APAP at supratherapeutic doses may negatively affect normal brain development.

Improvement in the in vitro oxidative folding of disulfide-containing proteins, such as extracellular and pharmaceutically important proteins, is required. Traditional folding methods using small molecule aliphatic thiol and disulfide, such as glutathione (GSH) and glutathione disulfide (GSSG) are slow and low yielding. Small molecule aromatic thiols and disulfides show great potentiality because aromatic thiols have low pKa values, close to the thiol pKa of protein disulfide isomerase (PDI), higher nucleophilicity and good leaving group ability. Our studies showed that thiols with a positively charged group, quaternary ammonium salts (QAS), are better than thiols with negatively charged groups such as phosphonic acid and sulfonic acid for the folding of bovine pancreatic trypsin inhibitor (BPTI). An enhanced folding rate of BPTI was observed when the protein was folded with a redox buffer composed of a QAS thiol and its corresponding disulfide. Quaternary ammonium salt (QAS) thiols and their corresponding disulfides with longer alkyl side chains were synthesized. These QAS thiols and their corresponding disulfides are promising small molecule thiols and disulfides to fold reduced BPTI efficiently because these thiols are more hydrophobic and can enter the core of the protein. Conformational changes of disulfide-containing proteins during oxidative folding influence the folding pathway greatly. We performed the folding of BPTI using targeted molecular dynamics (TMD) simulation and investigated conformational changes along with the folding pathway. Applying a bias force to all atoms versus to only alpha carbons and the sulfur of cysteines showed different folding pathways. The formation of kinetic traps N' and N* was not observed during our simulation applying a bias force to all atoms of the starting structure. The final native conformation was obtained once the correct antiparallel β-sheets and subsequent Cys14-Cys38 distance were decreased to a bond distance level. When bias force was applied to only alpha carbons and the sulfur of cysteines, the distance between Cys14-Cys38 increased and decreased multiple times, a structure similar to the confirmation of N*, NSH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.

NMR spectroscopy is a very powerful technique for the characterization of structure and dynamics of a variety of systems starting from small organic molecules to large biological macromolecules. In solids, the study of protons becomes more interesting because they are very sensitive to inter-molecular packing and are directly involved in hydrogen-bonding and aromatic π-π interactions, etc. The present thesis is devoted essentially to utilizing information from proton resonances obtained using multinuclear and multiple-quantum approaches. The thesis has two parts. The first part deals with methodological developments in the area of solid-state NMR, relevant to the study of rigid powder samples as well as partially ordered liquid crystalline materials. Methods have been proposed to investigate the structure of small molecules at moderate spinning frequencies and thermotropic liquid crystals at static conditions. Proton detected heteronuclear experimental methods based on both first and second-order cross polarization at moderate and ultra-fast magic angle spinning rates are also proposed. The second part of the thesis deals with the application of both newly proposed and existing solid state NMR methods to the study of several biologically relevant systems. These include the study of several designed as well as naturally occurring peptides. The use of first-principles calculations based on GIPAW method for supporting the experimentally obtained results has also been made. The thesis is divided into five chapters. In the second chapter, a new pulse sequence to correlate Double Quantum (DQ) proton frequencies to carbon Single Quantum (SQ) chemical shifts in the solid state has been proposed. In this sequence, named as MAS-J-1H (DQ)-13C-HMQC, the correlation between 1H and 13C is achieved through scalar coupling, while the double-quantum coherence among protons is generated through dipolar couplings. This experiment is particularly suited for the study of 13C in natural abundance. The advantages of the technique with applications to alanine, histidine and a model liquid crystalline material have been demonstrated. The assignment of 13C spectra of partially ordered systems has also been considered. In this case the assignment of the spectrum is a major challenge due to the interplay of anisotropic order and chemical shift parameters. The DQ-SQ correlation experiment described in the thesis has been applied to a well known liquid crystal and also to a novel thiophene based liquid crystal and the local order parameters of the liquid-crystal have been obtained. The thesis also presents results on the azelaic acid -isonicotinamide co-crystal as well as the drug ibuprofen obtained by using novel methodologies. In the case of the former, the problem of overlap of resonances was overcome with the use of the REVERSE-CP approach to separate out the carbon attached protons from the rest of the protons. Subsequently, by the use of several combined approaches, the structural features were identified. A new heteronuclear correlation pulse sequence for solids under fast MAS conditions has also been tested. With low r.f powers, a second-order dipolar term mediated transfer of magnetization between I and S spin known as second order cross-polarization (SOCP) was exploited to obtain the entire spin system connectivity. Both carbon detected and proton detected experiments have been carried out and their utility evaluated. Similar approaches to shed light on the structure and conformation of a set of proline and pseudoproline based designed β-turn peptides that are used as templates for understanding protein folding have been made. Results of studies on two biologically important forms of the short-chain peptides namely glutathione reduced (GSH) and oxidized (GSSG) tripeptides are also presented.