Cette thèse concerne le développement d’approches spectroscopiques infrarouge et Raman exaltées de surface: la spectroscopie infrarouge exaltée de surface (SEIRAS) combinée avec une cellule de perfusion et la spectroscopie Raman exaltée de surface (SERS) couplée avec l’électrochimie. Dans le cadre du premier projet, différentes protéines ont été étudiées : lactose perméase (LacY), complexe I et IM30. Nous avons déterminé le pKa de Glu325 dans LacY sauvage et dans différents mutants portant des mutations dans le centre actif de translocation des protons. Sauvage complexe I a été oxydé avec différents agents oxydants et réduit avec NADH. Spectres différentiels correspondants ont été analysés. Des changements conformationnels dans la protéine IM30, induits par la présence des ions Mg2+, ont été observés.Dans le cadre du deuxième projet, une cellule spectroélectrochimique contenant une grille d’or a été adaptée pour étudier des protéines redox actives. Cette grille d’or sert à la fois de substrat SERS et d’électrode de travail. Cyt c, Hb et Mb ont d'abord été utilisés pour valider la configuration, puis l'approche a été étendue pour étudier une protéine membranaire. / This thesis concerns the development of surface-enhanced infrared and Raman spectroscopic approaches: surface-enhanced infrared absorption spectroscopy (SEIRAS) combined with perfusion cell and surface-enhanced Raman spectroscopy (SERS) combined with electrochemistry. Within the first project different proteins were studied: Lactose Permease (LacY), complex I and IM30.The pKa of Glu325 in LacY WT and in different mutants carrying mutations in the proton translocation active center was determined. WT complex I was oxidized with different oxidizing agents and reduced with NADH. Corresponding redox-induced conformational changes were studied. The evidence was given that Mg2+ ions induce conformational changes in the protein IM30.Within the second project the spectroelectrochemical cell containing gold grid electrode was adopted for the studies of redox active proteins. This gold grid serves both as working electrode and as SERS active substrate. First Cyt c, Hb and Mb were used to validate the setup and then the approach was extended to study a membrane protein.

Στην παρούσα διπλωματική εργασία γίνεται χρήση τριών διαφορετικών ειδών λεπτών υμενίων διοξειδίου του τιτανίου (TiO2) ως στερεό υπόστρωμα για την ακινητοποίηση πρωτεϊνών με σκοπό την ανάπτυξη και σύγκριση αμπερομετρικών βιοαισθητήρων με ευαισθησία στο υπεροξείδιο του υδρογόνου (H2O2). Το πρώτο είδος των λεπτών υμενίων TiO2 είναι από την εμπορική πάστα της Dyesol η οποία παρασκευάζεται με τη μέθοδο sol-gel, το δεύτερο είδος είναι από μία πάστα δικής μας παραγωγής με τη μέθοδο sol-gel και το τρίτο είδος είναι από μία πάστα που παρασκευάσαμε χρησιμοποιώντας την έτοιμη νανοδομημένη σκόνη TiO2, Degussa P – 25. Τα υμένια TiO2 που έχουν παρασκευαστεί από την εμπορική πάστα της Dyesol χρησιμοποιήθηκαν και για τη δημιουργία υβριδικού υποστρώματος με νανοσωματίδια Αργύρου το οποίο μπορεί να βρει εφαρμογή τόσο στη φωτοκατάλυση όσο και στην ανάπτυξη πιο ευαίσθητων αμπερομετρικών βιοαισθητήρων. Αρχικά περιγράφεται η λειτουργία των βιοαισθητήρων καθώς και οι σημαντικότεροι τύποι βιοαισθητήρων που έχουν κατασκευαστεί μέχρι σήμερα. Σημαντικό ρόλο στην επιτυχή κατασκευή ενός βιοαισθητήρα παίζει η επιλογή του υλικού που θα χρησιμοποιηθεί ως υπόστρωμα / ηλεκτρόδιο (στη συγκεκριμένη περίπτωση, υμένια TiO2) καθώς και ο τρόπος που ακινητοποιείται το βιομόριο πάνω σε αυτό, γι’ αυτό και έχει δοθεί έμφαση στην ανάλυση των παραπάνω πληροφοριών. Στη συνέχεια περιγράφεται η δομή και η φυσική λειτουργία της πρωτεΐνης, (κυτόχρωμα c), που χρησιμοποιήθηκε ως το βιομόριο επιλογής για την ανάπτυξη του βιοαισθητήρα. Αναλύονται οι κρυσταλλικές δομές του διοξειδίου του τιτανίου και οι βασικές φυσικοχημικές τους ιδιότητες. Επίσης γίνεται περιγραφή του φαινομένου της φωτοκατάλυσης, ενώ αναφέρονται τα πλεονεκτήματα και τα μειονεκτήματα του TiO2 για την εφαρμογή αυτή. Στο τέλος του πρώτου κεφαλαίου γίνεται αναφορά και στα νανοσωματίδια αργύρου καθώς και στους λόγους που βοηθούν στην αύξηση της φωτοκαταλυτικής απόδοσης του TiO2. Στη συνέχεια περιγράφονται οι πειραματικές διατάξεις που χρησιμοποιήθηκαν τόσο για τον χαρακτηρισμό των υμενίων TiO2 όσο και για την αναλυτική μελέτη της ακινητοποίησης του κυτοχρώματος c και των νανοσωματιδίων αργύρου πάνω σε αυτά. Περιγράφεται επίσης, η ηλεκτροχημική κυψελίδα 3 ηλεκτροδίων και οι τεχνικές της κυκλικής βολταμμετρίας και της φασματοηλεκτροχημείας που επιλέχθηκαν για τη μελέτη των ηλεκτροχημικών ιδιοτήτων των υμενίων TiO2 με ή χωρίς ακινητοποιημένη πρωτεΐνη, των υμενίων TiO2 με ακινητοποιημένα νανοσωματίδια αργύρου καθώς και για την ανάπτυξη των αμπερομετρικών βιοαισθητήρων με ευαισθησία στο H2O2. Τέλος, περιγράφεται η σύνθεση των τριών διαφορετικών παστών TiO2, η πειραματική διαδικασία εναπόθεσης των υμενίων του TiO2 σε υποστρώματα αγώγιμου υάλου και κατόπιν, ακολουθεί η ανάλυση των πειραματικών αποτελεσμάτων. / In this report 3 different types of TiO2 films were used as solid substrates for the immobilization of proteins in order to be used for the development and evaluation of amperometric biosensors for hydrogen peroxide (H2O2). The first type of thin TiO2 films were made using a Dyesol sol gel commercial TiO2 paste, the second type was produced from a TiO2 paste produced in our lab following a standard sol-gel procedure and the third type of films were produced from a paste prepared using Degusa P25 TiO2 powder. In addition, the thin TiO2 films produced from the Dyesol paste were modified with Ag nanoparticles in order to examine their electrochemical behavior which could lead to enhanced photcatalytic and/or biosensing performance. In the first chapter, a general decription of the different types of biosensors developed so far is presented and emphasis is given to their function and applications. In order to develop a successful biosensor, the choice of the material to be used as the solid substrate is very important as well as the type of the biomolecule used as the recognition element. The sensitivity and response of the biosensor is greatly enhanced by the method used to immobilize the biomolecule on the solid support in a stable and functional way. Therefore in this work both the material, TiO2 films, and the biomolecule of choice, Cytochrome-c, are presented in detail and in particular their physicochemical properties, their functions and applications. Moreover the different methods that have been used for the successful immobilization of biomolecules on solid surfaces are well documented. Furthermore, the photocatalytic properties of the TiO2 films are discussed and how they are enhanced by the deposition of silver nanoparticles on their surfaces that could also lead to the development of more sensitive and accurate amperometric biosensors. In the second chapter, the experimental techniques and procedures used for the characterization of the resulting TiO2 films and for the adsorption process of protein and/or silver nanoparticles on their surfaces are well presented. Furthermore, details are given of the electrochemical techniques (cyclic voltammetry and spectroelectrochemistry) used to evaluate the electrochemical performance of the resulting films with or without protein or silver nanoparticles. A detailed description of the 3 electrode electrochemical used to perform these experiments is also presented. Finally emphasis is given to the procedures used for the development of the electrochemical biosensors for H2O2. Finally, a description of the procedures used for the synthesis of the 3 different TiO2 pastes and of the method used for the production of thin TiO2 films on conducting glass is given followed by the presentation, analysis and discussion of the data collected.

Intracellular protein sorting plays a key role in the regulation of cellular metabolism, gene expression, signal transduction and a number of other cellular processes. Proteins targeted to specific cellular compartments contain organelle-specific targeting sequences which interact with various components of the import machinery that are often evolutionarily conserved. For example, proteins targeted to peroxisomes interact with specific receptor proteins through unique peroxisomal targeting signals (PTS) which results in their import into peroxisomal matrix or insertion into peroxisomal membrane. Peroxisomal protein import has been studied in a number of species and several conserved PTS and receptor proteins have been identified. In our study, we report the unexpected finding that cytochrome c (cyt c), which lacks a canonical PTS, is targeted to peroxisomes of the methylotrophic yeast, Pichia pastoris. This is a unique feature of P. pastoris and is not observed in other yeast species such as the conventional yeast, Saccharomyces cerevisiae or other methylotrophic yeasts such as Hansenula polymorpha. Using S. cerevisiae cyc1 null mutant strain as a surrogate model, we demonstrate that P. pastoris cytochrome c (PpCyt c) is targeted to S. cerevisiae peroxisomes indicating that peroxisomal targeting is a unique and inherent property of PpCyt c and the machinery required for this is conserved in S. cerevisiae as well. We further demonstrate that Ppcyt c targeted to the fatty acid-induced peroxisomes of S. cerevisiae is a hemoprotein with covalently attached heme suggesting that PpCyt c synthesized in cytosol is first targeted to mitochondria where heme is added to the apoprotein by cytochrome c heme lyase and the holoprotein is then re-targeted to peroxisomes through an unknown mechanism. Proteins imported into peroxisomes carry specific peroxisomal targeting signals (PTS) known as PTS1 and PTS2. PTS1 is a tripeptide sequence (SKL) at the carboxy terminus of peroxisomal matrix proteins. To investigate whether the carboxy terminus of PpCyt c contain PTS1 or PTS1-like sequences, we made GFP fusion proteins with PpCyt c carboxy terminal amino acids (GFP-ATK, GFP-LAKATK) and examined their ability to localize to peroxisomes. Neither of these two proteins is targeted to peroxisomes indicating that PTS1-like sequences are not involved in peroxisomal targeting of Ppcyt c. Two receptors known as Pex5 and Pex7 are known to be involved in peroxisomal protein import and we therefore examined PpCyt c import into peroxisomes of P. pastoris strains lacking pex5 and pex7. Peroxisomal import of PpCyt c is abolished in pex5 but not pex7 mutant strain indicating that PpCyt c is imported into peroxisomes by a pex5-dependent but PTS1independent pathway. Since we observed significant amino acid differences between PpCyt c and S. cerevisiae cytochrome c (ScCyt c) in their carboxy-and amino-termini, we interchanged these amino acids between PpCyt c and ScCyt c and examined their subcellular localization. Such studies revealed that swapping the N-terminal or C-terminal amino acids of PpCyt c with those of S. cerevisaie cytochrome c (ScCyt c) abolishes peroxisomal localization of PpCyt c. Thus, both N-and C-terminal amino acids of PpCyt c are essential for its import into peroxisomes. Interestingly, in a number of fungal species, the N-and C-terminal amino acid sequences of cytochrome c are identical to those of PpCyt c indicating that peroxisomal targeting of cytochrome c may be observed in other yeast species as well. S. cerevisiae cells expressing PpCyt c exhibit several unique biochemical properties. S. cerevisiae cells expressing PpCyt c grow more rapidly than those expressing ScCyt c when cultured on media containing oleic acid as the sole carbon source and uptake of C-oleic acid from the medium as well as its assimilation into neutral lipids is quantitatively higher in the former. Surprisingly, the phenotype of S. cerevisiae cells expressing PpCyt c is dramatically altered such that the kinetics of growth on fatty acid containing media as well as lipid profile appear to be identical to those of P. pastoris rather than S. cerevisiae. Thus peroxisomal targeting of cytochrome c dramatically alters the kinetics of growth of S. cerevisiae cells in fatty acid containing media as well as the lipid metabolism raising several interesting questions on the molecular mechanisms involved in the alteration of phenotype of S. cerevisiae. It is likely that peroxisomal targeting of cytochrome c results in quantitative as well as qualitative changes in fatty acid metabolism and this opens up new vistas for the bioconversion of fatty acids into value-added lipid products by metabolic engineering. Based on these studies, we propose a new role for cytochrome c in peroxisomal fatty acid metabolism. Our study demonstrates that evolutionarily conserved proteins such as cytochrome c can acquire unique, species-specific functions that may be of great physiological significance to that organism.

In this work, an electrochemical method based on a cyt c biosensor has been developed, for the detection of selected arsenic and cyanide compounds. Boron Doped Diamond (BDD) electrode was used as a transducer, onto which cyt c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide by inhibition mechanism. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH= 7) was found to be (1.087- 4.488 ×10-9 M) and the detection limits ranging from 0.0043- 9.1 μM. These values represent a big improvement over the current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines.

In this work, an electrochemical method based on a cyt c biosensor has been developed, for the detection of selected arsenic and cyanide compounds. Boron Doped Diamond (BDD) electrode was used as a transducer, onto which cyt c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide by inhibition mechanism. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH= 7) was found to be (1.087- 4.488 ×10-9 M) and the detection limits ranging from 0.0043- 9.1 μM. These values represent a big improvement over the current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines.

Magister Scientiae - MSc / In this work, an electrochemical method based on a cyt c biosensor has been developed, for the detection of selected arsenic and cyanide compounds. Boron Doped Diamond (BDD) electrode was used as a transducer, onto which cyt c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide by inhibition mechanism. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH= 7) was found to be (1.087- 4.488 ×10-9 M) and the detection limits ranging from 0.0043- 9.1 μM. These values represent a big improvement over the current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines. / South Africa