Cell division in the Gram-positive bacterium Streptomyces coelicolor starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of S. coelicolor exploit two types of cell division with strikingly different outcomes depending on the developmental stage. The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery. By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The ftsZ17(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation. An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of ftsZ expression and the turnover of the FtsZ protein. S. coelicolor is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like Escherichia coli. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves.

La mucoviscidose est une maladie génétique due à des mutations du gène CFTR, conduisant à une altération de la fonction de canal à ions chlorure de la glycoprotéine transmembranaire CFTR associée à une atteinte pulmonaire sévère. Plusieurs études récentes ont amené à reconsidérer l'utilisation des vecteurs adénoviraux (Ad) de sérotype 5 (Ad5) dans la mucoviscidose, lesquels induisent non seulement des réactions immunes anti-adénovirales mais aussi des effets cytopathiques indésirables. (1) Dans une première partie de notre étude, nous avons étudié l'entrée et le transit intracellulaire de l'Ad5/F35, vecteur chimérique portant les fibres de l'Ad sérotype 35 sur une capside de sérotype 5. Nous avons montré que la protéine fibre est déterminante dans l'internalisation et le trafic intracellulaire de ce vecteur. Le vecteur Ad5/F35 exprimant la fusion GFP-CFTR s'est révélé (i) être dépourvu de cytotoxicité, (ii) transduire efficacement les cellules épithéliales pulmonaires par voie apicale, et (iii) restaurer l'activité de canal à chlorure dans les cellules CFTR(-). Il constitue donc un vecteur de transfert du gène CFTR potentiellement utilisable en thérapie génique de la mucoviscidose. (2) Dans une seconde partie, nous avons exploré une stratégie alternative de transfert de la protéine CFTR par trogocytose. Nous avons fait l'hypothèse que le canal CFTR pouvait être véhiculé par des microvésicules ou microparticules membranaires (MP) émanant de la membrane cellulaire et libérées dans le milieu de culture. En utilisant un système d'expression stable de la protéine CFTR étiquetée par la protéine fluorescente GFP (GFP-CFTR) dans des cellules donneuses, nous avons pu démontrer que les MP sont capables de prendre en charge et délivrer la protéine GFP-CFTR à des cellules réceptrices, mais ce transfert n'est assuré que par une population réduite de MP (≤ 8 %), et la durée de vie du GFP-CFTR n'est que transitoire (≤ 24h). En fait, la majorité des MP transfèrent des molécules d'ARN messager ou polysomal GFP-CFTR. La protéine GFP-CFTR néosynthétisée à partir de ces ARNm est exprimée plus tardivement (> 48h) mais de façon prolongée (≥ 10 jours). La fonctionnalité du canal CFTR ainsi néosynthétisé est en cours d'évaluation. Les MP constituent donc un nouveau type de vecteurs de transfert non génique du CFTR qui pourraient être employés en thérapie de la mucoviscidose.

New methodologies for the efficient syntheses of 4,4-difluoro-4-bora-3a,4adiaza- s-indacenes (BODIPYs) and rosamines were developed. A serendipitous discovery led to a new reaction which afforded BODIPYs in high yields. Systematic studies of the kinetics and mechanisms of the new reaction were performed. A series of BODIPYs were successfully prepared using the new approach. A simple and efficient synthesis of rosamines with cyclic-amine substituents was devised. These new rosamines showed interesting anti-tumor activities. Several types of novel fluorescent compounds were prepared. Highly fluorescent GFP-chromophore analogs were designed and synthesized. The correlation between the optical properties and the structures was investigated. New pyronin dyes with mesoheteroatom substituents were efficiently prepared. The fluorescence properties of these compounds were highly dependent on the nature of the meso-substituents. A set of BODIPY dyes that fluoresce brightly above 600 nm were made. They were then used as acceptors to prepare water-soluble through-bond energy transfer cassettes. All the cassettes had complete energy transfer and high quantum yields in MeOH. A few also had good fluorescence properties in aqueous media and even on proteins. The through-bond energy transfer cassettes were used to monitor protein-protein interactions. In order to test our hypothesis, an artificial protein interaction system was built by utilizing the biotin/(strept)avidin interactions. Thus Atto425-BSA-biotin, streptavidin-cassette1 and avidin-cassette2 were prepared. The interactions between Atto425-BSA-biotin and cassette labeled (strept)avidin were successfully detected in vitro and in living cells by fluorescence techniques.

Many multicellular cyanobacteria produce specialized nitrogen-fixing heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function to accommodate the oxygen-sensitive process of nitrogen fixation. This dissertation focuses on my research on heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation. We using microarray experiments we found that conR (all0187) gene is necessary for normal septum-formation of vegetative cells, diazotrophic grow, and heterocyst morphogenesis. In our studies we characterized the expression of sigma factors genes in Anabaena PCC 7120 during heterocyst differentiation, and we found that the expression of sigC, sigG and sigE is localized primarily in heterocysts. Expression studies using sigE mutant showed that nifH is under the control of this specific sigma factor.

The development of hybridoma technology nearly forty years ago set the foundation for the use of antibodies in the life sciences. Subsequent advances in recombinant DNA technology have allowed us to adapt antibody genes to various screening systems, greatly increasing the throughput and specialized applications for which these complex biomolecules can be adapted. While selection systems are a powerful tool for discovery and evolution, they can be slow and prone to unintended biases. We see computational approaches as an efficient process for rapid discovery and engineering of antibodies. This is particularly relevant for biodefense and emerging infectious disease applications, for which time is a valuable commodity. In the first chapter of this work, we examine computational protocols for ‘supercharging’ proteins. This process resurfaces the target protein, adding charged moieties to impart specialized functions such as thermoresistance and cell penetration. Current algorithms for resurfacing proteins are static, treating each mutation as an event within a vacuum. The net result is that while several variants can be created, each must be tested experimentally to ensure the resultant protein is functional. In many cases, the designed proteins were severely impaired or incapable of folding. We hypothesize that a more dynamic approach, keeping an eye on energetics and the consequences of mutations will yield a more efficient and robust method for supercharging, successfully adding charges to proteins while minimizing deleterious effects. We continue on this theme applying the successful algorithm to supercharging antibodies for increased function. Utilizing the MS2 model biosensor system, we rationally engineer charges onto the surface of an antibody fragment, increasing thermoresistance, minimizing destabilizing effects, and in some cases actually increasing affinity. Finally, we apply next-generation sequencing approaches to the rapid discovery of antibodies directed against the Zaire Ebolavirus species. We utilize a local immunization strategy to generate a polarized antibody repertoire that is then sequenced to provide a database of antigen-specific variants. This repertoire is probed in silico and individual antibodies selected for analysis, bypassing time- and resource-consuming selection experiments. / text

Η παρούσα εργασία είχε ως αντικείμενο, αρχικά, τη δημιουργία υβριδικών μορφών της πρωτεΐνης Geminin- ενός αρνητικού ρυθμιστή του κυτταρικού κύκλου- με το φθορίζον μόριο GFP (Green Fluorescent Protein). Πραγματοποιήθηκε τόσο αμινοτελική όσο και καρβοξυτελική σύντηξη της Geminin με το μόριο-ιχνηθέτη, έτσι ώστε να μελετηθεί η συμπεριφορά του νέου, υβριδικού μορίου και στις δύο περιπτώσεις και να εξεταστεί εάν ο προσανατολισμός της GFP πρωτεΐνης δύναται να μεταβάλλει το χαρακτήρα της σημασμένης πρωτεΐνης. Ακολούθησε ανίχνευση και παρακολούθηση καθενός υβριδικού μορίου σε ανθρώπινα καρκινικά κύτταρα μετά από διαμόλυνση για μικρό (παροδική διαμόλυνση διάρκειας 22 ωρών) ή μεγαλύτερο χρονικό διάστημα (σταθερά διαμολυσμένες κυτταρικές σειρές). Ο χαρακτηρισμός των φθοριζουσών μορφών της Geminin περιελάμβανε μελέτη τόσο του υποκυτταρικού εντοπισμού όσο και της χρονικής έκφρασης των μορίων και σύγκριση με τα αντίστοιχα χαρακτηριστικά του ενδογενούς μορίου. Ανάλυση του ενδοκυτταρικού εντοπισμού τόσο της καρβοξυτελικής όσο και της αμινοτελικής σύντηξης της Geminin με τη GFP, αποκάλυψε, εκτός του πυρηνικού φαινοτύπου, που παρατηρείται φυσιολογικά για το ενδογενές μόριο, σημαντικό ποσοστό παροδικά διαμολυσμένων κυττάρων με τα υβριδικά μόρια εκτός του πυρηνικού διαμερίσματος. Ο φαινότυπος αυτός εμφανίζεται – σε μικρότερο ποσοστό- ακόμη και στην περίπτωση σταθερά διαμολυσμένων κυττάρων, όπου τα επίπεδα έκφρασης της πρωτεΐνης Geminin-GFP είναι παρόμοια με τα αντίστοιχα του ενδογενούς μορίου. Στη σειρά αυτή, το μεγαλύτερο ποσοστό κυττάρων, παρά την ισχυρή μεταγραφική δραστηριότητα που προσδίδει ο CMV υποκινητής, εκφράζει το εξωγενές μόριο σύμφωνα με το πρότυπο που παρουσιάζει η ενδογενής Geminin, δηλαδή κατά τις φάσεις S και G1. Επομένως, η ρύθμιση της Geminin-GFP υβριδικής πρωτεΐνης πραγματοποιείται σε μεγάλο βαθμό σε μετα-μεταγραφικό επίπεδο. Ιδιαίτερο ενδιαφέρον παρουσιάζει το γεγονός ότι η αλλαγή στην ενδοκυτταρική κατανομή του υβριδικού μορίου δεν παρουσιάζει τυχαία χρονική εμφάνιση. Το γεγονός ότι εμφανίζεται αποκλειστικά σε αρνητικά για κυκλίνη Α κύτταρα, φανερώνει την ύπαρξη συσχέτισης με τη φάση του κυτταρικού κύκλου και μάλιστα υποδηλώνει την επιλεκτική έξοδό της από τον πυρήνα κατά τη φάση G1. Πιθανόν, η έξοδος της Geminin από τον πυρήνα κατά τη φάση αυτή, να διασφαλίζει τη διεκπεραίωση της διαδικασίας της αδειοδότησης του γενετικού υλικού –αναστολέας της oποίας είναι η Geminin και κατ’ επέκταση την πρόοδο του κυτταρικού κύκλου Το φαινόμενο αυτό χρήζει περαιτέρω διερεύνησης μιας και η αλλαγή στην ενδοκυτταρική κατανομή πολλών ρυθμιστικών μορίων αποτελεί έναν αποδοτικό τρόπο ελέγχου της γονιδιακής έκφρασης στους ευκαρυωτικούς οργανισμούς, ενώ παράλληλα φαίνεται να επηρεάζει πολλές ζωτικής σημασίας λειτουργίες. / The aim of the present study was the construction of fused forms of Geminin- a negative cell cycle regulator-with GFP (Green Fluorescent Protein). Amino- and carboxy terminal fusions of Geminin and GFP were performed, in order to study the behaviour of the hybrid molecule in both cases and to examine if the orientation of the reporter gene can interfere with the character of the new molecule. Each fused molecule was either transiently or stably transfected in human cancer cells and its behaviour was studied. The characterization of the fluorescent molecules of Geminin was performed at the level of subcellular localization and regulation throughout the cell cycle and was compared to the endogenous characteristics. Analysis of the subcellular distribution of both fused forms, revealed -apart from the nuclear phenotype, normally observed- a significant percentage of transiently transfected cells, where the hybrid molecules were excluded from the nucleus. This phenotype was observed- though less strongly- in the case of stably transfected cells, where the expression levels of Geminin-GFP protein are similar to the endogenous molecule. The majority of these cells, despite the strong CMV driven transcriptional activity, express Geminin-GFP during S and G1 phase, like the endogenous protein, suggesting that the regulation of Geminin-GFP is performed mainly at post-transcriptional level. This nuclear exclusion was observed according to a cell cycle- dependent manner. The phenomenon of nucleocytoplasmic shuttling of many proteins seems to be a very efficient way of controlling gene expression in eukaryotes. In order to elucidate the role of sucellular distribution of Geminin-GFP in cell cycle regulation, further examination of this phenomenon is needed.

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule’s reduction potential compared with a dithiol redox buffer like lipoic acid. From this study, we concluded that the glutathione redox couple in ER significantly raised the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in ER with high reliability in real-time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggest a range of ER redox potential consistent with those determined by earlier approaches that did not involve fluorescent sensors. Our primary aim in developing eroGFP1-iL as a redox-sensing tool was to be able to assess whether redox changes represent an early initiator of ER stress in obesity-induced reduction in high molecular weight (HMW) adiponectin in circulation. Hypoxia is a known mediator of redox changes. We found that oligomerization of HMW adiponectin was impaired in the hypoxic conditions observed in differentiated fat cells. The redox-active antioxidant ascorbate was found capable of reversing hypoxia-induced ER stress. Lastly, we demonstrated that changes in ER redox condition is associated with ER stress response and is implicated in the mechanism of action of the insulin-sensitizing agent troglitazone and desensitizing agent palmitate. Using the redox sensing property of eroGFP1-iL, palmitate was found to be an effective modulator of redox changes in the ER and troglitazone was found to cause oxidizing changes in the ER. The action of palmitate in causing aberrant ER redox conditions was associated with aberrant HMW adiponectin multimerization. Palmitate-induced ER stress was ameliorated by troglitazone. Taken together, the data suggest a potential role of ER redox changes in ER stress and impaired protein folding in adipocytes.

No description available.

No description available.

La toxina-ε, produïda per la soca D de Clostridium perfringens, és la principal responsable de l’enterotoxèmia en animals de granja. Produeix un edema generalitzat, uns efectes citotòxics molt greus al ronyó (mort de les cèl•lules epitelials dels túbuls distals) i també efectes neurològics (mort neuronal provocada per excitotoxicitat). Tots aquests efectes porten finalment a la mort de l’animal. Actualment existeixen tractaments preventius de la toxina mitjançant la vacunació, però encara es desconeix el mecanisme d’acció letal, el seu receptor i els primers passos que porten a la citotoxicitat. Amb la finalitat d’estudiar el comportament d’aquesta toxina, al nostre laboratori vàrem produir una proteïna de fusió formada per la toxina-ε i la proteïna verda fluorescent GFP (toxina-ε-GFP). Aquesta eina va ser de gran utilitat, ja que es comportava igual que la toxina-ε nativa i podia localitzar-se de manera directa utilitzant microscòpia de fluorescència. De fet, injeccions i.v de toxina-ε-GFP a ratolí, varen revelar un edema generalitzat i una mort de les cèl•lules epitelials dels túbuls distals del ronyó de manera idèntica a la toxina nativa. A més, també mostrava citotoxicitat en cultius de la línia cel•lular MDCK, on heptameritzava a la membrana cel•lular, formava porus i provocava la mort cel•lular (Soler-Jover et al., 2004). L’objectiu d’aquesta tesi va ser aprofundir en el coneixement de les primeres etapes de la intoxicació per la toxina-ε, caracteritzant la seva unió i distribució en els diferents òrgans i sistemes, en especial el renal i nerviós. Per dur a terme aquest objectiu general, vàrem realitzar dues aproximacions experimentals: 1. Vàrem incubar la toxina-ε-GFP sobre seccions de teixits, on vàrem observar la seva unió a la mielina tant del SNC com del SNP. També vàrem veure que en el sistema renal, la toxina-ε-GFP reconeixia cèl•lules epitelials dels túbuls distals i col•lectors del ronyó, així com cèl•lules de l’uroteli. Els estudis realitzats en aquest treball (tractaments amb pronasa E, detergents, Nglicosidasa F i beta-eliminació) apunten que el receptor podria tractar-se d’una Oglicoproteïna tant en el sistema nerviós com en el renal, i que un ambient lipídic (o la integritat de la membrana) seria necessari per permetre la unió de la toxina-ε al seu receptor. A més, els estudis realitzats amb cross-linkers en les cèl•lules MDCK han permès identificar una proteïna d’uns 36 kDa (Annexina A2) que podria actuar com a correceptor o intervenir en l’oligomerització i formació de porus a la membrana plasmàtica. Mitjançant un assaig d’ELISA en cèl•lules MDCK, vàrem observar que només hi ha un sol lloc d’unió per a la toxina-ε, i aquest és saturable i d’alta afinitat. 2. Vàrem reproduir a ratolí els efectes de la toxina injectant i.v dosi letals de toxina-ε- GFP, i vàrem estudiar la seva distribució en el sistema nerviós. Vàrem veure que la toxina-ε-GFP a banda d’unir-se a les cèl•lules endotelials també era capaç de travessar la barrera hematoencefàlica (BHE) i arribar així al parènquima del cervell, on provocaria la mort neuronal ja fos d’una manera directa o indirecta (Soler-Jover et al., 2007). Donat l’interés en trobar nous vehicles capaços de travessar la BHE, vàrem analitzar aquesta propietat en diferents mutants de toxina-ε, tot i que encara no hem identificat cap mutant que hagi perdut la capacitat tòxica però no seva capacitat invasiva. La futura identificació del receptor de la toxina-ε, la formació dels heptàmers i la seva inserció a la membrana són, sens dubte, els grans reptes que ajudaran a caracteritzar el mecanisme d’acció de la toxina-ε de Clostridium perfringens. / ε-Toxin, produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. ε-Toxin accumulates specifically in the renal and nervous system were it produces the death of the epithelial cells from the distal tubules and neurons, respectively. Vaccines are very useful in the prevention of the ε-toxin intoxication, but nothing is known about the receptor and the first intoxication steps. We produced a recombinant ε-toxin protein with the green fluoresence protein GFP (ε- toxin-GFP) which is a useful tool because it behaves as the native ε-toxin and it can be detected by fluorescence (Soler-Jover et al., 2004). The aim of this thesis is to go into detail in the knowledge of the first steps in the intoxication pathway, characterizing the binding and distribution of the ε-toxin in different organs and tissues, especially in the renal and nervous system. To achieve this aim we performed two experimental approaches. On one hand, we incubated the ε-toxin-GFP on tissue sections, were its binding to myelin from CNS and PNS was observed. We also observed binding of ε-toxin to the urothelium and epithelial cells from distal and collecting tubules in the kidney. Our work revealed that the receptor in the nervous and renal system could be an Oglycoprotein, and that a lipidic environment (or the membrane integrity) would be required for the binding of ε-toxin to its receptor. In addition, an ELISA-based binding assay revealed a single high-affinity binding site for ε-toxin in MDCK cells. Moreover, cross-linking experiments identified a 36 kDa protein (Annexin A2) which could be involved in the membrane pore formation step. On the other hand, we reproduced in mice the in vivo effects by injecting i.v ε-toxin- GFP. The toxin crossed the BBB and penetrated the brain parenchyma producing neuronal death (Soler-Jover et al., 2007). We are interested in the finding of new vehicles to cross the BBB. In fact, some ε-toxin mutants have been studied but no one is able to cross the BBB without producing cell damage and animal death.