No description available.

No description available.

Gap junctions are communicating junctions between cells that allow small molecules to pass from the cytoplasm of one cell to the cytoplasm of an adjacent cell. The pores of gap junctions are comprised of two adjacent connexons on neighboring cells, and each connexon is comprised of six connexin proteins. The eye lens of vertebrates is an avascular tissue that is dependent on gap junctions for the distribution of nutrients as well as the removal of waste products. In addition, as the lens cells develop into fibers, they lose their intracellular organelles including the membrane-bound organelles, and are highly dependent on connexons for movement of metabolites and waste materials. Only two connexins, in Bos Taurus Cx44 and Cx49, are highly expressed in lens fiber cells. Thus, the lens offers an excellent system for studying gap junctions. In this study, high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) techniques were used to isolate and characterize connexin proteins from the eye lens of the cow and mouse. Despite over 300 proteins being identified from bovine lens using MS techniques, it was still possible to identify the two connexin proteins following proteolytic digests and MS analysis of the resultant peptides. Several post- translational modifications (PTMs) were identified and characterized in lens fiber connexins, including phosphorylations, acetylations and deamidations and proteolytic cleavages. Changes in phosphorylation of several other lens proteins upon the activation of protein kinase C were also identified. Detection of the orthologous proteins in mouse lens proved more challenging, but peptides derived from both connexin proteins were also detected from this tissue and PTMs of mouse connexins were also observed. Glutathione-S-transferase fusions to mouse Cx44 and Cx50 were used to identify a number of proteins that may interact with the mouse connexins, and the relevance of those interactions was considered. The utility of mass spectrometry to the identification of specific proteins from complex mixtures was clearly demonstrated, and its application to understanding the functional relevance of PTMs was discussed.

Gap junctions are communicating junctions between cells that allow small molecules to pass from the cytoplasm of one cell to the cytoplasm of an adjacent cell. The pores of gap junctions are comprised of two adjacent connexons on neighboring cells, and each connexon is comprised of six connexin proteins. The eye lens of vertebrates is an avascular tissue that is dependent on gap junctions for the distribution of nutrients as well as the removal of waste products. In addition, as the lens cells develop into fibers, they lose their intracellular organelles including the membrane-bound organelles, and are highly dependent on connexons for movement of metabolites and waste materials. Only two connexins, in Bos Taurus Cx44 and Cx49, are highly expressed in lens fiber cells. Thus, the lens offers an excellent system for studying gap junctions. In this study, high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) techniques were used to isolate and characterize connexin proteins from the eye lens of the cow and mouse. Despite over 300 proteins being identified from bovine lens using MS techniques, it was still possible to identify the two connexin proteins following proteolytic digests and MS analysis of the resultant peptides. Several post- translational modifications (PTMs) were identified and characterized in lens fiber connexins, including phosphorylations, acetylations and deamidations and proteolytic cleavages. Changes in phosphorylation of several other lens proteins upon the activation of protein kinase C were also identified. Detection of the orthologous proteins in mouse lens proved more challenging, but peptides derived from both connexin proteins were also detected from this tissue and PTMs of mouse connexins were also observed. Glutathione-S-transferase fusions to mouse Cx44 and Cx50 were used to identify a number of proteins that may interact with the mouse connexins, and the relevance of those interactions was considered. The utility of mass spectrometry to the identification of specific proteins from complex mixtures was clearly demonstrated, and its application to understanding the functional relevance of PTMs was discussed.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Melinda K. Duncan, Dept. of Biological Sciences. Includes bibliographical references.

No description available.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. EMBARGOED - This thesis is subject to a 12 month embargo (07/03/06 to 07/03/07) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 236-266.

Λόγω της διαφάνειας των οφθαλμικών ιστών η σκέδαση φωτός αποτελεί ιδανικό εργαλείο για την ανίχνευση των αρχικών σταδίων ορισμένων παθολογικών τους καταστάσεων. Για παράδειγμα, η θόλωση του φακού των θηλαστικών λόγω ηλικίας ή/και άλλων εξωγενών αιτίων καλείται καταρράκτης. Ο καταρράκτης δεν μπορεί να διαγνωστεί κλινικά σε πρώιμο στάδιο με αποτέλεσμα την δημιουργία σοβαρών προβλημάτων στην όραση. Το γεγονός ότι το φως έχει την ικανότητα να ανιχνεύει τις μοριακές αλλαγές οι οποίες είναι πρόδρομα συμπτώματα του καταρράκτη αναδεικνύει την σημασία της έγκαιρης διάγνωσης στην αντιμετώπιση διάφορων οφθαλμικών παθήσεων. Ο φακός θεωρείται ως ένα πυκνό διάλυμα πρωτεϊνών (κρυσταλλίνες, ~40 % wt) σε νερό και η αδιαφάνεια η οποία αποτελεί την εκδήλωση του καταρράκτη προκαλείται ουσιαστικά από την συσσωμάτωση των πρωτεϊνών. Στόχος αυτής της διατριβής είναι η διερεύνηση των μοριακών μεταβολών οι οποίες λαμβάνουν χώρα κατά την ανάπτυξη του καταρράκτη. Ιδιαίτερη σημασία δίνεται επίσης στην ανάπτυξη μιας μη-επεμβατικής μεθοδολογίας για έγκαιρη διάγνωση οφθαλμικών παθήσεων με τη βοήθεια της δυναμικής σκέδασης φωτός. Με την βοήθεια της τεχνικής αυτής, κατάλληλα τροποποιημένης για την μελέτη οφθαλμικών ιστών, μελετήθηκαν οι δυναμικές ιδιότητες των πρωτεϊνών χοίρειων φακών (π.χ. οι συντελεστές διάχυσης, η θερμοκρασιακή τους εξάρτηση σε διάφορα μέρη του φακού, κλπ.) χρησιμοποιώντας το πειραματικό μοντέλο του “ψυχρού” καταρράκτη. Στο μοντέλο αυτό η ελεγχόμενη ψύξη φακών επιφέρει βαθμιαία καταρρακτογένεση. Ιδιαίτερη έμφαση δόθηκε σε τέσσερα κυρίως είδη περαμάτων. (α) Μελέτη της εμφάνισης του ψυχρού καταρράκτη στον πυρήνα του φακού. (β) Μελέτη της επίδρασης του μήκους κύματος της ακτινοβολίας στην εμφάνιση και στην έκταση του φαινομένου του ψυχρού καταρράκτη. (γ) Μελέτη του φαινομένου του ψυχρού καταρράκτη κατά μήκος μιας διαμέτρου του φακού, δεδομένης της βαθμίδας συγκέντρωσης των πρωτεϊνών του φακού (μεγάλη συγκέντρωση στον πυρήνα και μικρή συγκέντρωση στην περιφέρεια του φακού). (δ) Μελέτη του επίδρασης της προθέρμανσης του φακού σε θερμοκρασίες υψηλότερες της φυσιολογικής στο φαινόμενο του ψυχρού καταρράκτη. Τα βασικά συμπεράσματα της παρούσας διατριβής συνοψίζονται ως εξής. Υπάρχουν σαφείς συσχετισμοί μεταξύ των φασματικών χαρακτηριστικών (συναρτήσεις αυτοσυσχέτισης) και των ιεραρχικών σταδίων ανάπτυξης του καταρράκτη. Ποιοτικές και ποσοτικές αλλαγές στην θερμοκρασιακή εξάρτηση διαφόρων παραμέτρων, οι οποίες σχετίζονται με τις μοριακές διαμορφώσεις των αρχικών σταδίων του καταρράκτη, εμφανίζονται ήδη από τους 17 oC όπου ο πυρήνας του φακού είναι ακόμα διαυγής. Η χρήση ακτινοβολίας κοντά στο υπεριώδες μέρος τους φάσματος ενισχύει την ανάπτυξη του ψυχρού καταρράκτη στον πυρήνα του φακού. Ο ψυχρός καταρράκτης δεν αναπτύσσεται στην περιφέρεια του φακού. Η προθέρμανση του φακού σε συγκεκριμένη θερμοκρασία καθώς και ο χρόνος παραμονής σε αυτήν επηρεάζει σημαντικά την ανάπτυξη του ψυχρού καταρράκτη στον πυρήνα αλλά όχι στην περιφέρεια του φακού. Όλα τα παραπάνω δείχνουν πως η δυναμική σκέδαση φωτός μπορεί να παρέχει παραμέτρους οι οποίες μπορούν να χρησιμοποιηθούν με επιτυχία ως ευαίσθητοι και αξιόπιστοι δείκτες της έγκαιρης, μη-επεμβατικής, και in vivo διάγνωσης του καταρράκτη. / On account of the transparency of ophthalmic tissues, light scattering is an ideal tool for detecting the early stages of some of their pathological conditions. For example, the opacity of the mammalian lens due to age or other external causes is called cataract. Cataract cannot be detected clinically at early stages and as a result serious vision problems appear. The fact that, light has the ability to detect molecular changes that are related to the mechanism of cataract formation draws attention to the importance of early diagnosis in ophthalmic disorders. The lens can be considered as a dense colloidal protein dispersion (crystallins, ~ 40% wt) in water where the opacity that leads to cataract formation how its basis to the aggregation of proteins. This dissertation is aimed at studying the molecular changes that take place upon cataract development. Particular emphasis is paid to the development of a non-invasive methodology for early diagnosis of ocular diseases with the aid of dynamic light scattering. By means of this technique, suitably modified for the study of ophthalmic tissues, the dynamic properties of the proteins of porcine lenses (e.g. diffusion coefficients and their temperature dependence at various parts inside the lens, etc.) were studied by using the experimental model of ‘cold’ cataract. In cold cataract the controlled cooling of the lens at temperatures below the physiological one induces gradual cataractogenesis. In particular, we focused on four kinds of experiments. (a) Detailed study on the cold cataract onset in the lens nucleus. (b) Study on the effect of the laser light wavelength in the onset and the extent development of cold cataract. (c) Study of the cold cataract effect along an equatorial diameter of the lens, considering the gradual concentration of the lens proteins (high protein concentration in the nucleus and low concentration in the cortex). (d) Study on the effect of thermal history, i.e. by warming up the lens at temperatures higher than the physiological one on the cold cataract effect. The basic conclusions of the present dissertation are summarized as follows: There are clear correlations between the spectral characteristics (autocorrelation functions) and the hierarchical stages of the onset of cataract. Qualitative and quantitative changes in the temperature dependence of several parameters, which are related with the diffusive motions of proteins at the early stages of cataract, appear already at 17 oC while the nucleus is still clear and highly transparent. The use of laser radiation close to the ultraviolet part of the spectrum seems to enhance the formation of cold cataract in the lens nucleus. Cold cataract does not develop at the cortex of the lens, in view of the low protein concentration. The lens pre-heating at a certain temperature for various time periods affects significantly cold cataract formation in the lens nucleus but not in lens cortex. The above mentioned make clear that dynamic light scattering can indeed provide useful parameters that can be successfully used as sensitive and reliable indicators for the early, non-invasive diagnosis of cataract in mammalian lenses and in vivo.