Call number: LD2668 .T4 1964 E21 / Master of Science

The ventricular epithelia of adult worker honey bees were investigated biochemically and ultrastructurally. The midgut tissues were shown to produce an endoprotease with trypsin-like activity. Enzyme activity was highest in the midgut tissues and the ectoperitrophic space of free-flying honey bees and of caged bees fed pollen. Lower levels of activity occurred in caged bees restricted to sucrose or fed artificial diets. The trypsin-like activity declined as the protein intake of the bees decreased with age. Ultrastructural studies revealed columnar cells in the posterior midgut engaged in the synthesis and release of membrane-bound vesicles. The apical cytoplasm of the epithelial cells in this region contains numerous electron dense vesicles which are released into the ectoperitrophic space of the midgut lumen. The microvilli in the crypts of this region are short, branching, and microvesiculate. Throughout the remainder of the midgut, the microvilli are profuse and elongate. The presence of the endogenously produced endoprotease and the regional variation in cell ultrastructure suggest that the honey bee may rely on countercurrent flow to distribute enzymes and nutrients efficiently throughout the midgut. Ultrastructural cytochemistry localized acid phosphatase and nonspecific esterase activity in primary and secondary lysosomes dispersed throughout the midgut tissues. Alkaline phosphatase activity was localized within large electron lucent microbodies that are present in all midgut columnar cells. The peroxisomal marker enzymes, catalase and L-α-hydroxy acid oxidase, were also localized in the same microbodies which previously had been described as holocrine secretory granules involved in dietary mineral regulation. Morphological and cytochemical assays suggest that the holocrine secretory granule arises from a microperoxisomal compartment involved in intermediary metabolism in the midgut of adult honey bees.

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Limbal stem cells maintain the corneal epithelium through a process of clonal growth and ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6 weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to represent chords of clonally related, inwardly migrating cells. By measuring the number and width of stripes and correcting for the effects of different proportions of LacZ-positive cells, an estimate of the number of coherent stem cell clones maintaining the tissue can be derived. Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks. An automated method was developed using image analysis software to analyse these striping patterns. This method produced results that did not differ significantly from the above. The dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6 heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at 15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77 transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics, estimated clone number was similarly reduced to ~50 with no further decline. Mice heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an increase in coherent clones. When the corneal epithelium is injured the surrounding epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged using time-lapse confocal microscopy. This technique demonstrated that clones remain contiguous throughout their migration. Healing of peripheral wounds was observed to form de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away from and towards the limbal region.

Sialic acids are involved in many cellular interactions. They can serve as an adhesion ligand or act as an inhibitor to cellular adhesion by charge repulsion or by masking potential ligands. Although sialic acids are implicated in the process of blastocyst implantation, their expression and regulation in uterine epithelium of mice have not been studied. The lectin, Sambucus nigra (SNA) specifically recognizes ��2,6-linked sialic acids, which are involved in cell recognition phenomena. It was used to probe frozen uterine sections from mice during days one through six of pregnancy. SNA staining was most intense at the apical surface of uterine epithelial cells on day one of pregnancy, decreased gradually through day four, and was undetectable by day five. The role of the steroid hormones, estrogen and progesterone, in regulating the expression of ��2,6-linked sialic acids was studied in uterine sections from mice during the estrous cycle and in ovariectomized mice given hormone replacement using SNA. SNA staining of these sections during the estrous cycle showed that the expression of ��2,6-linked sialic acids was stage dependent. Staining was most intense in uterine sections from mice in estrus, and was not detected in sections from mice in diestrus. In ovariectomized mice, staining was most intense in mice injected with estradiol alone, and no staining was evident in mice injected with progesterone alone. These results suggest that the expression of ��2,6-linked sialic acids decreases during the time of implantation and that estrogen stimulates and progesterone inhibits its expression. ��-Galactoside ��2,6-Sialyltransferase (��2,6-ST) is the enzyme that links sialic acids to Gal��1-4GlcNAc termini of N-linked oligosaccharides. In order to investigate the mechanism behind the hormonal regulation of ��2,6-linked sialic acids, the expression of ��2,6-ST was followed in uterine sections from mice during early pregnancy, during the estrous cycle, and in ovariectomized mice given hormone replacement. In-situ hybridization was performed using digoxigenin labeled RNA probes to characterize ��2,6-ST mRNA levels in uterine sections. Expression of ��2,6-ST protein was also measured in uterine sections with a polyclonal antibody against ��2,6-ST. The expression of ��2,6-ST mRNA and protein correlated well with the timing of the appearance of ��2,6-linked sialic acids. These results show that the expression of ��2,6-linked sialic acids on the surface of mouse uterine epithelium decreases at the time of implantation and furthermore, that this decrease is due to the regulation of ��2,6-ST by the steroid hormones. ��2,6-linked sialic acids may serve to inhibit cellular adhesion by creating a charge repulsion, or by masking potential binding sites. Removal of this inhibition may permit blastocyst implantation. / Graduation date: 2001

Epithelial wound healing is a common occurrence in many organisms. In spite of a long history of study in this field, we do not have a complete understanding of the molecular and cellular mechanisms of wound healing, which is a key element to appreciate in order to modulate this process for better clinical outcomes. Optimal outcomes are especially critical in the cornea as a failure to regenerate can result in blindness and a huge decline in quality of life. Matrix Metalloproteinases (MMPs) are a family of zinc dependent proteases that have been shown to be both regulators and effectors of the corneal wound healing process. Strict regulation of MMP-9, the most extensively studied member of the MMP family, has been shown to be critical for efficient wound regeneration. While we now know that MMP-9 is important, and we even have evidence defining some of the roles it plays in the corneal wound healing process, the mechanism by which MMP-9 is regulated is still under debate. Possible extracellular regulatory mechanisms range from cell-cell interactions to cell-matrix interactions to secreted factors. However, the detailed mechanism of events that takes place on the extracellular surface and the downstream signals that mediate MMP-9 are unknown. Therefore, one of the objectives of the presented work is to define the external mechanisms which mediate MMP-9 expression in resurfacing epithelial cells and to link these external signals to internal signaling pathways in vitro. Furthermore, while MMP-9 acts to slow the resurfacing phase of wound healing, other MMPs seem to cause opposing effects. The second objective of the presented work is to provide the first global spatial and temporal MMP expression profile for an in vivo epithelial wound healing scenario and to define possible macroscopic roles of these heretofore unknown MMPs. Finally, this thesis will look at the expression of many MMP family members in a penetrating model which is an increasingly more common wound scenario due to the increase in corrective surgery. The final objective is to examine human post-LASIK corneas and correlate MMP expression with age, post-operative time, or histopathological abnormalities. The knowledge obtained from all aspects of these studies will contribute to the current understanding and knowledge about the roles and regulatory mechanisms of MMPs in the corneal wound healing process.

published_or_final_version / Statistics / Master / Master of Philosophy

Μελετήσαμε την δράση της ακτινοβολίας στο εντερικό επιθήλιοκαι αν η χορήγηση αυξητικής ορμόνης και IGF-I μπορεί να αναστρέψει αυτήν την δράση.Συμπερασματικά η ακτινοβολία προκαλεί ατροφία και εξέλκωση του επιθηλίου και διαταραχή του εντερικού φραγμού μέσω επαγωγής της απόπτωσης των κυττάρων του εντερικού επιθηλίου και οδηγεί σε ατροφία, εξελκώσεις και διαταραχή της λειτουργικής ακεραιότητας του εντέρου ενώ η χορήγηση GH ή IGF-I αναστέλλει την απόπτωση των κυττάρων και διατηρεί την ακεραιότητα του εντερικού επιθηλίου. / We studied the effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on intestinal mucosal integrity and bacterial translocalion after abdominal radiation in rats.We have shown that abdominal radiation causes intestinal epithelial cell damage mainly through the induction of apoptosis and the treatment with GH and IGF-I inhibits apoptosis of the cells and preserves the mucosal integrity and also reduce the bacterial translocation that follows intestinal radiation.