No description available.

It is well established that failure of the lamellar basement membrane (BM) occurs during the development of equine laminitis. This is due to loss of the crucial BM components; laminins and collagens along with loss of attachment complex, the hemidesmosome, of the basal cell to the underlying BM. Previous studies have suggested that Ln-332 may be the primary protein involved in lamellar failure. However, the details of the progression and mechanism involved in this pathology are not currently fully known. This thesis aimed to refine the proteolytic processes and mechanisms occurring during the development of oligofructose induced laminitis. Through the use of novel temporal lamellar biopsies obtained during the development of laminitis induction, it was determined that loss of both Ln-332 and collagen type IV occurs as early as 12 hours post induction. This loss of reactivity initially occurred in a focal pattern with increasing loss as the disease progressed in severity. At the later stages of laminitis, separation of the basal epithelial cell from the dermal tissue was also observed, however at these points the BM still appeared intact. This suggests that more than one mechanism may be involved in disease pathology; one resulting in fragmentation of the BM while a second results in loss of the cell attachment allowing the intact BM to slip away. Immunohistochemical analysis of lamellar tissue revealed a unique pattern of reactivity for the Ln-332 γ2 antibody D4B5, in which no reactivity was observed in normal lamellar tissue, yet the epitope recognized by this antibody becomes apparent during disease development. This initially led to the hypothesis that cleavage of the γ2 subunit and the release of biologically active fragments may occur. However, at the molecular level, no γ2 fragments were detected by western blotting. In vitro cleavage of partially purified equine Ln-332 revealed that both MMP-2 and MT1-MMP were able to process the molecule to produce fragments corresponding to the biologically active counterparts. This suggests that the change in reactivity with this antibody may be due to other mechanisms such as decreased interaction of Ln-332 with other BM components resulting in loss of structural stability of the BM allowing for a change in the orientation of Ln-332. Increased MMP-2, MMP-9 and MT1-MMP expression has been demonstrated in laminitis and this was assumed to be the causative agent resulting in tissue destruction and failure. However, work in this thesis found no increase in gene expression of MMP- 2 and MT1-MMP, as well as no activation of pro MT1-MMP. Increased pro MMP-9 gene and protein expression was observed early in the disease progression yet no MMP- 9 activation occurred. Additionally, activation of MMP-2 was found to occur late in laminitis progression at least 12 hours following BM degradation, thus MMP-2 activation is a secondary effect of laminitis development. Thus, other proteases are expected to result in BM processing. Gene expression of the metalloprotease ADAMTS-4, was observed to increase early during laminitis development, suggesting this is a putative factor involved in intensifying the degradation of the lamellar BM. Work in this thesis also revealed that both Ln-332 and collagen type IV are widely distributed throughout organs in the equine body and localized primarily to BM structures. A novel finding of this thesis is that not only does BM degradation occur in the lamellar BM, it also occurs in organs remote from the hoof. At both the onset of lameness and the acute phase of laminitis, fragmentation of both Ln-332 and collagen type IV also occurs in both the skin and stomach. Recent studies have indicated that both leukocyte emigration and increased cytokine expression occurs in the lamellar tissue during laminitis. Work in this thesis added to this knowledge as leukocyte infiltration into the lamellar tissue occurs early during oligofructose laminitis induction as does increased IL-6 gene expression. Overall, work conducted in this thesis has added to the knowledge of the events occurring during laminitis development. Even though the complete mechanism of tissue destruction and lamellar failure was not established, the progression of events is now more clear in that BM degradation is one of the first events to occur, while MMP-2 activation occurs secondarily. Thus, other mechanisms must be at work early during laminitis development and discovering what they are must remain a research priority for the realization of effective therapeutic strategies.

Thesis (Ph. D.)--University of Adelaide, 1986. / Includes bibliographical references (leaves 119-136).

Abstract This study was initiated to elucidate the complete genomic structures of type XV collagen in man and mouse and the functional properties of their promoters, as well as to obtain knowledge of the biological role of type XV collagen during development and maturity using immunofluorescence and transgenic techniques. The cloning and characterization of genomic clones revealed that the human COL15A1 gene is 145-kb in size and consists of 42 exons, and the mouse Col15a1 gene is 110-kb with 40 exons. The genomic organization of the two genes was found to be highly conserved, except for two regions of divergence. The nuclease S1 protection analysis revealed multiple transcription initiation sites in both genes, which is in accordance with the overall genomic structures of their 5'-flanking sequences. Transient cell transfection experiments with varying lengths of 5'-deletion constructs identified the fragments necessary for basic promoter activity in both genes and those implicated in the positive and negative regulation of the mouse Col15a1 gene. Furthermore, the involvement of transcription factor Sp1 in the gene regulation of the human COL15A1 gene was demonstrated. A mouse specific polyclonal antibody against type XV collagen was generated and utilized in the localization of type XV collagen protein in developing and mature mouse tissues. Type XV collagen was deposited early in the development and was particularly prominent in capillaries. Spatio-temporal differences in the expression of type XV collagen in various capillary types was demonstrated. Early expression was also detected in the skeletal muscle and peripheral nerves, while expression in the heart, lung, and kidney appeared to be developmentally regulated. Transgenic mice lines expressing truncated type XV collagen driven by either short or long endogenous type XV collagen promoters were generated. The two promoters conferred different tissue-specificities and expression levels, the longer one resulting in more endogenous-like expression. Despite some expression at both mRNA and protein levels, the truncated type XV collagen did not cause any obvious phenotypic or histological changes in any of the lines driven by the shorter promoter fragment. In heterozygote matings of one of the lines driven by the longer promoter fragment, however, a portion of the transgene positive mice appeared to be lost prenatally. Furthermore, pregnancy terminations in this line indicated a high number of abortions beginning at about 11 days of development. Further studies are needed before detailed conclusions on the consequences of the generated mutation can be drawn. The elucidation of the genomic structure of the human COL15A1 gene provides the necessary database for screening mutations in patient samples for candidate diseases caused by this collagen. The genomic clones and the mouse-specific antibody against type XV collagen are valuable tools also in future projects. The knowledge of the developmental dynamics of type XV collagen is of great value, as it helps to understand the physiological consequences that the as yet unidentified mutations in type XV collagen may cause in humans.

Abstract In this work the type XVIII collagen has been studied by using several approaches, such as different animal models. The primary structure of frog, Xenopus laevis, type XVIII collagen and the expression pattern of its variants during early embryogenesis have been elucidated. The gene structure of human type XVIII collagen was characterized and the localization and processing of its longest variant was studied by generated antibodies. In addition, the function of the proteolytically released C-terminal part of type XVIII collagen, endostatin, was studied by generating transgenic mice expressing endostatin. The primary structure of X. laevis type XVIII collagen is comprised of three N-terminal variants resembling their mammalian counterparts. The sizes of the polypeptides are 1285, 1581, and 1886 residues. The most conserved regions are the C-terminal endostatin region and the cysteine-rich domain in the N-terminus. Whole-mount in situ hybridization reveals different expression patterns for variants during embryogenesis. The short variant is the most abundant, whereas the two longest variants exhibit more restricted expression. The gene structure of human type XVIII collagen reveals an exon-intron organization that is conserved with mouse. The length of the human gene is about 105 kb and contains 43 exons. The third variant of type XVIII collagen has a conserved cysteine-rich domain with homology to the extracellular part of frizzled proteins. This third variant is localized to developing muscle and lung, and is also found in serum. In cell culture, the proteolytic fragments of the N-terminus, including the cysteine-rich motif, are also detected. Endostatin function was studied by generating mouse lines expressing endostatin under the keratin-14 promoter, which drives the expression mainly in the skin. Three independent transgenic mouse lines were achieved with varied expression levels. The phenotype was seen in the eye with lens opacity and abnormal morphology of epithelial cells in the lens. In the skin, a broading of the basement membrane in the epidermis dermis junction was detected. Immunoelectron microscopy analysis revealed a polarized orientation of type XVIII collagen in the basement membrane. In transgenic mice, altered localization of endogenous type XVIII collagen was seen, suggesting displacement of the endogenous type XVIII collagen with transgenic endostatin leading to disorganized basement membrane.

Abstract Lysyl hydroxylase (LH, EC 1.14.11.4) catalyzes the post-translational hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. The hydroxylysyl residues participate in the formation of collagen cross-links, and some of the hydroxylysyl residues are further glycosylated. Three lysyl hydroxylase isoforms LH1, LH2 and LH3, encoded by three individual genes have been characterized and one isoform, LH3 is a multifunctional enzyme containing lysyl hydroxylase, collagen galactosyltransferase (GT, E.C. 2.4.1.50) and glucosyltransferase (GGT, E.C. 2.4.1.66) activities in vitro. In this thesis the genes for the mouse lysyl hydroxylases were each mapped to a different chromosome. In addition, the roles of the lysyl hydroxylase isoforms were characterized in mice by studying their expression during development and the distribution of LH2 and LH3 in adult mice. The results revealed a widespread expression of the mouse lysyl hydroxylases during embryonic development whereas LH2 and LH3 showed tissue- or cell-specific expression patterns in the adult. Alternative splicing of the gene for LH2 also showed developmental and tissue-specific regulation. The different functions of LH3 were studied in vivo by generating three different LH3 manipulated mouse lines. Analysis of the mouse lines revealed that LH3 has lysyl hydroxylase and glucosyltransferase activities in vivo, and that, in particular, the glucosyltransferase activity of LH3 is essential for normal development. The loss of glucosyltransferase activity caused disruption of basement membranes leading to embryonic lethality while the absence of lysyl hydroxylase activity led to ultrastructural alterations in muscle and basement membranes and disorganization of collagen fibrils. The disruption of basement membrane was due to an intracellular accumulation of unglycosylated type IV collagen, whereas the ultrastructural alterations were related to the abnormal aggregation and distribution of underglycosylated type VI collagen. The results demonstrate that hydroxylysine-linked glycosylations are critical for the secretion of type IV collagen and its assembly into basement membranes, and for the assembly and distribution of type VI collagen.

The work for this thesis was conducted on the basement lithologies of the Limpopo Province in South Africa. An investigation into the correlation between structural geology and the flow/occurrence of groundwater was conducted on these lithologies. Field measurements of geological structures were recorded and compared graphically and statistically to existing groundwater borehole data. Data analysis revealed that the structural geology in the basement lithologies of the Limpopo Province of South-Africa does not have a clearly identifiable influence in terms of spatial patterns in groundwater flow and occurrence at a regional scale. Groundwater targets created through weathering rather than tectonics were evidently more easily recognized. Structural controls on groundwater in the granitic aquifers from this specific area are not totally negligible, although it is believed that such influences will be better identified through intensive local scale investigations. As drilling for groundwater is an expensive practice, the knowledge gained through this study and the possibility of a correlation between groundwater flow/occurrence compared to structural geology might improve the odds of finding groundwater for the communities and farmers in the area. / Dissertation (MSc)--University of Pretoria, 2009. / Geology / unrestricted

The aim of the diploma thesis submitted is preparation of project documentation for construction of new municipal authority building. The building will be situated in the center of municipality Dlha nad Kysucou. The building is divided into the municipality office and hall for social occasions. The building is detached, with partial basement and two floors. Small and large halls are situated on the ground floor. Municipality offices can be found on the first floor. Foundations of the building are formed by foundation strips of regular concrete. Wall construction system, Porotherm blocks are used for vertical structures of the building. Horizontal structures were built using celling panels Spiroll, made out of ferroconcrete. Flat single ply roof has standard order of layers.

The topic of this master thesis is the elaboration of the project documentation of the new construction of a multifunctional building of the registered office of the company. New building is situated on the land in Ružomberok in the area intended for production and warehouse function. The proposed facility represents the background for a company that is a leading supplier of spare car parts. The building consists of a three floors with a partial basement. 1.PP and the part of 1.NP are storage areas. The rest of the first overhead floor is the shop. The second floor represents the administrative part of the business. The last, third floor, has 3 corporate apartments of various inclinations. The object is based on the concrete strip foundations. The structural system uses the ceramic blocks of type Therm. The walls of the basement are made of a concrete structural system. The ceilings of the building are designed as cast-in-place reinforced slabs. Screed to fall of the flat roof is thermal insulation and the finish is made of pea gravel.

This diploma thesis aims to design a project documentation for a realization of a newbuild hotel. The building is in Sigord recreation ground, near Kokosovce village in Slovakia. The object is a detached building with three storeys and a partial basement. An entrance hall, offices, bar and a restaurant are situated on the ground floor. The hotel is on the first and the second floors. Foundations of the building are made of jamb blocks and a reinforced concrete beam foundation. Construction system is skeletal, consisting of concrete poles and reinforced concrete beams, filling and vertical constructions are made of Ytong aerated concrete blocks. Horizontal constructions are made from Spiroll precast panels, with reinforced concrete ceilings. The building is finished off with a single ply roof with a standard order of layers.