Thesis (Ph.D). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2009/06/08). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Medical Physics Unit. Title from title page of PDF (viewed 2009/06/23). Includes bibliographical references.

Thesis (M.S. in Biomedical Engineering)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 102-122).

Abstract In this thesis study, the primary structure of the human α1(XVIII) polypeptide was elucidated, its tissue distribution was studied, and the phenotypic changes in the mouse eye due to lack of type XVIII collagen in a knock-out mouse model were studied further. In addition, the consequences of simultaneous lack of both type XVIII and XV collagen were studied in a mouse model lacking both of these proteins. Two variant forms of human α1(XVIII) polypeptide were identified in this study, although, to date, a third form has also been characterized. The analysis of tissue distribution of the two polypeptide forms revealed differences in their tissue distribution, since the longest variant occurs prominently in the liver, while the short form is the major transcript in other tissues studied, e.g. in the kidney. The study of the type XVIII single null mouse eyes revealed abnormalities in the anterior eye segment in addition to the previously reported defects in the posterior eye part. In the type XVIII single null mice the iris was fragmented, pigment deposits could be seen in the pupil, and the pupillary ruff in the edge of a normal mouse iris was missing in these mice. The ciliary body was also abnormal, since the ciliary processes start to show regression in adult animals and eventually the basal infoldings of the non-pigmented ciliary body epithelia become flattened in the null mice. The intraocular pressure stabilizes to a lower level in adult mutant mice compared to controls, most likely reflecting the atrophied ciliary epithelia. The BM zones were also defective in the type XVIII null mouse eyes. The absence of an immunosignal with one of the antibodies detecting laminin γ2 chain in the type XVIII null mouse eyes may implicate conformational changes in the laminin γ2 chain due to lack of type XVIII collagen, and subsequently interaction between type XVIII collagen and laminin γ2 chain in normal mouse eye BMs. The study of the type XVIII and XV double null mice revealed that these mice were viable and fertile and had no major additional abnormalities compared to both single null mice. However, the regression of hyaloid capillaries (vasa hyaloidea propria, VHP) was studied in these mice, and a slight delay in the detachment of these vessels from the retina was noticed. Thus, the two collagens do not function entirely independently from each other. The studies with type XVIII collagen single null mice indicate that in addition to the posterior eye phenotype, this collagen is needed for the normal structural integrity of the anterior eye segment and basement membranes of the eye. The mouse model lacking both type XVIII and type XV collagen indicates that the roles of the two collagens are essentially diverse, although a slight compensatory effect was observed in the detachment of the hyaloid capillaries from the retina.

Abstract Lysyl hydroxylase (E.C. 1.14.11.4) catalyzes the formation of hydroxylysine in collagens and other proteins with collagenous domains. The resulting hydroxylysine residues participate in the formation of collagen crosslinks, and serve as attachment sites for carbohydrate units. They have been regarded as non-essential, since the absence of lysyl hydroxylase 1 activity is not lethal, although it leads to the kyphoscoliotic type of Ehlers-Danlos syndrome, and since recombinant collagens I and III lacking any hydroxylysine form native-type fibrils in vitro. A novel human lysyl hydroxylase isoenzyme, lysyl hydroxylase 3, was identified, cloned and characterized here. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to catalyze hydroxylation of lysine residues in vitro. No differences were found in the catalytic properties between the recombinant lysyl hydroxylases 3 and 1. The human lysyl hydroxylase 3 gene was shown to be 11.6 kb in size and to contain 19 exons. The introns contain 15 full-length or partial Alu retroposons, which are known to be involved in most human gene rearrangements that occur by homologous recombination. The three recombinant human lysyl hydroxylase isoenzymes were isolated here for the first time as homogenous proteins. Limited proteolysis data suggested that the lysyl hydroxylase polypeptides might consist of at least three distinct domains, A-C. The N-terminal domain A was found to play no role in lysyl hydroxylase activity as a recombinant B-C polypeptide was a fully active hydroxylase. This work also confirmed that lysyl hydroxylase 3 has collagen glucosyltransferase activity as well as trace amounts of collagen galactosyltransferase activity. However, the levels of these activities were so low that their biological significance remains to be determined. In the last part of this work, lysyl hydroxylase 3 knock-out mice were produced and analyzed. The homozygous null embryos were found to die at a very early stage of development due to lack of type IV collagen in the basement membranes. The data demonstrated that hydroxylysine formed by lysyl hydroxylase 3 is essential for early mouse development and that lysyl hydroxylase 1 or 2 cannot compensate for the lack of its function.

Thyroid carcinomas are the most common endocrine tumors in humans. There are three major types of carcinomas of thyrocyte origin, including papillary, follicular, and anaplastic carcinomas. Papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancy accounting 80% of thyroid cancer cases, and present several histologic variants, namely classical (45%), follicular (18%), solid, diffuse-sclerosing, cribriform, … .Specific genetic events represent early initiating and late triggering events. Several genetic lesions have been identified in various thyroid carcinomas and some of them are specifically associated to one type thyroid cancer. For instance, RET/PTC is the most common molecular event in the radiation-associated PTC in childhood. <p>In the first part of the work, we studied two transgenic mouse models: the Tg-RET/PTC3 (Tg-RP3) mouse and the Tg-E7 mouse. Both strains express the human origin transgene (RET/PTC3 rearrangement or E7) exclusively in the thyroid under the control of the bovine thyroglobulin promoter. <p>Our study of these two models showed:<p>In both E7 and RET/PTC3 mouse models, the thyroids exhibited hyperplasia with own 'oncogene-dependent' follicular cell characteristics. Small follicular cells with hyperchromatic nuclei with an increased nucleus/cytoplasm ratio were numerous in the E7 mice, and large cells with convex apical border, a decreased nucleus/cytoplasm ratio, a pale nucleus and dispersed chromatin were found in the RET/PTC3 mice. <p>At 6, 10 months and later on, E7 mice developed huge heterogeneous, normal functional thyroid goiter, with no tumor formation. <p>As in previous studies on transgenic RET/PTC3 mouse models, the generally encountered features such as solid tumours were present. We also observed conventional variant of human PTC at late age (since 11 month-old) with quite low incidence (4%). In addition to solid and conventional variant PTCs, 28% of mice developed a peculiar big size thyroid tumor pattern with “proliferative papillary cystic changes with spindle cells and remodelling” and macrophage infiltration in the cysts at as early as 2 month of age; this kind of tumor histologically resembles the rare human young age 'diffused sclerosing' variant PTC (DSVP), but disappeared after 6 month. The other peculiar tumor exhibits morphological similarity with another rare human FAP-associated (Familial Adenomatous colonic polyposis) cribriform PTC, which showed a mixed architecture of several histological patterns (solid, follicular, cribriform). At 6 months, 26% of mice presented the cribriform tumor pattern.<p>From the analyse of the proliferation index in the two models, we conclude that RET/PTC3 fusion protein over stimulates MAPK and Akt/PKB-signalling pathways, through Ras-Raf-Mek-Erk, Ras-PI3-K/Akt/PKB, particularly in the large cells which were strongly positive for three proliferation markers. E7 bypasses these two pathways, by directly binding to Rb1 protein and releasing the E2F transcription factor which induces cell proliferation. <p>So RET/PTC3 and E7 mice present several morphologic features which mimic human PTC tumors; RET/PTC3 could therefore be used as a partial model for human PTCs. <p>Further investigation of gene expression will allow the characterization of the molecular phenotype of the observed variants.<p>In the second part of the work, we attempted to generate by xenobiotic administration an in vivo model of thyroid carcinoma. Chronic exposure of CD1 mice to acrylamide in the drinking water during 6 and 8 months at doses of 3mg/kg per day similar to those causing thyroid tumorigenesis after 2 years in rats, did not induce any thyroid tumors whatever the level of thyroid stimulation. <p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Abstract Type XIII collagen is a type II oriented transmembrane protein with a short intracellular domain, a single transmembrane domain and a large, mostly collagenous extracellular domain. Tissue localization and cell culture studies have implicated that it is involved in cell adhesion. The spatio-temporal expression of type XIII collagen mRNA and protein during murine development is studied here. Type XIII collagen mRNAs were expressed at a constant rate during development, with an increase of expression towards birth. The strongest expression was detected in the central and peripheral nervous systems of the developing mouse fetus. Cultured primary neurons expressed this collagen, and recombinant type XIII collagen was found to enhance neurite outgrowth. Strong expression was also detected in the heart, with localization to cell-cell contacts and perinatal accentuation in the intercalated discs. Other sites of type XIII collagen expression included cartilage, bone, skeletal muscle, lung, intestine and skin. Clear developmental shifts in expression suggest a role in endochondral ossification of bone and the branching morphogenesis in the lung. To elucidate the function of type XIII collagen transgenic mice were generated by microinjection of a cDNA construct that directs the synthesis of truncated α1(XIII) chains with an in-frame deletion of the central collagenous COL2 domain. This construct was thought to disrupt the assembly of normal type XIII collagen trimers. Expression of shortened α1(XIII) chains by fibroblasts derived from mutant mice was demonstrated, and the lack of intracellular accumulation in immunohistochemical analysis of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to developmental arrest and fetal mortality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a failure in the fusion of the chorion and allantois membranes and subsequent disruption in placentation, while the late phenotype fetuses were aborted by day 13.5 of development due to cardiovascular and placental defects. Furthermore, it was shown that the heterozygous mice that were initially of normal appearance and bred normally had an increased susceptibility to develop T-cell lymphomas and angiosarcomas later in life. The results presented here increase the evidence that type XIII collagen is involved in cell adhesion, with several important tasks during development. A role of type XIII collagen in malignant transformation of certain mesenchymal cell populations is also implicated.

Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.