No description available.

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology.

The serotonergic system regulates numerous behaviours and disruptions in this system have been associated with disorders of mood and mind. Although molecular genetic analysis has dissected many of the genes involved in the specification of the serotonergic system, relatively little is known about the mechanisms that promote axonal outgrowth from serotonin-producing neurons and how these projections are directed to innervate and form synapses with their appropriate targets. The mouse anorexia mutation causes hypersprouting of serotonergic projections in target fields and has provided us the unique opportunity to examine the crucial events that lead to the establishment of these complex serotonergic networks. Through positional cloning, I have identified a candidate gene that is upregulated during a time in which innervation and synaptogenesis of serotonergic neurons are maximal. I have assessed the expression of this candidate gene in the brain and have found striking differences in the pattern of expression between the normal and the mutant mouse. Furthermore, by using transgenic methods, I have partially rescued several hallmark behavioural phenotypes in the mutant mouse. Thus, this candidate almost certainly represents the “Anorexia” gene.

The serotonergic system regulates numerous behaviours and disruptions in this system have been associated with disorders of mood and mind. Although molecular genetic analysis has dissected many of the genes involved in the specification of the serotonergic system, relatively little is known about the mechanisms that promote axonal outgrowth from serotonin-producing neurons and how these projections are directed to innervate and form synapses with their appropriate targets. The mouse anorexia mutation causes hypersprouting of serotonergic projections in target fields and has provided us the unique opportunity to examine the crucial events that lead to the establishment of these complex serotonergic networks. Through positional cloning, I have identified a candidate gene that is upregulated during a time in which innervation and synaptogenesis of serotonergic neurons are maximal. I have assessed the expression of this candidate gene in the brain and have found striking differences in the pattern of expression between the normal and the mutant mouse. Furthermore, by using transgenic methods, I have partially rescued several hallmark behavioural phenotypes in the mutant mouse. Thus, this candidate almost certainly represents the “Anorexia” gene.

Pregnancy succeeds because the fetal allograft survives in the presence of a fully functional maternal immune system. The placenta, especially its trophoblast, provides the initial barrier between the maternal and fetal environment and, due to their location, trophoblast cells could be expected to be immune-privileged. Yet in the ectopic sites tested thus far, trophoblast stem cell transplants have failed to show noticeable immune privilege and appear to lack physiological support. However in this study, portal vein injected green fluorescent protein-labeled trophoblast stem cells were able to survive for several months in the livers of allogeneic female (14/14), but not male (0/4), mice. Gonadectomy experiments revealed that this gender-dependent survival does not require the presence of ovarian hormones (4/4) but the absence of testicular factors (5/5). In contrast, similarly labeled allogeneic embryonic stem cells were reliably rejected (11/11); these same embryonic stem cells survived when mixed with unlabeled trophoblast stem cells (13/13). The protective effect offered by the trophoblast stem cells did not require any immunological similarity with the co-injected embryonic stem cells. Neither the trophoblast stem cells nor the co-injected embryonic stem cells gave rise to tumors during the study period. Thus, this study demonstrates that, provided a suitable location and hormonal context, ectopic trophoblast stem cells may exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as provide a new model for studying trophoblast physiology and immunology.

Phased array surface coils offer high SNR over a large field of view. Phased array volume coils have high SNR at the surface and centre of the volume. Most array coil designs typically employ a combination of geometrical and additional techniques, such as isolating preamplifiers for element-to-element decoupling. The development of array coils for small animal MRI is of increasing interest. However isolation preamplifiers are expensive and not ubiquitous at the field strengths typically employed for small animal work (4.7T, 9.4T, etc). In addition, isolating preamps complicates the designs of coils for transmit SENSE since they do not decouple during transmitting. Therefore, this thesis reexamines a "tried and true" method for decoupling coil elements. In this work five different coils for mouse imaging at 200MHz are presented: a 16 leg trombone design quadrature birdcage coil and four geometrically decoupled volume phased array coils. The first mouse array coil is a two saddle quadrature coil with a circularly polarized field. The second coil is a four channel transmit/receive volume array coil that is decoupled purely geometrically, without the need for other forms of decoupling. The third array coil is a modified 'open' configuration to facilitate the loading of animals. The fourth coil presented is a 'tunable' decoupling coil, where the geometric decoupling between elements is 'tunable', in order to compensate for different loading conditions of the coil. Tunable decoupling between elements was achieved using two mechanisms, a decoupling paddle for isolation of top to bottom elements, with a variable overlap mechanism for decoupling diagonal elements. Bench measurements demonstrate good decoupling (better than -20dB) of the coil elements and 'tunability' of both mechanisms. Phantom images from all coils are presented.

Retinoic acid (RA) is an active metabolite of vitamin A that is essential for embryonic development, and homeostasis of adult tissues. RA is a ligand for the nuclear retinoic acid receptor, and RA-mediated signaling is critical for regulation of cell proliferation, differentiation and apoptosis. There is a spatio-temporal distribution of RA in the developing embryo such that some tissues are rich in RA, while others are devoid. This patterned distribution of RA is tightly controlled through the coordinated expression of RA-synthesizing (retinaldehyde dehydrogenase) and RA-catabolizing (CYP26) enzymes. In this thesis, I describe the cloning of a mouse gene encoding one of the CYP26 proteins, Cyp26b1. Cyp26b1 was shown to be highly expressed in the embryo, with transcripts localized to the hindbrain, limb buds and branchial arches. We also used homologous recombination to generate a line of transgenic mice with a loss-of-function deletion in Cyp26b1. These mice die shortly after birth with severe malformations affecting the limbs, craniofacial structures and epidermis; phenotypes that are all reminiscent of RA teratogenesis. We present an extensive characterization of the craniofacial and epidermal abnormalities in Cyp26b1-/- animals, and examine several molecular pathways that may be deregulated. CYP26B1 null embryos exhibit a truncated mandible, lack numerous facial bones, and show reduced ossification of the calvaria. Molecular analysis of Cyp26b1-/- embryos indicates hindbrain and branchial arch patterning is largely unaffected in early to mid-gestational mutants. However, there appear to be some subtle abnormalities in neural crest cell migration, which may contribute to the development of some of the observed phenotypes. CYP26B1 null mutants also lack hair follicles, which appears to be due to a downregulation of -catenin mediated signaling. Thus, in addition to cloning and characterizing the expression of murine Cyp26b1, we have demonstrated in vivo, that regulation of RA distribution by CYP26B1 is essential for morphogenesis of the epidermis and craniofacial structures. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2007-09-28 13:49:35.17

Helicase-like Transcription Factor (HLTF) is a DNA helicase protein which is homologous to SNF/SWI family. It has been demonstrated to be a functional homolog of yeast Rad5, required for the maintenance of genomic stability. Although the physiologic role of HLTF is largely unknown,inactivation of HLTF by promoter hypermethylation has been found in more than 40% human colon cancers. In this study, we have applied mouse transgenic approaches to determine whether loss of HLTF function could be important for colorectal carcinogenesis. HLTF knockout mice were generated by the deletion of first 5 exons of the HLTF gene. The complete loss of HLTF expression in HLTF -/- mice was confirmed by northern blot and real time RT-PCR assays. HLTF -/- mice did not show any developmental defects within a 2-year observation indicating that HLTF is dispensable for mouse development. Furthermore, HLTF -/- mice were free of intestinal or colorectal tumors or other types of tumors, suggesting that loss of HLTF function alone is not sufficient to drive oncogenic transformation in intestinal track and other tissues. To determine whether loss of HLTF function could cooperate with other tumor suppressors in the formation of colorectal cancers, we have bred HLTF knockout mice with the mutant mice for APC (adenomtous polyposis coli) and P53. In HLTF -/-APC Min/+ mice, a significantly increased formation of intestinal adenocarcinoma and colorectal cancers were observed. Although very few HLTF -/-P53 -/- mice developed colorectal cancers, these mice had increased incidence of the formation of metastatic lymphomas. Cytogenetic analysis of colorectal cancer cells derived from HLTF -/-APC Min/+ mice demonstrated a high incidence of gross chromosomal instabilities, including Robertsonian fusions, fragments and aneuploidy. All these genetic alterations were not observed in the intestinal tumor cells from APC Min/+, implicating that loss of HLTF function could induce genomic instability which contributes to intestinal carcinogenesis. To further investigate the role of HLTF in colorectal carcinogenesis, we have also applied a shRNA knockdown approach to down-regulate HLTF expression in human HCT-116 colon cancer cells. HCT-116 cells highly express HLTF and show less chromosomal instability, making these cells as a very useful model to investigate the loss of function of HLTF in human colorectal carcinogenesis. Using Western blot approach, we confirmed that HLTF knockdown HCT-116 cells had less than 5% of HLTF expression as compared to the scramble controls. By inoculating HLTF knockdown HCT-116 cells to Rag1 -/-IL2 -/- immunocompromised mice, we further demonstrated that HLTF knockdown promote tumor growth and invasion. Moreover, spectral karyotyping analysis revealed that HLTF knockdown human colon cancer cells had significantly increased chromosomal instability, including both aneuploidy and chromosomal translocation. Taken together, our work strongly indicates that loss of HLTF function can promote the malignant transformation of intestinal or colonic adenomas to carcinomas by inducing genomic instability. Given the high frequency of epigenetic inactivation by hypermethylation of HLTF in human colon cancers, our studies strongly suggest that this epigenetic alteration could be directly involved in the development of colorectal cancer rather than a consequence of this carcinogenesis.

No description available.

No description available.