No description available.

Excessive drinking during early pregnancy is associated with fetal developmental anomalies [1], particularly affecting heart formation, known as Congenital Heart Disease (CHD). This health concern is underscored by studies that indicate a high incidence of binge drinking among expectant mothers [1]. This research delves into alcohol's role in altering epigenetic patterns across the embryonic cardiac genome, seeking to isolate genes and their specific sites influenced by in utero alcohol exposure. The theory posits that ethanol-exposed embryonic mouse hearts will exhibit distinctive patterns of methylation in contrast to unexposed controls [2]. To investigate the proposed theory, ethanol was orally delivered to mouse models at a gestational stage critical for heart development, specifically embryonic day 9.5 (E9.5). Subsequently, at embryonic day 11.5 (E11.5)—a pivotal moment in cardiac morphogenesis—the mice were euthanized to harvest the embryonic hearts. The genomic material was then meticulously extracted from these hearts to enable a comprehensive analysis through whole-genome bisulfite sequencing (WGBS). Preliminary results revealed a marginal shift toward lower methylation levels without broad genomic changes in the ethanol treated samples [7]. Nonetheless, particular genes, like WNT7A gene, have been pinpointed for their suppressed activity following alcohol exposure, guiding further inquiries into aimed methylation changes and epigenetic variations that may illuminate the mechanisms by which maternal alcohol consumption prompts cardiac anomalies in CHD [7]

Haptic mice, computer mice modified to have a tactile display, have been developed to enable access to computer graphics by individuals who are blind or visually impaired. Although these haptic mice are potentially very helpful and have been frequently used by the research community, there are some fundamental problems with the mouse, limiting its acceptance. In this paper we have identified the problems and have suggested solutions using one haptic mouse, the VT Player. We found that our modified VT Player showed significant improvement both in terms of the odds of obtaining a correct responses and the time to perform the tasks.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disease characterized by progressive muscle-wasting and loss of mobility. One-third of patients with DMD are also affected by cognitive impairments such as a lower than average IQ and impaired working memory, comorbid with neuropsychiatric disorders such as anxiety and autism-related behaviours. DMD is caused by mutations in the DMD gene resulting in the deletion of the full-length dystrophin protein (Dp427) and, dependent on mutation, other dystrophin isoforms. These isoforms are predominantly found in the brain and deletion may impact on cognition. The most commonly used animal model to study DMD is the mdx mouse which completely lacks Dp427 but no other DMD isoforms. Although the muscle phenotype is well-established, behavioural characterization of the mdx mouse model has been inconclusive. In this thesis I investigated the hippocampal and amygdala cellular and behavioural phenotypes of the mdx mouse. I show that post-natal neural stem-like cell division in the SGZ is altered in the absence of Dp427 resulting in enhanced symmetric division. I show in vitro that primary mdx cultures are fewer and smaller than wild-type, consistent with an increase in symmetrical self-renewal whereas secondary cultures are fewer and larger, consistent with a shift in symmetric division producing transit-amplifying type 2a daughter cells. I next characterized the mdx mouse model using a battery of behavioural tests. Data presented here show that mdx mice do not exhibit an anxious phenotype, do not display autism-related behaviours, and do not display impairments in and spatial learning or memory. However, associative learning, as measured in the fear conditioning paradigm is enhanced in mdx mice. Lastly, I attempted to generate three different brain-specific dystrophin knock-out mouse models to examine role of other dystrophin isoforms. While none of the models were able to deplete dystrophin from brain, given the inverse relationship between Cre-mediated efficiency and the genetic distance of the loxP sites in the fDMDH mouse employed, I do provide important insight into the presence and absence of the muscle-specific enhancers in constructs commonly used to generate brain-specific mouse models. Taken together, this thesis provides converging evidence to indicate that loss of Dp427 impacts on fear associative learning and stem-cell like division in the SGZ but likely does not underlie the non-progressive cognitive impairments affecting one-third of all DMD patients.

Research involved in modeling human lung disease conditions has provided insight into disease development, progression, and treatment. In particular, mouse models of human pulmonary disease are increasingly utilized to characterize lung disease conditions. With advancements in small animal imaging it is now possible to investigate the phenotypic differences expressed in inbred mouse strains in vivo to investigate specific disease conditions that affect the lung. In this thesis our aim was to generate a comprehensive characterization of the normative mouse lung phenotypes in three of the most utilized strains of mice, C57BL/6, A/J, and BALB/c, through imaging techniques. The imaging techniques that we utilized in this research included micro-CT, a custom Large Image Microscope Array (LIMA) system for 3D microscopy, and classical histology. Micro-CT provided a non-destructive technique for acquiring in vivo and fixed lung images. The LIMA 3D microscopy system was utilized for direct correspondence of the gold standard histology images as well as to validate the anatomical structures and measurements that were extracted from the micro-CT images. Finally, complete lung histology slices were utilized for assessment of the peripheral airspace structures that were not resolvable using the micro-CT imaging system. Through our developed imaging acquisition and processing strategies we have been able to successful characterize important phenotypes in the mouse lung that have not previously been known as well as identify strain variations. These findings will provide the scientific community with valuable information to be better equipped and capable of pursuing new avenues of research in investigating pulmonary disease conditions that can be modeled in the mouse.

This research project deals with the characterization of self-replicating Kirsten murine viral DNA. The replication of this viral DNA and tetracycline resistance conferred to bacteria by this viral DNA will be studied. The restriction endonuclease and Southern blot analysis revealed a fragment of pBR322 from the Hind III and Pst I site that is located in the 3' end of the MLV-K:E molecule. Single stranded sequencing of the two terminal ends of this fragment verified that the 3' end of MLV-K:E contains identical sequence homology to pBR322. The presence of this pBR322 fragment explains the unusual properties of the MLV-K:E molecule. However, tetracycline resistance is less in E. Coli containing MLV-K:E than E. coli containing pBR322 as determined by zone of inhibition assay. This may be due to alteration in the promoter region of the tetracycline gene.

The feasibility of the Alb-uPA/SCID/Bg chimeric mouse as a model for Hepatitis C Virus (HCV) infection was assessed experimentally by (1) the infection and treatment with another hepatotropic virus, Hepatitis B Virus (HBV) and (2) the infection of the model with HCV and the subsequent treatment of that infection with a pro-apoptotic factor (BID) targeted to infected hepatocytes. In the former, the infected mouse responded favorably, and in the manner of human patients, to a standard imunoglobulin therapy. In the latter, HCV-infected hepatocytes were successfully targeted for cell death, with repeated doses of Adenovirus-delivered BID being the most effective at inhibiting virus spread. Efficacy and toxic side-effects of BID treatment could be reconciled by modulating the timing between doses, the most effective tested being three doses of BID at 7-day intervals. Analyses of chimeric model production were undertaken to improve the quality of human hepatocyte engraftment (typically only 25-35% of mice receiving grafts are currently used experimentally). Minor variations in success rates were experienced with respect to donor age or health status, or the age of recipient mice within an operational window of 5 to 13 days from birth. The greatest obstacle to useful engraftment (aside from technical challenges) was deemed to be the genetic/cellular integrity of the recipient mouse. This conclusion was based on variable engraftment success with ‘healthy’ donor cell preparations and a consideration of variability in immune deficiency arising in mice within a SCID/Bg mouse colony. / Experimental Surgery

No description available.

The phylogenetics of eight species of the Peromyscus truei and P. boylii species groups from 15 populations were analyzed based on mitochondrial DNA sequence differentiation, using 13 hexanucleotide specific restriction enzymes. P. difficilis, P. nasutus, and P. attwateri were found to be members of the same clade. P. leucopus was not found to be closely related to any of the species of the boylii or truei species groups. Phylogenetic interpretations for the remaining species differed based on Wagner and Dollo parsimony analyses. P. true appears to be most closely related to P. gratus based on Wagner parsimony and the phenetic analysis, while the relationship of P. gratus to other species could not be resolved based on Dollo parsimony.

The preimplantation stages of mammalian development are dedicated to the differentiation of two extraembryonic epithelia, the trophectoderm (TE) and the primitive endoderm (PrE), and their segregation from the pluripotent embryonic lineage, the epiblast. The TE and PrE are responsible for implantation into the uterus and for producing the tissues that will support and pattern the epiblast as it develops into the foetus. PrE and epiblast are formed in a two step process that involves random cell fate specification, mediated by fibroblast growth factor (FGF) signalling, and cell sorting through several mechanisms. In the present work I have addressed aspects of both steps of this process. Chimaera assays showed that epiblast precursors transplanted onto a recipient embryo rarely differentiate into PrE, while PrE precursors are able to switch their identity and become epiblast. Transient stimulation or inhibition of the FGF4-ERK pathway in the chimaeras can modify the behaviour of these cells and restore the plasticity of epiblast precursors. This work shows that epiblast precursors are refractory to differentiation signals, thus ensuring the preservation of the embryonic lineage. I have also found that atypical Protein Kinase C (aPKC) is a marker of PrE cells and that pharmacological inhibition of aPKC impairs the segregation of PrE and epiblast precursors. Furthermore, it affects the survival of PrE cells and can alter the subcellular localisation of the PrE transcription factor GATA4. These data indicate aPKC plays a central role for the sorting of the PrE and epiblast populations and links cell position within the embryo to PrE maturation and survival. Lastly, I have found that aPKC can directly phosphorylate GATA4 in vitro. Knockdown of GATA4 affects cell position within the embryo, whereas aPKC knockdown reduces the number of GATA4-positive cells. These results indicate GATA4 plays an important role in cell sorting during preimplantation development and suggest phosphorylation by aPKC could determine its presence in the nuclei of PrE cells. My work, in the light of the current knowledge, supports a model where the earliest cell fate decisions during mammalian development depend on cellular interactions and not on inherited cell fate determinants. This robust mode of development underlies the plasticity of the preimplantation embryo and ensures the formation of the first mammalian cell lineages, critical for any further progression in mammalian development.