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The rat has been an established experimental animal model within many areas of biological investigation for over one hundred years due to its size, breeding characteristics, and knowledge of its physiology and behaviour. In recent years its status as a leading biomedical model has been somewhat surpassed by the mouse. This is largely the result of the isolation and application of mouse embryonic stem (ES) cells. Mouse ES cells have the capacity for unlimited self-renew in vitro whilst maintaining pluripotency and germline competence, and most importantly are amenable to sophisticated reverse genetics strategies such as gene targeting, which have provided a route to germ line modification. Thus far, the derivation of rat ES cells has proved elusive. The generation of rat ES cells would therefore facilitate equivalent applications to rat genetics and significantly strengthen the rat as an experimental model system. Previous attempts to derive rat ES cells led to the isolation of rat ES-like cells. However, whilst these cells exhibit extensive self-renew in vitro, it was known that they fail to maintain significant levels of the key functional ES cell marker Oct4 and do not contribute to chimeras. Rather, these cells express the trophectoderm markers Cdx2 and CyclinD3, and have been termed ExS cells due to their probable extra-embryonic nature. In the work described in this thesis, further investigation of ExS cells revealed the absence of expression of the key pluripotency gene Nanog, although the expression pattern of Nanog in the rat embryo was shown to be similar to that of mouse. It was hypothesised that expression of exogenous Oct4 and Nanog or Sox2 genes could facilitate reprogramming of ExS cells into a 'true' ES cell state. Initial work described in this thesis demonstrated that it was possible to introduce transgenes into rat ExS cells and obtain stable transformants with long term transgene expression. On this basis Oct 4, Nanog and Sox2 transgene expression vectors were constructed and stably integrated into ExS cells, and transgene expression verified. However, no reactivation of an endogenous gene expression profile, characteristic of a true ES cell-like state, was observed in any of the transgenic lines produced. Concurrent with work on ExS cells, investigations by others using chemically defined, serum-free medium containing small molecule inhibitors of MEK and GSK3 (called 3i/2i medium) had demonstrated that it was possible to readily isolate mouse ES cells, even from strains known to be refractory to ES cell isolation. Therefore, the ability of this culture system to facilitate rat ES cell derivation was investigated. Rat 3i/2i cell lines were established from ICM outgrowths of Fischer, DA and Sprague Dawley E4.5 rat embryos. These cells maintained expression of Oct4 and Nanog and could generate complex teratomas consisting of all three germ layers. They were distinct from epiblast stem cells (EpiSC) in that they expressed Klf4, Rex1 and Stella and most importantly, they could contribute to the formation of adult chimaeras and demonstrated germline competency. Isolation of these authentic rat ES cells paves the way for gene targeting in the rat, a development that should greatly facilitate new biomedical discoveries.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Inflammation has modulatory effects on the brain, particularly during development. These plastic changes can hold severe functional consequences. Perinatal hypoxia-ischemia (HI)-induced inflammation can result in cerebral palsy and cognitive impairment. In an attempt to reduce inflammation in the brain, we assessed the probiotic Bifidobacterium (B.) infantis as an HI intervention, using a rat model. Rat pups, developmentally equivalent to preterm infants, were exposed to chronic hypoxia from postnatal (PND) 3 –PND 10. Inflammation was assessed through hippocampal concentrations of the cytokine interleukin-6 (IL-6). Tissue was collected from pups on PND 10 and analyzed via enzyme-linked immunosorbent assay (ELISA). Results showed lower IL-6 concentrations in hypoxic groups , regardless of B. infantis administration. Qualitative observations suggested poor gut health in association with hypoxia and probiotic exposure. These preliminary findings support the chronic hypoxia exposure model of HI and suggest the association with IL-6 and HI events is less straightforward than expected. / October 2016

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

1. The induction of CYP3A enzymes was investigated using a range of structurally unrelated drugs using in vivo and in vitro models. Hepatic microsomal testosterone 6(3-hydroxylation, anti-CYP3A immunoblot analysis, and molecular biology approaches were utilised in the investigation. 2. Using the rat as an in vivo model, potent induction of CYP3A enzymes was observed after administration of the synthetic glucocorticoid dexamethasone (at 150mg.kg.day for 4 days) and pregnenolone 16?-carbonitrile (at 150mg.kg-1.day-1 for four days). However, no induction was observed after administration of rifampicin (at 50 g.kg-1.day-1 for 4 days, a dose which causes potent induction in the rabbit). 3. Investigations into the effects of drug exposure on testosterone 6?- hydroxylation in cultured female rat hepatocytes revealed a positive in vivo/in vitro correlation. Cultured cells were treated with the same drugs (at 50 M concentration) for 72hrs. Dexamethasone was shown to be more potent than PCN, and rifampicin again had no effect. Dexamethasone-mediated induction of testosterone 6?- hydroxylation was dose-dependent and was shown to be maximal after 72hrs exposure. 4. The presence of the differentiating agent dimethylsulphoxide at 2% (v/v) in the cultiure medium enhanced CYP3A induction by the synthetic steroids by approximately 100% (p 0.05). 5. The potent glucococorticoid antagonist RU 486 induced testosterone 60- hydroxylation 5-fold when administered at 50mg.kg-1.day-1 for 4 days. Induction of the CYP3A protein was confirmed by immunoblot analysis of liver microsomes. Administration of RU 486 at 50mg.kg-1.day-1 did not antagonise the induction of testosterone 6?-hydroxylatiomn by dexamethasone at 150mg.kg-1.day-1. 6. Dexamethasone (0.1 to 10 M) -mediated induction of testosterone 6(3- hydroxylation in cultured rat hepatocytes was attenuated in the presence of RU 486. It is not known whether this was due to effects on CYP3 A gene expression or inhibition of enzyme mediated activity at the active site of the enzyme. 7. The lipid lowering drug SK F 98016 (150mg.kg-1.day-1) induced testosterone 6?-hydroxylation 10-fold when administered at 150mg.kg-1.day-1 for 4 days. This was confirmed by immunoblot analysis. Co-administration of RU 486 with SK F 98016 attenuated induction of CYP3A-mediated enzyme activity. The mechanism of induction of the CYP3A genes by SK F 98016 may therefore involve 'steroidal' compounds, the action of which is antagonised by RU 486. The dexamethasone- mediated increase in spectrally determined cytochrome P450 levels was also attenuated after co-administration with RU 486. As CYP3A induction was not affected by co-administration of dexamethasone with the anti-glucocorticoid RU 486, this result suggests that the glucocorticoid receptor may be involved in the induction of other P450 genes. 8. Treatment of rat hepatocytes with SK F 98016 (50 M) for 72 hours did not result in an increase in testosterone 6?-hydroxylation. In fact testosterone 6?-, 16?- and 17-oxidation activities were reduced to 50% of the activities measured in untreated hepatocytes. This pointed to some P450 inhibitory potential of SK F 98016. Investigation of the inhibitory potential of SK F 98016 on testosterone 60- hydroxylation in hepatic microsomes from PCN-treated rats showed an inhibitory effect with an IC50 of 50 M. The inhibitory effect seen in hepatocytes is similar to the effects of exposure to clotrimazole (50 .M) for 72 hours where testosterone metabolism at the 60 and 17 positions were inhibited by >90%. 9. To investigate whether the lack of inducing effect of SK F 98016 was due to the very high lipophilicity and extensive partitioning into the cultured hepatocyte, therefore resulting in a non-physiological state, cultured hepatocytes were exposed to the same drugs with albumin (from bovine serum, at the concentration present in human blood-36g/litre) in the medium in attempt to encourage an equilibrium of drug concentration between the medium and the inside of the hepatocyte. No significant induction of testosterone 60-hydroxylation was observed in the presence of albumin.

Master of Science / Department of Anatomy and Physiology / Mark Weiss / Artificial insemination and cryopreservation of sperm are important components of any transgenic animal facility because they allow for the reduction in animal colony size and the safe storage of germplasm from valuable strains. In addition, they allow long-term storage of these strains and easy transportation of the genetic material to other research facilities internationally. Thus far, only one laboratory has created live rat pups after sperm cryopreservation and intrauterine insemination. Another laboratory made advances in cryopreservation media that improved sperm motility post-thawing, but no pups resulted from this work. In my study, these two cryopreservation media were utilized to perform intrauterine inseminations with both fresh samples of rat sperm as well as samples that were cryopreserved in liquid nitrogen to replicate and extend these studies. Pharmacoejaculation was tested as a means to obtain spermatozoa without euthanizing the male to collect the epididymis, but results were inconsistent and the samples were not useful for intrauterine inseminations or cryopreservation. Epidiymal sperm was then collected into the various media and frozen in liquid nitrogen. In my hands, the frozen/thawed rat sperm achieved motility of less than 1%. Next, the impact of altering the freezing rate on sperm motility was evaluated. Epididymal sperm was collected and processed using a modified protocol and were then frozen at 2, 4 or 6 cm above the level of liquid nitrogen. Four to six days after freezing, samples were thawed and post-thaw sperm motility was evaluated. Sperm motility was measured prior to freezing as well as after-thawing. The sperm motility was correlated with LIVE/DEAD® staining. Sperm motility did not differ between the groups as a result of the freezing rate (Friedman test p=0.23). The published techniques are not robust and require further development to improve the motility of rat sperm after cryopreservation and achieve pregnancy via intrauterine insemination.

Alpine environments are poorly studied ecosystems, largely due to their inaccessibility and severe climatic conditions. Nonetheless, a better understanding is needed of the ecological processes shaping these habitats, particularly the interactions between plants and animals. Recent studies indicate that the levels of soil erosion have increased in parts of Lesotho, possibly because of overgrazing by domestic livestock and the activities of the African ice rat Otomys sloggetti robertsi, whose population numbers have increased in recent times. O. s. robertsi is a diurnal, herbivorous, burrowdwelling, murid rodent, endemic to the southern African Drakensberg. The aim of my study was to establish whether and how the ice rat influences the vegetation and the soil characteristics in its habitat, and to determine whether ice rat population numbers have increased. I conducted three experiments. 1) Enclosures/plots were erected in the Sani Valley to measure the impact of; i) ice rats alone; ii) both ice rats and livestock on vegetation and soil loss and gain (which was used as a proxy for soil erosion). 2) I also ascertained ice rat numbers (colony sizes) at three different locations in Lesotho (Katse Dam, Oxbow and Sani Valley) by conducting monthly censuses of discrete colonies at each locality. 3) Finally, questionnaire surveys were used to ascertain the perception of, and influence on, ice rats by the local human inhabitants in Lesotho. The enclosure/plot experiments showed that the plots accessed by ice rats only had higher levels of vegetation change (loss of cover, decrease in height) and soil movement than other plots from which they were excluded or could access together with livestock, which was contrary to my prediction that the combined influence of ice rats and livestock would have a greater impact. The size of ice rat colonies showed a three-fold increase in my study compared to those a decade ago. The interviews of the local human inhabitants supported this finding, with people also claiming that ambient temperatures had increased and snowfall had decreased. The interviewees did not express any meaningful opinion about how they influenced the biology of ice rats, but claimed that ice rats were responsible for land degradation in the high Drakensberg. In conclusion, the results suggest that ice rats are responsible for large scale damage at my study sites as a result of their foraging and burrowing activities, and erosion is likely to be exacerbated by the increasing numbers of ice rats. Nonetheless, soil erosion is a complex problem involving several biotic and biotic contributing factors, and long term studies are required to fully understand the underlying determinants of erosion in the Lesotho Highlands.

Ovarian hyperstimulation is achieved through ovarian gonadotropin stimulation, and thus associated with supraphysiological levels of oestrogen and progesterone. To investigate the effects of exogenous gonadotropins on the expression of TGF b1 and TGF b2, which have been recognized as possible modulators of many endometrial functions, FSH and hCG were superimposed upon the normal hormonal milieu of the cycling rat, prior to mating. Endometrial tissue was collected at 4.5, 5.5 and 6.5 days after mating. ELISA was performed to estimate blood oestrogen and progesterone levels and immunohistochemistry was undertaken to localize TGF b1 and TGF b2 in the uterine endometrium. Apart from the known detrimental effects of hyperstimulation on gross morphology, hormone levels and endometrial histology, the hyperstimulation was also found to affect TGF b expression. An increase in the expression of TGF b2 was distinct in the glandular epithelium of the hyperstimulated animals, while regionalized expression of both TGF b1 and TGF b2 was prominent in the stroma. In conclusion, hyperstimulation affects the expression of both TGF b1 and TGF b2, which may contribute to the disruption of the endometrial environment required for successful embryo implantation.