The placenta provides a protective barrier for the developing foetus during gestation. Physiological barriers including the placenta, liver, kidney, intestine and blood-brain barrier are known to express ATP-Binding cassette transporters (ABC transporters) and metabolising enzymes. These specialised proteins have the ability to transport or metabolise xenobiotics. There is evidence to suggest that ABC transporters and metabolising enzymes are located at the interface between the maternal and foetal blood supplies (a cell layer referred to as the syncytiotrophoblast) and therefore may help protect the foetus from harmful xenobiotics. During new compound development prenatal developmental toxicity testing forms an important part of safety assessment. In order to predict potential toxicity of a new chemical entity to humans, rodent and non-rodent species are currently used. This thesis investigates the rat and human placental barrier properties in order to help facilitate our knowledge of species differences and contribute to our understanding of the limitations of these surrogate models. The approaches taken include: genomic analyses using microarray data to compare the overall expression of ABC transporters and metabolising enzymes throughout gestation in both species, immunohistochemical techniques to localise transporters and metabolising enzymes in the rat placenta, and in vitro functionality assays of selected transporters performed in rat and human placental cell line models. The main findings have shown a similar mRNA expression level of ABCG2/BCRP (breast cancer resistance protein) throughout gestation in the rat and human, however different mRNA expression levels of other transporters (slco4a1/oatp4a1 in particular) and metabolising enzymes were also highlighted. Immunohistochemistry localised selected transporters to the syncytiotrophoblast region of the rat placenta (the interface of maternal and foetal circulations). Functional in vitro assays were successfully utilised in rat and human placental cell lines which showed functional ABCB1/P-gp in both species. Overall, these findings provide a genomic characterisation of the rat and human protective placental barrier properties and show transporter functionality in in vitro cell-based assays which will prove useful in prenatal and developmental toxicity tests. Alternatives to using animals have been explored by using functional in vitro assays which could potentially be implored during the new compound discovery phase. This could help to make animal testing more selective for given compounds and ensures the new chemical entity is being tested in the model closest resembling the human.

Home ranges of dusky-footed wood rats (Neotoma fuscipes) were determined by the exclusive boundary strip method in two colonies 630 m apart in Klamath County, Oregon. The mean home range area was 1800 - 1900 m2 . Densities or trees and shrubs were determined on selected home ranges and densities of wood rat houses and nests were determined on all home ranges. No significant correlation was found between home range areas and densities of various trees and shrubs, home range areas and densities of woodrat houses and nests, or home range areas and weights of woodrats in those areas. No movement between colonies was observed.

The objectives of this research were to elucidate the involvement of constrictor prostanoids in the vascular reactivity to vasopressin (VP) and the role of estrogen in the regulation of the constrictor prostanoid mechanism in the female rat. Aortas obtained from male, intact (InT)-, ovariectomized (OvX)- and OvX + estrogen-replaced (OvX+Est)-female rats were studied. Contractile responses to VP were examined in the presence of nonselective and selective cyclooxygenase (COX) inhibitors. Basal and VP-stimulated release of thromboxane A2 (TxA2) and prostacyclin (PGI2) from the aortic wall were measured. Concentration-response curves to exogenous TxA2 were also obtained. To elucidate the regulatory effects of estrogen on the constrictor prostanoid pathway, the expression of COX-1, COX-2, thromboxane synthase (TxS) and thromboxane receptor (TP) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Further, immunohistochemistry was employed to determine COX-1, COX-2 and TxS protein expression in aortic endothelium and vascular smooth muscle. The major findings of this research are that: 1) The contractile responses of the female rat aorta to VP were enhanced by COX-2-mediated production of constrictor prostanoids (PGH2/TxA2), and this mechanism is potentiated by estrogen; 2) Vascular reactivity to exogenous TxA2 was higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored vascular reactivity to TxA2 in the female aorta; 3) VP-stimulated release of endogenous TxA2 and PGI2 were higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored VP-stimulated release of these endogenous prostanoids by the female aorta; and 4) The expression of COX-2 and TxS mRNA and protein, and the expression of TP mRNA were higher in InT-female than in male, and were reduced by OvX and restored by estrogen replacement therapy. In conclusion, estrogen potentiated contractile responses of the female rat aorta to VP by upregulating the expression of COX-2, TxS and TP; thereby enhancing VP-induced release of TxA2, as well as the vascular reactivity to endogenous TxA2.

Infantile exposure to xenobiotics, e.g. from breastfeeding, poses a serious toxicity risk. Since the toxicokinetic mechanisms that principally determine exposure outcomes undergo a significant developmental maturation, infants may respond to exposures in a different way than adults. Hence, suitable model systems are required to provide risk relevant information in pediatric populations. This dissertations primary goal was to provide a critical evaluation of two such model systems; first, a pharmacokinetic model that may predict an infants capacity to eliminate toxicants by cytochrome P-450 (CYP) mechanisms and second, the developing rat as a model of human CYP2E1 and CYP1A2 ontogeny.<p>The first objective was to evaluate underlying assumptions of a pharmacokinetic model that describes the ontogeny of hepatic CYP activity using the rat. The study recognized some discrepancies with the stated assumptions. The impact of these discrepancies on the potential applicability of the model is discussed. As proof-of-concept, the observed data were fit to a model describing rat CYP2E1 and CYP1A2 ontogeny. A reasonable correlation (r = 0.75) was observed between observed and predicted oral clearance values of a CYP2E1 substrate indicating the potential applicability of such a model in risk assessment. <p>The second objective was to conduct an extensive characterization of rat hepatic CYP2E1 and CYP1A2 ontogeny at mRNA, protein, activity and intrahepatic expression levels. The results were compared to available human data to determine the appropriateness of the rat for assessment of toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity. Similarities in age-dependent changes in mRNA, activity and zonal hepatic expression patterns were noted between the rat and human prior to weaning. Unlike human data, rats show good correlation between changes in CYP2E1 and CYP1A2 activity and transcript levels, but not with the immunoquantifiable protein. Recognizing such similarities and differences between rats and human regarding onset, rate and pattern of CYP ontogeny will improve the accuracy of rat-to-human extrapolation of developmental toxicokinetic data. <p>Overall, the dissertation research provides mounting and supportive evidence for the use of such model systems in providing risk-relevant information in pediatric populations and to identify toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity.

A fundamental challenge in evolutionary biology and medical genetic research is to connect the phenotype (a disease in humans or an adaptive trait in animals or plants) with the genotype. Using a classical example of an adaptive trait with a strong Mendelian genetic basis - warfarin resistance in the Norway rat (Rattus norvegicus), my dissertation tests the main hypothesis that speculated ‘simple’ adaptive trait has a more complex genetic architecture. Warfarin is an anticoagulant rodenticide used since the 1950s, and also is a widely prescribed blood-thinning drug in human. As a rodenticide, warfarin has initially been very effective. However, resistant rodents have evolved quickly and Vkorc1 (vitamin K epoxide reductase complex subunit 1) is the known resistance gene. As a popular drug, warfarin has a narrow therapeutic window with several genes VKORC1, CYP2C9, CYP4F2 established as biomarkers predicting warfarin dose in humans, suggesting a complex genetic architecture of warfarin resistance in rodents. In my thesis I performed network-guided genomic association studies (NetGWAS) and gene expression analysis to identify candidate genes involved in warfarin resistance based on a sample of ~600 wild rats from 19 populations in Germany. My thesis work revealed that the resistance mutation in Vkorc1 likely is under balancing selection and was recently introduced to the rat population in our study area. A key innovation of my thesis is adopting a NetGWAS approach to prioritize true associations and conducting co-expression network analysis to detect expression changes related to warfarin. My work shows that additional candidate genes are connected to the vitamin K pathway of which Vkorc1 is an essential component. While the validation of identified genes remains a challenge, the value of my thesis for future investigation is shown: one candidate gene Calu (Calumenin) is associated with warfarin resistance in multiple populations and is an essential part of the vitamin K cycle. Finally, my thesis briefly examines the genetics underlying a newly postulated cost of resistance, arterial calcification. This dissertation provides us an innovative framework in which we learned the genetic architecture of an adaptive trait in multiple dimensions: nucleotide or expression variation, genomic distribution and gene-gene interactions.

Infantile exposure to xenobiotics, e.g. from breastfeeding, poses a serious toxicity risk. Since the toxicokinetic mechanisms that principally determine exposure outcomes undergo a significant developmental maturation, infants may respond to exposures in a different way than adults. Hence, suitable model systems are required to provide risk relevant information in pediatric populations. This dissertations primary goal was to provide a critical evaluation of two such model systems; first, a pharmacokinetic model that may predict an infants capacity to eliminate toxicants by cytochrome P-450 (CYP) mechanisms and second, the developing rat as a model of human CYP2E1 and CYP1A2 ontogeny.<p>The first objective was to evaluate underlying assumptions of a pharmacokinetic model that describes the ontogeny of hepatic CYP activity using the rat. The study recognized some discrepancies with the stated assumptions. The impact of these discrepancies on the potential applicability of the model is discussed. As proof-of-concept, the observed data were fit to a model describing rat CYP2E1 and CYP1A2 ontogeny. A reasonable correlation (r = 0.75) was observed between observed and predicted oral clearance values of a CYP2E1 substrate indicating the potential applicability of such a model in risk assessment. <p>The second objective was to conduct an extensive characterization of rat hepatic CYP2E1 and CYP1A2 ontogeny at mRNA, protein, activity and intrahepatic expression levels. The results were compared to available human data to determine the appropriateness of the rat for assessment of toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity. Similarities in age-dependent changes in mRNA, activity and zonal hepatic expression patterns were noted between the rat and human prior to weaning. Unlike human data, rats show good correlation between changes in CYP2E1 and CYP1A2 activity and transcript levels, but not with the immunoquantifiable protein. Recognizing such similarities and differences between rats and human regarding onset, rate and pattern of CYP ontogeny will improve the accuracy of rat-to-human extrapolation of developmental toxicokinetic data. <p>Overall, the dissertation research provides mounting and supportive evidence for the use of such model systems in providing risk-relevant information in pediatric populations and to identify toxicokinetic mechanisms underlying age-dependent differences in susceptibility to toxicity.

The objectives of this research were to elucidate the involvement of constrictor prostanoids in the vascular reactivity to vasopressin (VP) and the role of estrogen in the regulation of the constrictor prostanoid mechanism in the female rat. Aortas obtained from male, intact (InT)-, ovariectomized (OvX)- and OvX + estrogen-replaced (OvX+Est)-female rats were studied. Contractile responses to VP were examined in the presence of nonselective and selective cyclooxygenase (COX) inhibitors. Basal and VP-stimulated release of thromboxane A2 (TxA2) and prostacyclin (PGI2) from the aortic wall were measured. Concentration-response curves to exogenous TxA2 were also obtained. To elucidate the regulatory effects of estrogen on the constrictor prostanoid pathway, the expression of COX-1, COX-2, thromboxane synthase (TxS) and thromboxane receptor (TP) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Further, immunohistochemistry was employed to determine COX-1, COX-2 and TxS protein expression in aortic endothelium and vascular smooth muscle. The major findings of this research are that: 1) The contractile responses of the female rat aorta to VP were enhanced by COX-2-mediated production of constrictor prostanoids (PGH2/TxA2), and this mechanism is potentiated by estrogen; 2) Vascular reactivity to exogenous TxA2 was higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored vascular reactivity to TxA2 in the female aorta; 3) VP-stimulated release of endogenous TxA2 and PGI2 were higher in the female than in the male rat aorta, and OvX attenuated and estrogen replacement therapy restored VP-stimulated release of these endogenous prostanoids by the female aorta; and 4) The expression of COX-2 and TxS mRNA and protein, and the expression of TP mRNA were higher in InT-female than in male, and were reduced by OvX and restored by estrogen replacement therapy. In conclusion, estrogen potentiated contractile responses of the female rat aorta to VP by upregulating the expression of COX-2, TxS and TP; thereby enhancing VP-induced release of TxA2, as well as the vascular reactivity to endogenous TxA2.

In order to study the expression of MMP2, MMP 3 and MMP9 in breast cancer brain and lung metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain and lung. Immunohistochemistry and Western blotting analyses showed that MMP 2, -3 and -9 protein expression is consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. Lung metastases express abundant MMP2, -3 and -9 in neoplastic cell cytoplasm. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed an increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain and lung metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. TIMP2 overexpression also decreased the development of breast cancer lung metastasis in our model. Our results suggest that MMP2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain and lung. Because astrocytes have been associated with breast cancer brain metastasis we evaluated the role of astrocytes and ERK2 pathway in MMP2 up-regulation in BC brain metastasis. A significant decrease in brain metastases development, and orthotopic tumor size and weight were observed in animals inoculated with ENU1564-TIMP2 cells. These were associated with decreased MMP2 activity, as demonstrated by gel zymography. Rat astrocyte-conditioned media increased expression of MMP2 in ENU15645 cells and increased in vitro cell invasion of ENU1564 and ENU1564-TIMP2 cells. Blockage of ERK1/2 phosphorylation by treatment with PD98059 decreased the expression of MMP2 in cancer cells grown in rat astrocyte-conditioned media. We determine that MMP2 plays a role in in vivo development of breast cancer brain metastases. Additionally, we conclude that astrocytes are associated with expression of MMP2 in cancer cells via ERK1/2 signaling pathway.

The effects of high levels of radiation are frequently studied, but the effects of very lowdose irradiation are still unknown even in today?s technology-driven world. A study recently carried out at Texas A

The conductance catheter technique is commonly used in research to assess cardiac hemodynamics through measurement of ventricular pressure and volume. In order to perform chronic cardiac studies in murine rodents, a small low-power device capable of performing these measurements is necessary. This thesis presents the design, implementation, and test of such a device, coupled with a radio that allows for the telemetry to be transmitted to a base station. Multiple low-power design techniques were employed in this device, which is surgically embedded in the animal. The total mass of the device with battery is 4 grams, and the device volume is 10cm3. Results show that it is capable of periodic monitoring of pressure volume loops for up to 60 days on a single charge. / text