Thesis (M.S. )--University of Tennessee Health Science Center, 2008 / Title from title page screen (viewed on Sept. 17, 2008). Research advisor: Lorraine M. Albritton, Ph.D. Document formatted into pages (x, 138 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 40-48).

Parathyroid hormone is a vital mediator of bone metabolism and studies have shown that exogenous treatment can enhance the fracture repair process in murine models. Bone remodeling is a complex process that necessitates multiple molecular and cellular interactions that are affected by genetic variations. These differences contribute to both histological and whole organ level differences of fracture healing. This study was performed to determine the effect of genetic variability of fracture healing in mice treated with parathyroid hormone during two time windows. The first window was the first 14-day period post fracture associated with chondrogensis and the second was the day 15 to day 28 post fracture, which is associated with osteogenesis. Three inbred strains of mice A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3) that have material and structural differences in bone quality were given Femoral shaft fractures and healing was evaluated at different time points post fracture using quantitative real-time polymerase chain reaction (qRT-PCR) and qualitative radiographic analysis. Chondrogenic genes Sox9, ColIIa, aggrecan, and ColXa and osteogenic genes ostrix, osteocalcin, BSP, and DMP1 were examined. The temporal analysis of mRNA expression revealed that PTH treatment given in the first 14 days post fracture enhanced osteogenic and chondrogenic expression in B6 mice, but hindered expression in AJ mice. Treatment with PTH from post fracture day 14 to day 28 greatly affected the osteogenic expression of B6 mice, but had little affect on other animals. Radiographic analysis showed that each strain presents callus formation at approximately day 7 and reaches maximum size at day 21 post fracture. Additionally B6 mice appear with the largest callus and AJ the smallest. Taken together, these results are consistent with past studies in showing that different strains of mice express a unique temporal and mRNA expression pattern of chondrogenic and osteogenic differentiation. Furthermore, these variations affect the biomechanical properties of the fracture callus during bone remodeling.

Genome wide association studies (GWAS) have transformed the study of the heritability of complex diseases such as type 2 diabetes (T2D), with the current tally of established risk loci close to ninety. Each of these loci has the potential to offer novel insights into the biology of this disease, and opportunities for clinical exploitation. However, the complexity of T2D has often frustrated efforts to achieve these functional and translational advances. This thesis aims to delve into the functional characterisation of two known susceptibility loci, KLF14 and ADCY5, and describe findings relevant to disease pathology. KLF14 and ADCY5 are two loci associated with T2D predisposition working through disparate mechanisms. Variants at the maternally imprinted KLF14 locus are associated with measures of insulin resistance and expression data has implicated KLF14 as a master regulator of genes in adipose tissue. In contrast, variation at the ADCY5 locus is associated with impaired beta cell function, high fasting glucose, and low birth weight suggesting ADCY5 is having an effect on insulin secretion. In this thesis, ENU mouse models of these two genes are investigated functionally to elucidate more about the pathology of common human variation at these loci. A mouse model was derived with an ENU point mutation at Adcy5 Y1064C. Phenotyping of this model revealed improved oral glucose tolerance, and secretion studies from isolated islet cells demonstrated impaired glucagon secretion from mice homozygous for the Y1064C mutation in the presence of adrenaline. These results suggest that Adcy5 is involved in glucagon regulation in the alpha cell. The Adcy5 Y1064C confers a protective effect against hyperglycaemia in mouse indicating that the T2D risk allele at the ADCY5 locus in humans may have the opposite direction of effect. A mouse model containing the ENU point mutation Klf14 R238L predicted to be disruptive to KLF14 protein function showed no significant difference in body weight, measures of insulin resistance, or blood cholesterol. However, expression of several genes associated in trans with variation near KLF14 in humans was changed in adipose tissue and skeletal muscle when the R238L mutation was inherited maternally compared to mice which had inherited the mutation paternally or carried two wild type alleles. This result suggests a mechanism by which Klf14 is regulating genes across metabolic tissues.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Murine hepatitis virus type 3 (MHV-3), a member of the coronavirus family, induces a response that varies with the age and genetic background of the host mouse strain. A/J mice are fully resistant to the virus, while Balbc/J are fully susceptible and C3HebFe/J are semi-susceptible, making it possible to predictably reproduce the major human responses to hepatitis viruses. Although there has been considerable discussion of viral pathology in the literature, there has been much less emphasis on pathogenesis. In the experiments described here, histological, biophysical, and immunological techniques have been used to define the processes and cells involved. Transmission electron microscopic observations have confirmed that Kupffer and endothelial cells of hepatic sinusoids show clear changes by 12 hrs post-infection (p.i.), which are more advanced than hepatocellular changes. No replicating virus was seen in altered hepatocytes up to 3 days p.i. Scanning electron microscopy demonstrated that areas of necrosis are focal in nature and at 2-3 days p.i. consist of small spherical areas without flow. In vivo microcirculatory studies confirm the localized nature of the lesion and have shown that red cell velocity can be recorded in individual sinusoids . Velocities were found to vary from zero within a lesion to a normal velocity of 69±31 um/sec over a distance of not more than 3 sinusoids. In-vivo microcirculatory studies also revealed the ability of macrophages to move upstream (against flow) in the hepatic sinusoids. Using fluorescein labelled antibodies to cell surface markers (Thy-1, Lyt-2, and L3T4) it was shown that no T-cells of any subset were present in the areas of hepatocellular necrosis. Furthermore, treatment with cyclosporine A, which would be expected to decrease necrosis due to cell mediated cytotoxicity, did not significantly alter the course of the disease. The only cells which increased in number in the liver post infection were cells of the monocyte/macrophage lineage (Mac 1+), which had increased twofold at 12 hrs (p<.025) p.i. and to greater than twenty fold (p<.005) by 3 days p.i. Resistance in the A/J strain did not reflect an inability of the immunocompetent cells to present and respond to viral antigen. It was demonstrated that MHV-3 infected macrophages from resistant A/J mice are better able to stimulate proliferation of allogeneic and syngeneic lymphocytes than those from the sensitive Balb/cJ strain. In contrast, MHV-3 infection caused a significant enhancement of chemiluminescence from Balb/cJ macrophages, which did not occur in A/J animals. In vivo studies demonstrated a significant increase in free radical reaction products, including conjugated dienes (of long chain free fatty acids and aldehydes), thiobarbituric acid reactive substances, and lipid soluble fluorescent products between 12-72 hours p.i. with MHV-3 in the livers of susceptible Balb/cJ strain mice. All of these are products of oxidative cleavage of cellular and membrane polyunsaturated fatty acids, and result from the action of oxygen free radicals. Free radical inhibitors, or quenchers of free radical reaction products, were able to significantly reduce the liver necrosis in the susceptible mouse strain following infection. Radioimmune assays for antibody to MHV-3 have confirmed the presence of preformed antibodies to (or cross-reactive with) MHV-3 in the sera of both susceptible and resistant mice, pre and post-infection. Immunofluorescent labelled antibodies have also been used to demonstrate the presence of IgG deposits in the sinusoids of the liver both pre and post infection. This suggests the possibility that these mice have been infected with a non-virulent MHV strain prior to these experiments. From these studies, we conclude that the hepatic injury caused by MHV-3 infction in Balb/cJ mice is mediated predominantly by fixed and migratory cells of the mononuclear phagocytic series. Susceptibility and resistance are related to strain dependant differences in the response of macrophages (and Kupffer cells) to infection, and include the release of procoagulant activity (previously shown) and reactive oxygen radicals (and possibly other macrophage activation products such as PAF) that act together to induce hepatocellular necrosis. Preformed non-neutralizing antibody and an intact complement cascade may enhance viral uptake and activation of macrophages in the Balbc/J mice. Resistance to necrosis may be enhanced by a genetic deficiency of C5 in the A/J mice, preventing the formation of the membrane attack complex and hence complement dependant cell lysis, or macrophage activation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The choroid plexuses are largely responsible for cerebrospinal fluid (CSF) secretion and therefore play a fundamental role in brain homeostasis. The membrane proteins involved in CSF secretion are not fully known. Several electroneutral transporters have been identified by molecular methods in choroid plexus epithelial cells but there is a lack of functional data to support their expression making it impossible to elucidate their role in CSF secretion fully. The activity of many of these transporters can be observed in cell volume regulation. Thus, the main aim of the present study was to determine the ability of mammalian choroid plexus epithelial cells to regulate their volume in response to anisosmotic challenge and to investigate the transporters involved.Experiments were performed on cells isolated from the mouse fourth ventricle choroid plexus. Cells were isolated using a combination of manual perturbation, the enzyme dispase and a Ca2+ free incubation to disrupt tight junctions. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pL.Cells exhibited a HCO3- dependent regulatory volume increase (RVI) in response to hypertonic challenge. Strong evidence is presented that the Na+/H+ exchanger (NHE1) and the Cl-/HCO3- exchanger (AE2) contribute to the RVI but the Na+K+2Cl- cotransporter (NKCC1) and the epithelial Na+ channel (ENaC) do not. Choroid plexus cells exhibit a HCO3- dependent regulatory volume decrease (RVD) in response to hypotonic challenge. The RVD was unaffected by DIOA (an inhibitor of KCC activity), the K+ channel inhibitors TEA+, Ba2+ or 4AP or the Cl- channel inhibitors DIDS or NPPB. However removal of extracellular Ca2+ completely abolished cell swelling in response to hypotonic challenge. This sensitivity of volume change to Ca2+ was specific to cell swelling as cell shrinkage in hypertonic artificial CSF was unaffected by removal of extracellular Ca2+.Thus functional evidence is presented to further elucidate the role of several proteins in the choroid plexus cell volume regulatory response to anisosmotic challenge.

Methotrexate, a folic acid antagonist, has been used in the clinical treatment of a wide variety of malignant neoplasms for over 20 years, either as a single agent or in combination with other antineoplastic agents. It is a cell cycle specific inhibitor and kills cells only in the S phase of growth. MTX is a potent inhibitor of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3.), which catalyses the NADPH dependent reduction of dihydrofolic acid and folic acid to tetrahydrofolic acid: the metabolically active coenzyme form of folic acid essential in the biosynthesis of dTMP from dUMP by thymidylate synthetase. Inhibition of DHFR therefore leads to the inhibition of DNA synthesis and cell death. Methotrexate has many favourable properties; for instance, it interacts directly with intracellular sites without the need for prior metabolic transformation. It can be administered in large doses because toxicity to normal cells can be minimized by the administration of folinic acid (N5 formyl tetrahydrofolic acid) shortly after the administration of MTX. However, the effectiveness of MTX is inevitably compromised by the emergence of drug resistance, which can be either intrinsic, i.e. the tumour cells are resistant to MTX at the outset, or the tumour cells acquire resistance after exposure to MTX. An understanding of the mechanisms of resistance to MTX is therefore very important if treatment with this potent antineoplastic agent is to be improved. Three mechanisms of resistance to MTX have been determined from studies with experimental tumour systems: impaired uptake of MTX; increased levels of dihydrofolate reductase; and appearance of altered dihydrofolate reductase with a lower affinity for MTX. Impaired uptake of MTX and increased levels of OHFR can both theoretically be overcome by sustaining increased concentrations of free intracellular MTX. This can be achieved by exposing the cells to higher concentrations of MTX, and many chemotherapeutic regimens now use 'high-dose' MTX which can achieve plasma concentrations of MTX as high as 10⁻³M. However, resistance to MTX is still a major clinical problem and the use of 'high-dose' MTX has not significantly increased the therapeutic index of MTX treatment. Appearance of DHFR with a lower affinity for MTX suggests as an alternative the synthesis of an agent which would be a potent inhibitor of the altered enzyme, and requires the detailed characterization of the properties of this enzyme. If the altered enzyme retains some affinity for MTX, the administration of MTX and the more potent agent would result in better growth inhibition of the resistant tumour. In this thesis, a mouse leukemia cell line (L5178Y) grown in suspension culture was used to isolate two MTX-resistant cell lines and these were used to study the mechanisms leading to MTX resistance. Both resistant cell lines exhibited impaired MTX uptake when exposed to 10⁻⁶M MTX but not when exposed to 10⁻⁴M MTX, Both lines also had elevated DHFR levels (7 to 9 fold). A variant form of DHFR present in small amounts in both cell lines was isolated by MTX-sepharose affinity chromatography. The altered DHFR differed from the major form of reductase present in these cells in its markedly lower affinity (100,000 fold) for MTX. The two forms of the enzyme were purified from the most resistant cell line and their properties compared. They were found to differ moderately in their Km for substrates, however, the Ki of MTX differed by a factor of 100,000 for the two forms. In addition there were marked differences in their heat stability, isoelectric points and sensitivity to p-chloromercuriphenyl-sulphonate, and a minor difference in their molecular weights. It is concluded that the presence of a highly resistant form of DHFR in these cell lines represents an important mechanism in conferring a high degree of resistance to these cells. The importance of this form of DHFR in MTX resistance is discussed in relation to impaired transport and elevated DHFR levels. Experiments to determine the amino acid sequence of the altered enzyme are underway and once determined should facilitate the synthesis of specific inhibitors of its activity. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22331号 / 医博第4572号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 長尾 美紀, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Department of Microbiology / PhD (Microbiology)

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