Browsing by Author "Philip L. Sannes, Committee Member"

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Antiandrogens and Development of the Male Rat Reproductive Tract
(2004-02-18) Barlow, Norman James; C. Lee Robinette, Committee Member; Paul M.D. Foster, Committee Co-Chair; Talmage T. Brown, Committee Co-Chair; Philip L. Sannes, Committee Member
Di(n-butyl) phthalate (DBP) is an antiandrogen with known human exposure. The objectives of this thesis were to investigate the development of male reproductive tract malformations secondary to in utero DBP exposure from the fetus to the adult, to characterize the effects of DBP on fetal testicular gene expression for the steroidogenic enzymes, and to further explore DBP's potential for inducing Leydig cell adenomas following gestational exposure. In utero DBP exposure led to a characteristic set of fetal testicular lesions including large aggregates of fetal Leydig cells, multinucleated gonocytes, and increased numbers of gonocytes. In addition to the testicular effects, DBP also caused maldevelopment of the epididymides. During the early postnatal period the fetal testicular lesions became less apparent while decreased numbers of spermatocytes were observed. Underdeveloped epididymides noted in fetuses remained small or failed to fully develop resulting in epididymides with missing components. Malformed epididymides were fully manifest in the adult with absent portions observed both unilaterally and bilaterally. Testicular atrophy with loss of spermatocytes became more severe as gestationally exposed animals matured. Gene expression for the steroidogenic enzymes was examined in testes exposed to DBP in utero. Gene expression was decreased for P450 side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase, and P450c17; while mRNA expression for 17beta-hydroxysteroid dehydrogenase, which catalyzes the final step in testosterone biosynthesis, was not altered. In utero exposure to DBP failed to induce an increased incidence of classical Leydig cell adenomas. However, a dysgenetic lesion composed of numerous poorly differentiated Leydig cells surrounding immature seminiferous tubules was identified. Testicular dysgenesis was observed with a similar incidence between age groups in mature rats, which supports in utero induction by DBP rather than development over time. Together these data provide insight into the molecular mechanisms underlying the induction of DBP-initiated male reproductive tract malformations.
Characterization of Agrin Function in Chicken and Zebrafish Embryogenesis.
(2005-07-19) Kim, Min Jung; Robert R.H. Anholt, Committee Member; Brenda J. Brizuela, Committee Member; Philip L. Sannes, Committee Member; Gregory J. Cole, Committee Chair
Agrin is an extracellular matrix heparan sulfate proteoglycan that plays a key role in the development of the neuromuscular junction (NMJ) by inducing the clustering of acetylcholine receptors at synaptic sites of the NMJ. Although recent studies have extended our understanding of agrin's function in the nervous system, its function in the CNS is not clearly understood. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the basic fibroblast growth factor (FGF-2), using both PC12 cells, and chick retina neuronal cultures. Agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. Agrin augments and sustains a transient early phosphorylation of ERK (extracellular signal-regulated protein kinase) in the presence of FGF-2. Neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2. To overcome the lethality of agrin gene disruption and the difficulty of embryonic manipulation of agrin function in mice, a gene encoding zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS, the NMJ, and non-neural structures such as the pronephric duct, and endodermal tissues. A morpholino-based gene targeting against agrin significantly impair development of tail and the NMJ, and cause severe defects in motor neuron axon outgrowth and formation of the midbrain-hindbrain boundary, eye, and otic vesicles. Morphants subsequently develop paralysis, and die at larvae stages. Knockdown of agrin in zebrafish strikingly resembles phenotypes of zebrafish FGF-related mutants, such as disruption of the MHB, optic and otic vesicles during zebrafish development. Inhibition of FGFR synergizes defects from agrin knockdown resulting in MHB disruption, a shortened tail, small eyes and otic vesicles, which suggest that agrin modulates the activity of FGF signaling pathways. In conclusion, my studies show that agrin is essential for NMJ formation as well as sensory and motor neuron axonal growth and pathway formation in zebrafish development. Importantly, the HSPG agrin may be involved in regulation of early CNS development via maintenance and regulation of FGF signaling.
Evaluation of tumor hypoxia and proliferation in canine spontaneous solid tumors
(2004-07-04) Azuma, Chieko; Philip L. Sannes, Committee Member; James A. Raleigh, Committee Member; Marlene L. Hauck, Committee Member; Donald E. Thrall, Committee Chair
Tumor hypoxia and proliferation have been shown to be related to malignant tumor progression and treatment resistance. Studying tumor hypoxia and proliferation is important to address the biology of tumors and problems which may limit successful therapy. Tumor oxygenation is directly affected by oxygen consumption of tumors. Oxygen consumption is increased in actively proliferating cells. In this study the primary aim was to study the relationship between tumor hypoxia and proliferation in canine spontaneous solid tumors using immunohistochemical methods. Hypoxia was determined by detecting exsogenous hypoxia marker, pimonidazole adducts and proliferation was determined by the fraction of cells labeled with proliferating cell nuclear antigen. We hypothesized that hypoxia may be more pronounced in rapidly proliferating tumors due to increased oxygen consumption. Pimonidazole adduct formation occurred within 20 minutes after intravenous administration and cells with adducts were stable for several days in canine spontaneous tumors. Rapid and stable adduct formation in vivo are the important characteristics of using pimonidazole as a clinically useful hypoxia marker. There was no association between tumor hypoxia and proliferation evaluated by immunohistochemistry. The clinical outcome of the dogs studied in the project is unknown at this time, but these results to date suggest that tumor hypoxia and proliferation measurements are independent and may be potentially complementary predictive factors in canine spontaneous tumors. Classic diffusion limited hypoxia may be more related to intensive areas of pimonidazole labeling and transient perfusion limited hypoxia may not have been detected with our technique. Perfusion limited hypoxia being related to proliferation might be more complex because the areas of hypoxia are transient and such areas still might be actively proliferating under the transient hypoxic conditions. Methods need to be developed to differentiate acute versus chronic hypoxia to know the true hypoxic fractions and their effects on tumor biology. The type of cells may affect estimates of hypoxia by pimonidazole labeling and it is necessary to evaluate histologic features before designing a study. The hypoxia marker pimonidazole is a powerful tool that can be used in a clinical setting to study tumor biology with minimum invasiveness.
Heparan Sulfate Proteoglycan Agrin in CNS Development and Aging
(2008-04-06) Liu, I-Hsuan; Philip L. Sannes, Committee Member; Jane L. Lubischer, Committee Member; Jeffrey A. Yoder, Committee Member; Robert R. H. Anholt, Committee Chair
Agrin is an extracellular matrix heparan sulfate proteoglycan and is best known for its role as the organizer of the neuromuscular junction. Emerging in vitro evidence shows that it plays many possible roles in the central nervous system. The studies present in the Chapter 1 examine the hypothesis that agrin also plays a role in Parkinson's disease. In support of the hypothesis, this study demonstrates that agrin binds to α-synuclein via its heparan sulfate glycosaminoglycans chains, potentiates conformational changes in α-synuclein into β-sheet structure, and enhances insolubility of α-synuclein. Furthermore, agrin is also found colocalized with α-synuclein in neuronal Lewy bodies in the substantia nigra of Parkinson's disease human brain. These results suggest that agrin is capable of potentiating the formation of α-synuclein amyloidosis in Parkinson's disease brain and may indicate shared molecular mechanisms leading to the pathophysiology in Alzheimer's disease and Parkinson's disease, two most common neurodegenerative diseases. Zebrafish has been an attractive animal model for neural development. To begin to address agrin's function in central nervous system, a study on agrin knockdown in zebrafish is present in Chapter 2. Agrin mRNA is detected as a maternal message in embryonic zebrafish, and is expressed in the developing central nervous system and in nonneural structures such as somites and notochord. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon–Beard sensory neurons are also observed, which included truncation of axons and increased branching of motor axons, suggesting roles of agrin in both axon outgrowth and guidance. Moreover, agrin morphants exhibit significantly inhibited tail development, as well as defects in the formation of the midbrain–hindbrain boundary and reduced size of eyes and otic vesicles. These results show that agrin plays an important role in the development of both peripheral and central nervous system in zebrafish. Among the phenotypes that result from agrin knockdown using morpholino antisense oligonucleotides is reduced eye size in agrin morphants. The studies presented in Chapter 3 show that retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted in a dose-dependent manner following agrin morpholino treatment. Pax6.1 gene expression, a marker of eye development, is markedly reduced in agrin morphants, providing support for agrin's role in retinal development. Increased apoptosis is detected suggesting the reason for microphthalmia. Both pax2a and atoh7 gene expressions are decreased suggesting that both FGF8 and sonic hedgehog signaling are affected. Confocal micrographs of HuC-GFP transgenic zebrafish and immunostaining showed impaired differentiation and disorganization in all three cellular layers in the retina in agrin morphants. MHB formation, and expression of mRNAs in this organizer region, is disrupted in agrin morphants. The retinotectal topographic projection to the optic tectum is also perturbed in agrin morphants suggesting the role of agrin for FGF8 signaling. Collectively, these phenotypes in agrin morphants provide support for a crucial role of agrin in retinal development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.
Molecular mechanisms of etomoxir-induced toxicity
(2002-12-16) Merrill, Christine Lee; Richard T. Miller, Committee Member; Talmage T. Brown, Committee Co-Chair; Kevin T. Morgan, Committee Co-Chair; Tony R. Fox, Committee Member; Philip L. Sannes, Committee Member
Etomoxir (ET) is a member of a family of substituted 2-oxirane-carboxylic acids that inhibit mitochondrial long-chain fatty acid beta-oxidation (FAO), ketogenesis and gluconeogenesis. Once converted to its CoA ester, ET irreversibly binds to the CPT-1 catalytic site and prevents long chain fatty acids from entering the mitochondrion. Along with this inhibition of FAO, ET causes a shift in energy substrate utilization from fatty acids to glucose, leading to systemic hypoglycemia, hypoketonemia, and hypotriglyceridemia. These effects make ET potentially useful in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). The compound has been shown to induce cardiac and hepatic hypertrophy in animals and, therefore, has not been fully developed as an antidiabetic agent to date. It is well established that ET activates the peroxisome proliferator activated receptor-alpha (PPARα) which can cause both oxidative stress and dysregulation of the cell cycle control gene program. The goal of this research was to evaluate the ET-induced alterations in gene expression profiles in hepatocytes to elucidate the possible role of cell growth dysregulation and/or oxidative stress in ET-induced hepatic toxicity. In HepG2 cells treated with a high dose of ET, gene expression strongly suggestive of oxidative stress was observed and this was supported by decreased levels of reduced glutathione, reduced/oxidized glutathione ratio (GSH/GSSG), concurrent increase in oxidized glutathione (GSSG) and superoxide generation. A significant decrease in mitochondrial membrane potential and ATP levels implicated impairment of mitochondrial energy metabolism. Other gene expression findings suggested activation of p53, DNA repair and cell cycle arrest. In rats, ET induced a strong mitogenic response in the livers of rats 24 h after administration of one 25 mg/kg dose, that was consistent with the cell proliferation caused by peroxisome proliferators. This finding was coincident with a predominance of cell proliferation/growth-related gene expression alterations. Oxidative stress genes were down regulated; suggesting that this is not a viable etiologic mechanism for induction of ET-induced hepatic hypertrophy. PPARα appears to play a role in ET-induced hepatic hypertrophy, as shown by the early cell proliferation followed by increased level of PPARA mRNA, peroxisome proliferation and increase of PPARα-related genes.