Upon aging, the intervertebral disc (IVD) inevitably undergoes degeneration characterized by biochemical and morphologic changes. IVD degeneration can lead to multiple clinical disorders such as back and neck pain, and myelopathy. Low back pain can disable up to 85% of the adult population and results in a significant restriction of social activities and inability to work. Such disorder incurs billions of dollars in medical expenditures each year. Despite advances in the detection and treatment of the degeneration, the regeneration of the IVD remains low because current therapies are limited by exogenous curing approaches. New strategies for the reversal of IVD degeneration, including gene, cytokine, and stem cell therapies that can influence the anabolic and catabolic pathways in disc cells have been under investigation. These therapies aim to rejuvenate or replace diminished nucleus pulposus cells in the degenerative IVD. Recent reports have put forth a proposal of “endogenous disc stem cells”, suggesting that cells derived from the degenerative IVD tissue possess stem cell properties. These putative stem cells are believed to regulate the development and homeostasis of the IVD tissue. In this study, we identified and characterized a stem cell population from the IVD of healthy Rhesus monkey, termed disc stem/progenitor cells (DSCs). We show that the DSCs possess clonogenicity, multipotency and self-renewal capacity. The DSCs are phenotypically similar to bone marrow mesenchymal stem cells (BMSCs) but they are not identical. The DSCs show a faster growth rate under hypoxia than normoxia. DSCs derived from nucleus pulposus (DSCNP) show a stable expression level of hypoxia inducible factor-1 alpha (Hif-1ａ) in response to hypoxia. DSCs derived from annulus fibrosus (DSCAF) are more resistant to apoptosis under hypoxia than DSCNP. More importantly, small leucine-rich proteoglycans (SLRPs) are identified as important DSC niche components. We show that biglycan (bgn) and decorin (dcn) reduce the susceptibility of DSCs to hypoxia-induced apoptosis by promoting the expression of hypoxia inducible factors (HIFs). Our findings suggest that DSCs rely on the unique niche components for survival. In summary, our findings propose the existence of endogenous stem cells in IVD. Further study of the DSCs may provide new insights into the biology of IVD and facilitate the design of new strategies to treat disc degeneration in future. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Benefiting from higher SNR as well as better spatial, temporal and spectral resolution, magnetic resonance imaging (MRI) at high field has proved to be a valuable neuroimaging modality which provides comprehensive evaluation of the central nervous system non-invasively. The objectives of this doctoral work were to develop MRI methodologies and to assess the functional, metabolic and structural alterations in rodent brains under normal and manipulated conditions. Firstly, to improve the functional sensitivity and spatial precision, a novel functional MRI (fMRI) method using balanced steady state free precession with intravascular susceptibility contrast agent was proposed and its feasibility was evaluated in rat visual system. This new approach was sensitized to cerebral blood volume (CBV) changes. It provided comparable sensitivity to conventional CBVweighted fMRI using echo planar imaging but with no severe image distortion and signal dropout. Robust negative responses during visual stimulation were observed and activation patterns were in excellent agreement with known neuroanatomy. As a promising alternative to conventional CBV-weighted fMRI, it was particularly suited for fMRI investigation of animal models at high field. Secondly, the relationship between anatomical connections and resting-state fMRI connectivity was explored using a well-controlled animal model of corpus callosotomy. Both complete and partial callosotomy resulted in significant loss of interhemispheric connectivity in the cortical areas whose primary interhemispheric connections via corpus callosum (CC) were severed. Partial restoration of interhemispheric connectivity and increased intrahemispheric connectivity were also observed. The experimental findings directly supported that anatomical connections via CC play a primary and indispensable role in resting-state connectivity, and that resting-state networks could be dynamically reorganized or acquired directly or indirectly through the remaining anatomical connections. Thirdly, proton magnetic resonance spectroscopy (1H MRS) was employed to monitor the longitudinal metabolic alterations elicited by exogenous stimulation and endogenous modification, respectively. Significantly lower hippocampal N-acetylaspartate (NAA) was observed in fear conditioning animals, indicating reduced neuronal dysfunction and/or integrity, which contributed to the trauma-related symptoms. Meanwhile, pregnant animals exhibited prominently higher hippocampal NAA level, reflecting the increased density of neurons in this region, which might facilitate supporting behaviors that involving learning and memory. The 1H MRS detection of ongoing neurochemical changes induced by fear conditioning and pregnancy, especially in the hippocampus, can shed light on the mechanisms of learning and memory and the neurochemical underpinnings of behavioral improvement in pregnant animals. Lastly, manganese-enhanced MRI (MEMRI) was employed to investigate the hypoxic-ischemic (HI) injury in the late phase and the neural response to conditioned fear. Significantly higher enhancement in T1-weighted images was found in the peri-lesional region 24 hours after manganese administration and it colocalized with the increase in glial cell density in histological staining, demonstrating the existence of reactive gliosis in the late phase after HI injury. In fear conditioned animals, higher manganese uptake was observed in amygdala, hippocampus, paraventricular nucleus of hypothalamus and cingulate cortex, which were all highly-involved in the process of fear. These findings suggested MEMRI approach were useful in investigation of post-injury cellular events and functional reorganization as well as for in vivo mapping of neuronal activity. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

　　Vad1.3 is an evolutionarily-conserved, testis-specific gene identified from a retinol-treated Vitamin A-deficiency (VAD) rat model. VAD1.3 is expressed throughout spermiogenesis at the acrosome of spermatids and epididymal spermatozoa, suggesting a role in acrosome biogenesis or acrosome reaction. The present study aimed to explore the functional role of VAD1.3 in spermatogenesis and sperm functions by the cellular and gene-knockout approaches. 　　Double immunofluorescent microscopy confirmed the co-localization of VAD1.3 and syntaxin 1 in mouse spermatids and spermatozoa. Deletion analysis of the Vad1.3 gene in transfected mouse spermatocyte GC2-spd and human cervical cancer HeLa cells revealed a polarized peri-nuclear/Golgi expression pattern for the N-terminal GFP-VAD fusion proteins which contain a bipartite nucleus localization (BNL) motif, but a nuclear expression pattern for the C-terminal GFP-VAD. The N-terminal sequences of VAD1.3 mediated its interaction with syntaxin 1, as demonstrated by both co-localization and co-immunoprecipitation studies. The full-length GFP-VAD co-localized with the Golgi markers and was redistributed into the endoplasmic reticulum after brefeldin A treatment, suggesting that VAD1.3 was recruited through the ER-Golgi-acrosome pathway. 　　Vad1.3+/- mice was previously generated by the conventional knockout approach. The heterozygous mice had normal spermatogenesis during postnatal days and adulthood (6-8 weeks). At the age of 8-19 months, 6 out of 17 heterozygous mice but no wild-type exhibited a decrease in the epididymal sperm count and testicular weight (p < 0.05). Histological analyses unveiled disarrangement of the seminiferous epithelium and sloughing of germ cells, predominantly spermatids, which was mediated partially by apoptosis as a higher percentage of TUNEL-positive cells were detected in these heterozygous mice (p < 0.05). This phenotype was associated with a decrease in the mRNA (p < 0.05) and protein levels of VAD1.3 in the testis. 　　Crossing of the Vad1.3+/- mice produced wild-type and heterozygous offspring in a ratio of 1:3, but no Vad1.3-/- mice were found. There was no significant difference between the heterozygous intercrosses and the wild-type intercrosses in the number of oocytes ovulated, the developmental rate of embryos from zygotes to blastocysts, the number of implantation site, resorption site or the offspring could result from defective fertilization between Vad1.3 null gametes rather than developmental lethality. The role of VAD1.3 in fertilization was supported by the inhibitory effects of the anti-VAD1.3 antibody on in vitro fertilization and progesterone-induced acrosome reaction. Immuno-staining revealed that VAD1.3 was present in the acrosome-intact spermatozoa but not in acrosome-reacted spermatozoa, indicating a role of VAD1.3 in ZP-binding or acrosome reaction rather than sperm-egg fusion. In oocytes VAD1.3 was distributed in the cytoplasm near the cortex. litter size. Only a few Vad1.3-/- embryos were found at the zygotic (3.7%) and 2-cell (3%) stages in the heterozygous intercrosses. These findings suggested that the absence of the Vad1.3-/- 　　In sum, VAD1.3 may play important roles in fertilization and spermatogenesis in mice. The BNL motif of VAD1.3 directs its Golgi expression and the N-terminal sequence of the protein mediates its interaction with syntaxin 1. The use of tissue-specific knockout approach may help to answer the functional role of VAD1.3 in future. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

The endothelium is important for the regulation of vascular tone. In diseases like hypertension, the endothelial cells become dysfunctional. This dysfunction is characterized by nitric oxide (NO) deficiency, impairment of endothelium-dependent hyperpolarization (EDH) and the overwhelming production of endothelium-derived contracting factor (EDCF). Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, producing carbon monoxide(CO), bilirubin and free iron. Up-regulation of the inducible isoform (HO-1) of the enzyme lowers blood pressure in animals. The purpose of the present study was to investigate whether or not up-regulation of HO-1by the pharmacological agent hemin improves endothelial function in arteries of spontaneously hypertensive rats(SHR). Twenty four hours after intraperitoneal injection of hemin (50mg/kg) in 36 weeks old SHR, the expression and activity of HO-1 were augmented, in both the endothelium and vascular smooth muscle. Hemin-treatment potentiated endothelium-dependent relaxations to the muscarinic agonist acetylcholine in both the aorta and the mesenteric artery, whereas the HO inhibitor protoporphyrin IX zinc (II) (ZnPP; 30 mg/kg) prevented the beneficial effect of hemin, suggesting that HO-1 induction improves endothelial function. Hemin-treatment did not augment acetylcholine-induced NO-mediated relaxations, and did not alter the expression level of either phosphorylated eNOS (Ser1177) or total eNOS, suggesting that the improvement of endothelial function by HO-1 induction cannot be attributed to an increased bioavailability of NO. In the mesenteric arteries, hemin treatment potentiated acetylcholine-evoked EDH-mediated relaxations in the presence of L-NAME and indomethacin. The IKCa channel blocker TRAM-34andthe Na+-K+-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34-and ouabain-sensitive relaxations were enhanced by hemin-treatment. K+-induced ouabain-sensitive relaxations and the expression of Na+-K+-ATPase were increased by hemin-treatment. Taken in conjunction, these observations imply that the improved EDH-mediated relaxations by HO-1 induction is due to an improvement of IKCa-Na+-K+-ATPase pathway. Treatment with an antioxidant apocynin (50mg/kg) showed a similar effect as hemin, and the combined treatment with hemin and apocynin did not cause a greater improvement. In vitro treatment with bilirubin, enhanced EDH responses and K+-induced ouabain-sensitive relaxations. These observations suggest that the effect of HO-1 induction on EDH-mediated relaxations is possibly due to its antioxidant properties and the production of bilirubin. In the aortae, hemin-treatment reduced endothelium-dependent contractions in response to acetylcholineor to a calcium ionophoreA23187. Production of reactive oxygen species (ROS) was suppressed by hemin-treatment, judging from the results of 2’,7’-dichlorodihydrofluoresein diacetate staining, dihydroethidium staining and lucigenin chemiluminescence, which was attributed to the decreased expressions of NADPH oxidase-2 (Nox2) and cyclooxygenase-1(COX-1). The production of prostacyclin was decreased, which was explained by a lower expression of COX-1. Contractions to vasoconstrictor concentrations of prostacyclin and its mimetic iloprost were attenuated, suggesting that the responsiveness of thromboxane-prostanoid receptors (TP receptors) to prostacyclin was decreased by hemin-treatment. The effects of HO-1 on the suppressed production of ROS and prostacyclin, and the decreased responsiveness of TP receptors, contribute to its inhibitory role on EDCF-mediated response. Thus, up-regulation of HO-1 improves endothelial function in the SHR by potentiating EDH response and impairing EDCF. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Magnetic Resonance Imaging (MRI) is a non-ionizing imaging modality that can provide images with excellent soft tissue contrast at high resolution. In particular, molecular and cellular MRI is a powerful imaging method that could provide a non-invasive way for assessing specific biological processes in vivo in living organisms. The ability to monitor and track biological structures and processes down to molecular and cellular level and the possibility to probe the development, survival, migration, and differentiation of cells in vivo, has opened up new ways for scientists to investigate the fundamental mechanisms of health and diseases. In this dissertation, novel applications of conventional MR contrast agents to study specific biological structures and processes are demonstrated. First, the potential of manganese enhanced MRI (MEMRI) for in vivo tract tracing and assessment of neuroarchitecture was investigated. Manganese was intracortically infused into the visual cortex along the border of the primary and secondary visual cortex and then imaged 8 and 24 hours later. A dynamic migratory path of manganese from the infusion site through the corpus callosum to the contralateral hemisphere was observed. Also, layer specific enhancement on the contralateral cortex and the connection of the visual cortex with other brain structures were shown and the results were consistent with established anatomical data. Secondly, MEMRI was performed to probe in vivo neuronal changes in the rodent brain following 72-hour rapid eye movement sleep deprivation. Significant reduction in manganese uptake was observed in the cortical and hippocampal region in the sleep deprived animals when compared to the normal group. In particular, the dentate gyrus substructure in the hippocampus exhibited the least uptake. This indicated the functional vulnerability of the hippocampus and the cortex to sleep deprivation. Lastly, in vivo tracking of endogenous neural stem and progenitor cell migration during neurogenesis in neonatal rat brain was performed by micron sized iron oxide particles (MPIO) labeling. Susceptibility weighted imaging was used for image processing to highlight the susceptibility contrast induced by the iron oxide particles. MPIO-labeled cells induced contrast was clearly enhanced in the susceptibility weighted images, particularly at day 3 after MPIO injection in which the MPIO-labeled NPCs became more dispersed in the olfactory bulb. The ventral migratory pathway of endogenous neural stem and progenitor cells, which could not be easily observed in conventional T2*W imaging, couldalsobe detected. Overall, various biological systems and processes have been successfully interrogated using MR contrast agents. Through these studies, the versatility and power of molecular and cellular MRI have been demonstrated. Looking ahead, the rapid development and combination of different molecular and cellular imaging techniques would certainly revolutionize the way we study health and diseases. In the end, this could foster our understanding of basic life sciences and hence improve the quality of healthcare. / published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

Foodborne toxins include a variety of biologically produced toxins and process-induced toxicants. Among them, marine algal toxins in polluted seafood can induce serious harmful effects on human, while heat-induced toxicants remain in a wide range of food and chronically affect health. In this present study, several representative toxins from these two categories were selected and studied, they are, brevetoxins (PbTxs), saxitoxin (STX) and acrolein (ACR). During past decades, the molecular actions of these toxins have been well studied, however, their effects and mechanisms corresponding to their sublethal toxicity in vivo still need more investigation. Therefore, in our current study, we adopted medaka fish (Oryzias melastigma) and zebrafish (Danio rerio) as models to study the toxicological responses and pathways of these selected toxins at the molecular and cellular level. In the first part of study, the targeted organs of medaka fish, gills and brains, were studied in medaka fish after exposure to the sublethal level of PbTx-1, the most potent neurotic shellfish poisoning (NSP) toxin. Facilitated by the two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ ionization tandem time-of-flight mass spectrometry (MALDI TOF/TOF MS), proteins affected by PbTx-1 in these organs were identified, including myosin like proteins, aldose reductase, gelsolin and keratin. Collectively, the proteins altered after exposure suggested the altered calcium ion binding process, and dysfunction in cytoskeleton assembly and metabolism. After successfully applying the fish model and proteomic approaches in the NSP toxin study, we put emphasis on the developmental toxicity of foodborne toxins, as children are more sensitive and vulnerable to foodborne toxins. Among the marine algal toxins, paralytic shellfish poisoning (PSP) toxins are the commonest and most lethal ones with STX as the most potent PSP toxin. Therefore, we examined the developmental effects of STX using medaka model. By exposing medaka embryos from the early blastula stage onwards, which covered the main developmental stage of the central nervous system and somites, we found newly hatched medaka fish exhibit abnormal growth with longer body length and relatively smaller yolk sac size. High cell proliferation, neuron development, and metabolism were confirmed by whole-mount immunostaining and 2-DE. In summary, STX disturbs the normal growth of medaka embryos probably by affecting the metabolic rate in the exposed medaka embryos. Opposite to STX, after exposure to ACR, a pollutant that ubiquitously exists in food and environment, the zebrafish exhibited delayed development. ACR is a known glutathione (GSH) depleting factor and oxidative stress inducer. Apart from developmental retardance, increased reactive oxygen species (ROS) and inflammation were found in newly hatched zebrafish, suggesting the developmental delay may be partly related to the ACR-induced oxidative stress. In summary, findings from the present study highlighted the molecular responses and possible pathways of some selected foodborne toxins. Developmental effects and toxicity were also found after exposing to both the biologically produced toxin and the heat-produced toxicant. Our current study makes contributions to the knowledge on the hazardous effects of foodborne toxins in vivo, and provides useful information for the further study on the human health. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Adrenal gland, as an important effector tissue of the sympathetic nervous system, has critical roles in cardiovascular system under both chronic hypoxia (CH) and intermittent hypoxia (IH) conditions. Nitric oxide (NO), synthesized by nitric oxide synthases (NOS), is the most important intracellular signaling molecule, as well as free radical in response to hypoxia. Yet the regulation and effects of endogenous NO production mediated in the adrenal medulla induced by hypoxia remains largely unknown. We first studied how endogenous NO production were regulated by different NOS in rat adrenal medulla in response to CH or IH. After CH, elevated levels of endogenous NO production, eNOS expression, and apoptotic chromaffin cells were observed in the adrenal medulla. However, a remarkable decreased endogenous NO production and nNOS expression were shown in the IH-treated adrenal medulla. These results suggested that, in the rat adrenal medulla, the elevation of NO production through increased protein level of eNOS may play a protective role in the adaptive response to CH; the reduction of NO production through decreased expression of nNOS is important for the pathophysiological response to IH. The oxidative stress and cellular injury in the adrenal medulla under chronic intermittent hypoxia (CIH) condition is undefined. We tested the hypothesis that melatonin, a potent antioxidant, is protective against CIH-induced oxidative stress and local inflammation in the rat adrenal medulla. Results showed that levels of oxidative stress, lipid peroxidation, and inflammatory mediators were significantly increased after CIH treatment. Also, the protein levels of antioxidant enzymes were significantly lowered in the hypoxic group. Co-treatment of melatonin with hypoxia significantly reduced oxidative stress and inflammatory responses in the adrenal medulla. Moreover, the amount of apoptotic cells in the hypoxic groups was significantly less in the melatonin-treated group. Thus, melatonin may act as a protective agent against adrenal damages in patients with severe obstructive sleep apnea syndrome. Previous studies have shown that CIH associated with recurrent apnea induced oxidative stress and pathophysiological changes in the cardiovascular system. Yet the mechanism of the CIH-induced oxidative stress and local inflammation in the adrenal medulla was undefined. We therefore determined whether the up-regulation of the expression of NADPH oxidase (NOX) mediated by renin-angiotensin system (RAS) may take part in the injuries caused by CIH in the rat adrenal medulla. We found that CIH treatment dramatically induced marker levels of adrenal oxidative stress, inflammation, macrophage infiltration, and apoptosis in rats. Co-treatment with NOX inhibitor, apocynin, counteracted such reactions. Furthermore, the mRNA levels of NOX subunits (p22PHOX, NOX2, and NOX4) and RAS components (ATG, AT1, and AT2) were increased significantly in the CIH group, but reduced in apocynin-treated CIH group, supporting the involvement of NOX and RAS in CIH-induced adrenal injury. In conclusion, we defined the roles of NO production, NOX, and RAS in the rat adrenal injury during hypoxic conditions. We also found that melatonin can protect adrenal medulla from hypoxia-induced damages in rat. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Dentistry / Master / Master of Orthodontics

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences