Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: phytochrome; shade avoidance; microarray; HFR1. Includes bibliographical references (p. 52-56).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xiii, 108 p.; also includes graphics. Includes abstract and vita. Advisor: Paul K. Herman, Dept.of Molecular, Cellular, and Developmental Biology. Includes bibliographical references (p. 96-108).

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 110-140).

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Melanoma is a malignant type of skin cancer arising from the combined effects of genetic alteration and extrinsic signaling, resulting in transformation of neural crest (NC)-derived melanocytes into metastatic melanoma. Current therapies against metastatic melanoma are merely effective with less than 5% 5-year survival rate of patients. Understanding the underlying molecular mechanism of how melanoma acquires metastatic behavior could formulate strategies for new therapeutic options. Features of metastatic melanoma resemble NC cells undergoing an epithelial-mesenchymal transition (EMT) suggesting similar regulators might be in place to control the process. Our previous studies showed that SoxE transcription factors (Sox8/9/10) play a crucial role in NC development, in particular Sox9 transactivates expression of Snail2 and co-operates with it to induce features of EMT. To examine the role of SOXE proteins in melanoma development and whether they regulate SNAIL expression, we first investigated the expression profile of SOXE and SNAIL in a human melanoma tissue array. The data showed that SOX8, SOX10, and SNAIL genes are highly expressed in metastatic melanoma whereas SOX9 and SNAIL2 transcript levels are low. Moreover, SNAIL transcript level was shown to have a positive correlation with SOX8 and SOX10 expression levels. SNAIL is well-known to be the key regulator of tumor invasiveness in various cancers. Our data raised the possibility that SOXE proteins may also regulate SNAIL expression in initiating melanoma metastatic behavior. The human metastatic melanoma cell line A375 exhibits similar SOXE and SNAIL expression profiles as the tissue array. Knockdown of SNAIL in A375 reduced its migratory ability and in vivo tumorigenecity, suggesting that SNAIL plays a crucial role in melanoma metastasis. How SNAIL transcription is regulated in melanoma has been poorly understood. Previous studies have identified a minimal enhancer region downstream of the SNAIL locus which contains YY1 and SOX consensus binding sequences. Chromatin immunoprecipitation assay revealed that SOX8 and SOX10 proteins could bind to the SNAIL 3’ minimal enhancer region specifically. Mutation of the SOX consensus binding sequence reduced the enhancer activity while mutations in both SOX and YY1 binding sites resulted in further reduction suggesting that YY1 and SOX protein binding is required and important for enhancer activity and SNAIL transcription. These findings provide a molecular basis to examine further whether metastasis of melanoma is regulated by SOXE proteins in which one of the potential mechanisms could act through regulation of SNAIL expression. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Neural stem cells (NSCs) exist in both embryonic and adult neural tissues and are characterized by their self-renewal capacity and multipotency that contribute to the generation of three major cell types in the vertebrate central nervous system (CNS):neurons, oligodendrocytes and astrocytes. The tremendous therapeutic potential of NSCs to treat the neurodegenerative diseases and repair brain injuries has provoked intensive study in the molecular regulation of their induction, maintenance and differentiation. Previous study reported that Sox9, a member of high-mobility-group(HMG) containing SoxE transcription factors family, plays important roles in regulating the formation and maintenance of NSCs in both mouse and chick CNS, as well as the cell fate switch between neuronal and glial. Whether it plays similar roles in human NSCs (hNSCs)is still unknown. My RT-qPCR analysis showed that SOX9is expressed at a basal level in human embryonic stem cells (hESCs) and up-regulated upon commitment into neural lineage and maintained at a high level in hESCs-derived hNSCs. I therefore hypothesized that SOX9 might also be involved in the induction, maintenance and differentiation of hNSCs. To test this, two stable hESC lines(HES2)were generated with each constitutively expressing short hairpin RNA (shRNA) against SOX9andGL2 Luciferase (Luc, as control) respectively. Upon neural induction, SOX9-knock-down(KD) hESCs were able to commit neural lineage and differentiate into NSCs/neurospheres (NSPs), however, these NSCs exhibited reduced multipotency and glial marker (GALC, CD44) expressions but enhanced self-renewal compared to the shLuc NSCs. Hence, SOX9 is required for both the induction and maintenance of multipotent hNSCs. Strikingly, extensive TUJ1+ neurites and advance groupings of these neurites into bundles were observed in SOX9-KD NSPs after three days and seven days neuronal differentiation respectively, suggesting premature neurogenesis as a result of SOX9 ablation. In addition, RT-qPCR analysis revealed down-regulated expression of NSC marker HES1but induced proneural basic helix-loop-helix transcription factor MASH1in shSOX9-1208 NSCs. The inhibitory role of HES1 on the expression and functions of MASH1 has been reported to be essential for the timely generation of neurons. Hence, ablation of SOX9 is likely to relieve the inhibition on MASH1activity via down-regulated HES1expression and leads to early neuronal differentiation. Expression of the potent neurite blocker NG2 was also found to be reduced in SOX9-KD NSCs which may explain the extensive neurite network observed. Altogether, similar to previous studies in mouse NSCs, SOX9 is also required for the induction and maintenance of hNSCs. However, this study further reveals a putative novel role of SOX9 in preventing premature neuronal differentiation by regulating the expressions of HES1 to counteract MASH1 function and NG2 to control neurite outgrowth. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Circadian rhythmsare biochemical, physiological, and behavioral processes display oscillations of oughly 24-hour, which existing in both prokaryotes and eukaryotes. Circadian rhythms improve fitness of organisms in both constant and changing environments. The cryptochrome (CRY)and PAS-containing proteins are light sensors and key elements of the circadian system in eukaryotic organisms. Photolyases and cryptochromes are evolutionarily related flavoproteins which perform distinct physiological functions. Photolyases are evolutionarily ancient enzymes that activated by light and repairing UV-induced DNA damage. Although cryptochromes share structural similarity with the DNA photolyases, they lack the DNA repair activity. CRYs are key elements of mammal circadian system, and play roles in light sensing in insects and plants to entrain circadian rhythms. The PAS domains are widely distributed in proteins across all kingdoms of life and act as signal modules. They are common in photoreceptors and transcriptional regulators of eukaryotic circadian clock components including bHLH-PAS proteins (BMAL, CYC,CLK and NPAS2) and PER in animals, PHY and ZTL in plants, WC-1, 2and VVD in fungi. They are mainly involved in protein-protein interaction and light sensing functions. The CRY/PHR superfamily consists of 7 major subfamilies: CPD class I and CPD class II PHRs, (6-4) PHR, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the superfamily evolved primarily under strong purifying selection (average ω = 0.0178), it experienced strong episodic positive selection at some periods of evolution. The level of variation is subfamily-and domain-specific. The homologs with apparent circadian functions (i.e., plant and animal CRY) are significantly more conserved than the other photolyases. Photolyases were lost in eukaryotic groups like placental mammals, suggesting that natural selection apparently became weaker in the late stage of evolutionary history. The phylogenetic trees of fish Cry features two major clusters, which correspond to Cry1and Cry2. Teleost species possess extra copies of Cry1 due to fish-specific genome duplication (FSGD), and formed 3 clades of Cry1. Clade1B of Cry1(π= 0.129 ±0.062) is more conserved than the other paralogs (πrange from 0.173to 0.195). Test of positive selection revealed that fish cryptochromes evolved under strong purifying selection (average ω= 0.0066).Different fishes preserved different Cry duplicates that associated with reciprocal gene loss, thus generated the diverse circadian molecular mechanisms. The level of DNA variation in the PAS-containing proteins appears to be subfamily-specific. The animal PAS-containing homologs are more polymorphic than the plant and fungal homologs. Although the whole superfamily evolved primarily under strong purifying selection (average ω range from 0.0030to 0.1164), it experienced strong positive selection at some periods of the evolution. Although the PAS domains from different proteins vary in sequence and length, they maintain a fairly conserved 3D structure. The 3D fold of PAS domains is determined by only 8 conserved residues which shared by all subfamilies. The evolutionary time estimates showed that plant and animal Cry, WC-1& 2, bHLH-PAS proteins and Per originated in the Neoproterozoic Era (~1000 –542 Mya), plant Phy and ZTL evolved in the Paleozoic (541 –252 Mya), which might be a result of adaptation to the global climate and light regime changes. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Sox2, a HMG box transcription factor, is well known for its role in stem cell maintenance, iPS (induced pluripotent stem cell) induction, and development of neural tissues such as central nervous system and sensory organs. Sox2 has been demonstrated to be essential for the development of inner ear sensory patches. It has been shown that Sox2 is under the regulation of multiple regulatory elements to obtain a tissue specific manner. Two allelic mouse mutants, yellow submarine (Ysb) and Light coat and circling (Lcc) show hearing and balance impairments with different severity. They were made by random insertions of a transgene (pAA2) and X-ray irradiation respectively. Ysb and Lcc are both localized to chromosome 3 and involve complex chromosomal rearrangements. The Sox2 coding region is intact in the mutants, while the Sox2 expression in the otocyst is greatly reduced in Ysb and totally lost in Lcc, which indicates the tissue specific reduction of Sox2 may be due to the rearrangement of Sox2 regulatory element(s). Since Sox2 null mutants die before implantation, the two Sox2 inner ear mutants are valuable models for studying Sox2 knock down (Ysb) and Sox2 knock out (Lcc) condition in the inner ear. To understand the molecular basis behind Sox2 regulation in the inner ear, this project aims to identify the Sox2 otic regulatory elements, and potential Sox2 downstream targets involved in the development of inner ear. Previous work has indicated that Nop1 and Nop2 are the otic specific regulatory elements of Sox2 in chicken ear. In this project, transgenic mice were generated using Nop1-Nop2, and the result showed Nop1-Nop2 could drive Sox2 expression to the dorsal side of the otiv vesicle, which is different from the endogenous Sox2 expression pattern. Therefore, Nop1 and Nop2 may require other regulatory element(s) to gain a correct regulatory pattern. BAC(RP23-335P23), which contained the DNA sequences close to Ysb integration site 1 was also been tested in transgenic mice. Interestingly, the result showed that BAC(RP23-335P23) could drive Sox2 expression to the ventral side of the otic vesicle, indicating that this BAC may contain the Sox2 otic regulatory element(s). In this project, the binding relationship between Sox2 protein and Math1 enhancer has also been identified using chromatin immunoprecipitation (Ch-IP). Results showed that Sox2 could bind to Math1 enhancer A in the inner ear cochlea. So Sox2 may regulate Math1 through binding to Math1 enhancer A in inner ear development. Using a bioinformatics approach, potential Sox2 target genes in inner ear development have been identified from public microarray data on E9 to E15 inner ear tissue by the presence of conserved Sox2 binding sites. Among these potential targets, 4 genes (Itga6, Erbb3, Sox10 and Mycn) have been selected based on their known functions. Their expression patterns in the cochlea of wild type, Ysb and Lcc were verified. The identification of Sox2 downstream target genes using a bioinformatics approach will help us to understand the molecular basis of Sox2 regulation, and also understand the role of Sox2 in the inner ear development. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Human embryonic stem cells (hESCs) are characterized by unlimited proliferation (self-renewal), capability to differentiate into derivatives of all three germ layers (pluripotency), and abbreviated cell cycle structure. Despite tremendous efforts in identification of important regulators, the complicated molecular mechanisms and essential effectors underlying the distinctive features of hESCs have not yet been fully elucidated. Forkhead box transcription factor M1 (FOXM1) has been demonstrated to be critical for the maintenance of pluripotency in mouse embryonic stem cells (mESCs) and mouse embryonal carcinoma cells (mECCs). The present study hypothesized that FOXM1 is important to the self-renewing capacity and pluripotency of hESCs. The objectives of this study were to characterize FOXM1 expression in undifferentiated and differentiating hESCs, and to study the effect of perturbing FOXM1 expression on pluripotency and proliferation. Undifferentiated VAL3 analyzed by bivariate flow cytometric analysis revealed that FOXM1 expression was regulated in a cell cycle phase-dependent manner, with expression level increased from G1 through S phase, and eventually reached peak levels in G2/M phase. To study the subcellular localization of FOXM1 with respect to cell cycle progression, VAL3 cells were synchronized by nocodazole-mediated cell cycle block, followed by immunocytochemical analysis. The result indicated that FOXM1 underwent nuclear translocation at late-S and early-G2 phase of the cell cycle. When VAL3 spontaneously differentiated as embryoid bodies (EBs), the mRNA expression of FOXM1 displayed profound fluctuation over the differentiation process. Retinoic acid (RA) treatment induced rapid differentiation of VAL3, yet differential expression pattern of FOXM1 was observed for cells grown in different culture media. FOXM1 mRNA expression persisted in differentiating VAL3 cultured in mTeSR. By contrast, RA-driven differentiation of VAL3 cultured in conditioned medium was accompanied by transient depletion and resurgence of FOXM1 protein expression. Differentiation of VAL3 driven by Definitive Endoderm kit did not alter FOXM1 expression, whereas induced differentiation by Bone Morphogenic Protein 4 (BMP4) led to repression of FOXM1. The functional role of FOXM1 in hESCs was investigated with the use of siRNA. Transient knockdown of FOXM1in VAL3 did not induce substantial repression of pluripotent marker (OCT4, SOX2, NANOG) expression nor significant morphological change of colonies, despite upregulation of early differentiation marker SSEA-1. Intriguingly, FOXM1 depletion led to altered cell cycle progression and delay in G2 phase progression, possibly attributed to the downregulation of Cyclin B1 and Cdc25B. Also FOXM1 knockdown impaired VAL3 proliferation, yet no prominent defect in mitosis was observed. In conclusion, the present study reported for the first time the expression and functions of FOXM1 in undifferentiated hESCs. Upon differentiation, FOXM1 expression varied in cells committing to different lineages. Depletion of FOXM1 did not interfere with hESCs pluripotency, but hindered cell cycle progression and cell proliferation, suggesting that FOXM1 is mainly involved in promoting rapid proliferation of hESCs. The functional role and regulatory mechanics of FOXM1 in hESCs cell cycle control and differentiation warrant further investigation. / published_or_final_version / Biochemistry / Master / Master of Philosophy