Thesis--Guo li Taiwan da xue, 1968. / Cover title. Colophon title also in English: The origin of Chʻu tzʻu: a survey of its literary and religious aspects. Includes bibliographical references (p. 113-116).

Small intestinal (SI) stem cells differentiate into short-lived progeny, except lysozyme-expressing Paneth cells. In vivo, the Tet-Op histone 2 B (H2B) - green fluorescent protein (GFP) fusion protein transgenic mouse has been employed to analyse the slow-cycling putative small intestinal epithelial stem cells, at cell position +4 (cp4 cells), through doxycycline-inducible transient expression of H2B-GFP. The aim of the study was to employ the same genetic mouse model in order to develop a culture system in which was possible to detect and investigate H2B-GFP-retaining putative SI stem cells. SI crypts isolated from 6-12 weeks old Tet-Op-H2B-GFP transgenic mice were established in culture (designated organoids) using growth factors and Matrigel. For in vitro transgene expression, doxycycline was added to the complete culture medium for 24/48 hours (pulse). H2B-GFP and lysozyme expression was studied by confocal and fluorescence microscopy. Percentages of H2B-GFP-retaining putative SI stem cells and of H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring GFP-immunoreactive cells. Ulex europaeus-I lectin (UEA-I) cell labelling, combined with flow cytometry, was employed in a pilot study aimed at establishing a protocol for the separation of H2B-GFP-retaining putative SI stem cells from H2B-GFP-retaining Paneth cells persisting in the small intestine of chased mice. After both 24 hours and 48 hours exposure (pulse) to the optimal concentration of doxycycline, the nuclei of all cells in SI organoids expressed H2B-GFP. During subsequent culture, in absence of doxycycline (chase period), there was gradual loss (due to cell division) of H2B-GFP. After 6 day chase, small numbers of H2B-GFP-retaining putative slow-cycling stem cells and Paneth cells were seen. Flow cytometric analyses indicated that to label SI crypt cells with UEA-I lectin is a promising approach for isolating putative SI stem cells for functional characterization. A method to induce ubiquitous transient expression of H2B-GFP in cultured SI organoids, compatible with regular organoid development, has been optimized. It allows detection, after a short chase period, of the slow-cycling H2B-GFP-retaining putative SI epithelial stem cells. The developed in vitro culture system will enable further characterisation of these cells and Paneth cells.

The SOX family of transcription factors are well-known regulators of diverse cellular events during development. SOX1, which belongs to the SOXB1 sub-family, is a key regulator of neural stem cell fate and a known specific marker of the neuroectoderm lineage. SOX1 plays an important role in early embryonic and postnatal CNS development. Recently, several studies have implicated SOX1 as a tumour suppressor gene in different cancer types. Conversely, SOX1 has also been reported to act as an oncogene in a prostate cancer model. In order to better understand SOX1 gene regulation, this project set out to gain a deeper insight into the regulation of SOX1 in the context of stem cells and cancer, and to identify potential regulatory mechanisms that can significantly regulate its function. Initially, SOX1 gene expression and its promoter DNA methylation pattern was analysed in a range of cancer cell lines to establish whether SOX1 epigenetic silencing was consistently found in cancer lines. Differential SOX1 expression across the analysed cancer cell lines suggests differential regulation of SOX1 in cancer, accompanied by cancer type dependent epigenetic silencing of SOX1 by DNA methylation. The second part of the study focused on the characterisation of the structure and expression of a newly identified SOX1 overlapping transcript (SOX1-OT), using RT-PCR and 5’5’RACE techniques. The SOX1-OT genomic locus was found evolutionary conserved across different species. SOX1-OT expression was further analysed in a human neuroprogenitor cell line across different time points of neural differentiation, highlighting its possible role in neural differentiation. Furthermore, the SOX1-OT gene expression profile was matched with SOX1 gene expression in a panel of different stem cell and cancerous cell lines. The co-expression profiles of SOX1-OT and SOX1 in stem cells and carcinogenesis indicated towards a potential role of SOX1-OT regulating SOX1 gene expression. Finally, a comprehensive bioinformatics analysis was performed to investigate evidence of SOX1 post translational modifications (PTMs). In silico prediction of phosphorylation, acetylation and sumoylation sites support SOX1 PTMs. The predicted PTMs within different SOX1 protein domains may affect its function through altering its DNA binding activities, cellular localisation and interaction with partner proteins. In conclusion, SOX1 expression in different stem and cancer cell lines is likely to be regulated by promoter DNA methylation, a long non coding RNA (SOX1-OT) and its function by different types of PTMs. These regulatory features may in the future advance the understanding of the SOX1 transcription regulatory network in stem cell developmental processes and its role in cancer development.

Cancers are amongst the leading cause of morbidity and mortality today. Besides the tremendous amount of research, it still appears to be a long way till we can fully understand the pathology and find its cure. Scientists are still striving to find out the precise pathogenesis, factors leading to progression and the mechanisms of spread of cancers. The ultimate objective is to find out how these can be prevented and treated. Many molecules are a focus of attention in this regard and CD24 is amongst them. CD24 is normally present on haematopoietic cells and embryonal epithelial cells but the expression is generally lost with cellular maturity and differentiation. Upregulation of CD24 has been documented in a large variety of cancers, besides non-malignant pathologies. In the recent literature, CD24 has been linked to significant cancer associated properies such as proliferation, metastasis and cancer stem cells. It is an interesting molecule with a very small protein core decorated with heavy glycosylation. The pattern and composition of CD24 glycosylation also varies within different tissues. This study attempted to explore the molecule a bit further regarding its structure, functions, molecular interactions and possible downstream signalling partners. The first part of the project was to evaluate the possible effects of CD24 in mediating cellular response to DAMPs (Damage Associated Molecular Patterns) in colorectal cancer cell lines. Malignancies have increased CD24 levels and there is increased tissue damage and necrosis thereby release of DAMPs (Damage Associated Molecular Patterns) in their microenvironment. The effects of CD24 on modulation of DAMPs response were analysed by exposing cells to autologous DAMPs and performing functional assays (migration and proliferation assays). The results of wound healing assay showed significant inhibition of colorectal cell migration by DAMPs but this was independent of CD24 status. Furthermore, transwell assays also showed significantly reduced directional motility, independent of CD24 status, in cells exposed to autologous DAMPs. The study also tested the effects of DAMPs on cellular proliferation in three colorectal cancer cell lines using Presto Blue assay. The results indicated a significant increase in cellular proliferation when exposed to DAMPs, irrespective of the CD24 status of the cell line. We proposed thereby that in colorectal cancer cells, CD24 does not appear to modulate cellular response to DAMPs unlike seen in immune cells. Nevertheless, DAMPs did show effects on autologous cell migration and proliferation. However it is acknowledged that the arguments can be strengthened by the use of purified DAMPs to demonstrate similar results and also by showing abrogation of effects after addition of anti-DAMPs antibodies to our functional assays. Another interesting aspect of CD24 is its localisation in the cell membrane. It is attached to the cell membrane via GPI anchor and resides in lipid rafts. Interestingly, it has no cytoplasmic or transmembrane domains that are used by most signalling molecules. It is unclear how CD24 molecule mediates diverse cellular properties and molecular responses in the absence of traditional signalling domains. The second part of this study was aimed at exploring some of the potential signalling partners and their functional relevance if any to CD24. The study explored Cten and CD24 interactions, as both molecules are proposed to be mediators of increased cellular motility. CD24 and Cten were also observed to have some common downstream signalling targets. In addition, the expression levels of both molecules also presented similar trends amid different cancer cell lines. The above observations led us to contemplate if the two molecules have mutual signalling and functional relationship. We observed that CD24 up regulation led to increased levels of Cten protein, whereas, knock down of CD24 resulted in down regulation of Cten protein. Also the results of our functional studies showed that the knockdown of Cten in co-transfection experiments abrogated the increased cell motility by CD24. Based on these observations we proposed that CD24 appears to modulate Cten levels and this regulation has significant functional relevance as well. The Co-immunoprecipitation experiments indicated that these two molecules did not seem to have physical interaction with each other, suggesting the possibility that the regulation of Cten by CD24 may be arbitrated by intermediate molecules. Similarly, we also investigated the molecular relationship between CD24, ILK and FAK using co-transfection technique. The results revealed enhanced cell migration through the membrane by CD24 but it was reduced after the knock down of both ILK and FAK. These findings provide further insight that not only these molecules are being regulated by CD24 as proposed by many recent studies but are also functionally relevant. The current study proposed that the increased cancer cell motility demonstrated by CD24 may well be mediated through FAK and ILK in addition to Cten. The argument needs further validation as the confirmation of successful transfection and expression levels by western blots are missing in this study. This is recognised as a limitation. Being heavily glycosylated CD24 is considered a mucin- like molecule and many studies point towards the possibility of its functional diversity to be related to these sugars. But to date not much is known about this aspect of CD24. Hence, the third part of our study was aimed at finding the functional and signalling significance of these sugar binding sites. CD24 has both O and N-sugar binding sites. While the molecule has multiple O-glycosylation sites, there are only two potential sites for N-glycosylation. Employing the fact that for CD24 molecule to be N-glycosylated, asparagine is a must and should be present in a specific sequence. We aimed at replacing the asparagine by glutamine, hence disabling the site to be decorated by N-sugars in CD24. We designed to mutate these N-glycosylation sites using Phusion-site directed mutagenesis kit. The procedure uses mutant primers in a PCR-based methodology to create the desired mutation. Once successful the next step of the study was to use this mutant CD24 in further experiments to explore the signalling and functional significance of these sugars in CD24 molecule. However, the results of our gene sequencing experiments showed that the attempt at generating the required clones was not successful and needed retrial. This step could not be carried out due to time limitation. In summary, it can be said the study aimed at exploring novel aspects of CD24 biology from three different perspectives with some exciting new facts and findings. Though these are undermined by a number of limitations that can be rectified as discussed in the thesis and would generate stronger and publishable contribution to the current understanding of CD24 biology.

Real time monitoring of stem cells has been a growing area of interest over the past decade because of new regenerative medicine approaches. Also, the effect of culture composition on stem cell metabolic pathways and their regulation of cellular fate are of increasing importance. In our project, non-destructive metabolomic analysis of stem cells and their physiological status during proliferation and differentiation stages will be investigated. This will be achieved through mass spectrometric analysis of released metabolites by LCMS and GC/MS; therefore stem cells should ideally be cultured in a specialized format to maintain their physiological properties during non-invasive live analysis while being compatible with real time GC/MS. To address this issue, mesenchymal stem cell attachment, proliferation and differentiation were tested in 20ml glass vials required for GC/MS analysis. Environmental conditions in which MSCs can proliferate under sealed condition and at room temperature were also optimized. Our data show the potential of MSCs to attach, proliferate and differentiate in 20ml glass vessels. Furthermore, cells could maintain their metabolic activity for approximately 6hrs not only under hypoxic but also hypothermic conditions. Therefore, live time analysis of their chemical volatiles could be performed in a format compatible for LC-MS and GC/MS which will be performed for undifferentiated MSCs and their differentiated populations (osteogenic and adipogenic lineages) in the next phase of the project. A specific treatment approach has emerged from hMSCs osteogenic study and identified a particular pathway suggested to be involved in chondrogenesis, and linked to the collagen II biosynthesis. The treatment is based on on natural products (L-lysine and ascorbic acid) and when tested on human cell cultures, was observed to significantly increase the morphological and functional markers of chondrogenesis, suggesting its potential to promote de novo cartilage formation in vitro.

El presente proyecto de tesis desarrolla un nuevo organocatalizador del tipo / prolinamida, adem��s posee simetr��a C2, este factor hipot��ticamente aumenta la / enantioselectividad de los organocatalizadores. Se prob�� su poder organocatal��tico en la / reacci��n ald��lica modelo, la condensaci��n de 4-nitrobenzaldeh��do con acetona, / obteni��ndose diversos resultados (Ver Cap��tulo 5 RESULTADOS Y DISCUSI��N). / Entre las condiciones que destacan son el uso del organocatalizador (1) dise��ado y / sintetizado en este proyecto en una concentraci��n al 10 mol%, 1 equivalente de 4- / nitrobenzaldeh��do, 5 equivalentes de acetona, sistema de disolventes DMSO/H2O en / proporci��n 3:1 y la adici��n de 75 mol% de ��cido ac��tico, para la obtenci��n del producto / ald��lico (R)-2 como mayoritario con un rendimiento del 96% y un exceso / enantiom��rico del 82% (Ver Cap��tulo 6 CONCLUSIONES).

Thesis (M.A.)--Guo li Taiwan shi fan da xue, 1979. / Cover title. On double leaves. Reproduced from typescripts. Includes bibliographical references (p. 141-146).

"Guo li Taiwan da xue Zhong wen yan jiu suo shuo shi lun wen". / On double leaves. Reproduced from typescript. Bibliography: leaves 68-70.

Thesis (M.A.)--Fu ren da xue, 1973. / Reproduced from typescript copy; on double leaves. Includes bibliographical references.

The heterochromatin-associated proteins are subject to several different posttranslational modifications; hence, their level must be tightly controlled; otherwise as transcription factor co-repressor(s) complexes with these proteins, it may lead to stable silencing. An obvious mechanism to limit the expression time of a protein is to destroy it via the ubiquitin-proteasome system. FBXW7 (F-box and WD repeat domain–containing 7) is an E3-ligase targets transcriptional modulators and proto-oncogenes for degradation with crucial functions in cell-fate determination and tumorigenesis. In addition, most of current studies focused on epigenetic modifications that influence on the core histones within the euchromatin-heterochromatin transition, whereas the heterochromatin proteins and their partners’ identity remained largely unclear. Dr Nateri’s lab have recently identified several proteins which are targeted by the FBXW7 E3 ligase for the ubiquitin-mediated degradation. Among others, my study was focused on the role(s) of heterochromatin protein– binding protein 3 (HP1BP3) protein in epigenetic-reprogramming and its underlining mechanisms, including EMT and cell cycle progression, in normal and cancer cells. It's worth mentioning that apart from the single publication (Hayashihara et al., 2010a), the role of HP1BP3 was unknown when I began my project. HP1BP3 modulates the entry/exit of nucleosomal-DNA through binding to HP1α protein. HP1α is enriched in the pericentromeric heterochromatin, and it has been reported that HP1α recruitment in this region depends on SUV39H1/2-mediated H3K9 trimethylation. Widespread epigenomic alterations, occurs during cell differentiation, cell cycle progression and malignant transformation, but how epigenetic mechanisms contribute to the transcriptional reprogramming that accompanies EMT is still poorly understood. Furthermore, chromatin modulation events are important to control the cell-cycle-dependant gene expression during development and differentiation. Dysregulated expression of upstream cell-cycle regulators can affect DNA replication, repair, and/or division, leading to carcinogenic. Herein, our data show that the loss of FBXW7 mediated HP1BP3 induction alters heterochromatin states, through rescuing HP1α from its repressive function, impairing SUV39H1-mediated the methylation of histone H3 lysine 9 (H3K9me3), and stimulating the acetylation of H3K9 (H3K9ac) that lead to activation of epithelial-mesenchymal transition (EMT) pathway in Tiger skin fibroblast and HCT116 human colorectal cell lines. This induction of HP1BP3 upregulates the level of mesenchymal markers/regulators (Ncadherin, ZEB-1, Vimentin, and Snail1) in Tiger fibroblast cells while downregulating the epithelial marker (E-cadherin) and upregulating mesenchymal markers (ZEB-1, Vimentin, and Snail1) in HCT116 cells. In addition, upregulated HP1BP3 is an inducer of both G2/M cell cycle arrest and G1 to S phase transition via downregulating Cyclin B1 and SUV39H1/H3K9me3 while upregulating H3K9ac mark, in human Tiger fibroblasts and HCT116 CRC cells. Taken together, these findings point towards the important biological functions of HP1BP3 and its contribution in regulation of chromatin/EMT associated genes expression which consequently can be implicated in the pathogenesis of different types of FBXW7-mutated cancer.