Ng, Chor Fung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Xu, Jiaying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 98-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Masayo Kotaka. / "May 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 179-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

While prompt activation of NF-κB is essential for optimal immune response, it is equally important to terminate the response to avoid tissue damage and perhaps even death resulting from organ failure. This thesis describes two inhibitors of NF-κB, A20 and IκBβ. A20 is an essential inhibitor of NF-κB mediated inflammation as mice lacking A20 die from multi-organ inflammation and cachexia. Multiple biochemical approaches have suggested that A20 functions as a deubiquitinase by disassembling K63-linked regulatory ubiquitin chains from upstream adapter molecules like RIP1. To determine the contribution of the deubiquitinase role of A20 in downregulating NF-κB, we generated and characterized a knock-in mouse lacking the deubiquitinase activity of A20. However, we find that these mice display normal NF-κB activation and show no signs of inflammation. Our results suggest that the deubiquitinase activity of A20 is dispensable for downregulating NF-κB. The second part of this thesis unravels a new biological pathway mediated by IκBβ. Unlike IκBα, which functions solely as an inhibitor of NF-κB, IκBβ can both inhibit and activate NF-κB depending on the physiological context. We hypothesized that this may be because IκBβ (unlike IκBα ) exists in two forms, a constitutively phosphorylated form and an unphosphorylated form. Prior work from our group has demonstrated that hypophosphorylated IκBβ complexes with p65:cRel and mediates the expression of certain inflammatory genes like TNFα . We report here that Glycogen Synthase Kinase 3β (GSK-3β ) interacts with and phosphorylates IκBβ at Serine-346. This phosphorylation masks the NLS of p65 in the phospho-IκBβ:p65:cRel complex, thereby sequestering the complex in the cytoplasm and mediating the anti-inflammatory role of IκBβ. We discovered a peptide that can inhibit this phosphorylation by abrogating the interaction between GSK-3β and IκBβ. Mice succumb to a sublethal dose of LPS when injected with this peptide because of increased production of TNFα (but not IL-6); thereby demonstrating the inflammatory role of unphosphorylated IκBβ in upregulating specific genes like TNFα. We propose a signaling model by which phosphorylation by GSK-3β can regulate the functions of IκBβ in response to LPS.

The nuclear factor-kB (NF-kB) signaling cascade is comprised of two branches, the canonical and alternative NF-kB pathways. Signaling through the alternative NF-kB pathway culminates in the activation of the downstream transcription factor subunits, RELB and NF-kB2. The biological roles of RELB and NF-kB2 within the B cell lineage have been obscured in constitutional knockout mice by the diverse functions of these subunits in non-B cell types. To overcome these limitations, conditional alleles were generated to investigate the roles of RELB and NF-kB2 in B cell development. These alleles allowed the identification of complex functional requirements for RELB and/or NF-kB2 in naïve B cells, germinal center (GC) B cells and plasma cells (PCs). These functional requirements may have implications for B cell malignancies that display mutations that constitutively activate the alternative NF-kB pathway. A large body of work has demonstrated that B cell activating factor (BAFF) signaling is critical for the maintenance of mature B cells. However, the contribution of the alternative NF-kB subunits that are activated downstream of BAFF remained unclear, especially in regards to their specific target genes. We have identified critical, B cell-intrinsic roles for RELB and NF-kB2 in the maintenance of mature B cells. In response to BAFF, these subunits were found to control the expression of anti-apoptotic genes, genes that ensure correct positioning within the B cell niche, and genes involved in promoting B–T cell interactions that allow effective antigen-mediated activation. During the GC B cell reaction, light zone (LZ) B cells undergo affinity-based selection mediated by T follicular helper (Tfh) cells. A subset of LZ B cells show activation of the NF-kB signaling cascade, suggesting a critical role for NF-kB in the selection of high-affinity GC B cells. We here report that GC B cell development occurred normally in mice with conditional deletion of either relb (RELB) or nfkb2 (NF-kB2) in GC B cells. In contrast, the simultaneous ablation of both subunits caused rapid involution of established GCs, similar to what has been observed for ablation of the canonical NF-kB transcription factor subunit c-REL. Intriguingly, RNA-sequencing analysis of relb/nfkb2-deleted GC B cells revealed no overlap between the genes controlled by RELB/p52 and c-REL within GC B cells. This suggests that signaling through the separate NF-kB pathways in GC B cells results in the expression of different biological programs that are independently required for the maintenance of the GC reaction. In addition, we observed that human PCs and PC precursors within the LZ showed high protein levels of NF-kB2 compared to surrounding lymphocytes, suggesting a biological role for this subunit in PCs. Indeed, ablation of nfkb2 alone in GC B cells led to a dramatic decrease in antigen-specific serum IgG1 and antigen-specific IgG1-secreting cells. Interestingly however, the mice developed normal frequencies of PCs, suggesting a role for NF-kB2 in PC physiology rather than differentiation.

Many aspects of biology depend on the ability of DNA-binding proteins to locate specific binding sites within the genome. This search process is required at the beginning of all site-specific protein-DNA interactions, and has the potential to act as the first stage of biological regulation. Given the difficulty of pinpointing a small region of DNA, within even simple genomes, it is expected that proteins are adapted to use specialized mechanisms, collectively referred to as facilitated diffusion [Berg et al., 1981], to effectively reduce the dimensionality of their searches, and rapidly find their targets. Here, we use a combination of nanofabricated microfluidic devices and single-molecule microscopy to determine whether facilitated diffusion contributes to all DNA target searches. We investigate promoter binding by E. coli RNA polymerase, foreign DNA recognition by CRISPR-Cas complexes, and Rad51’s homology search during recombination. In each example, we observe that the target searches proceed without extensive use of facilitated diffusion; rather, consideration of these non-facilitated target searches reveals an alternative search strategy. We show that instead of reducing the dimensionality of their searches, these proteins, reduce search complexity by minimizing unproductive interactions with DNA, thereby increase the probability of locating a specific DNA target.

Breast cancer is the most commonly occurring cancer in women, with incidence rates approaching 1.38 million cases per year worldwide. Over the last few decades, there have been numerous attempts to develop, synthesise and advance into the clinic novel and selective breast cancer therapies. Research work has shown that bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) exerts potent in vitro anti-cancer activities and strong DNA binding properties. The aim of this thesis was to synthetise novel bisnaphthalimidopropyl derivatives (BNIPs) and investigate their subsequent modes of action within two human metastatic breast cancer cell lines, MDA-MB-231 and SKBR-3. A series of novel BNIPs, bisnaphthalimidopropyl-piperidylpropane (BNIPPiProp), bisnaphthalimidopropyl- ethylenedipiperidine (BNIPPiEth) and (trans(trans))-4,4’-methylenebis-cyclohexylamine (trans,trans-BNIPDaCHM) were synthesised, characterised and studied in comparison to BNIPDaCHM for their DNA binding and anti-cancer activities against MDA-MB-231 and SKBR-3 cells. Thermal denaturation studies have shown that BNIPs can intercalate and stabilize the double helix of Calf Thymus, each BNIP can competitively displace EtBr from DNA in a dose dependent manner and by UV binding studies, high affinity was found for the three novel BNIPs. After 24 hours treatment, all novel BNIPs, exhibited strong cytotoxicity with IC50 values ranging from 1.4 μM to 3.3 μM in MDA-MB-231 cells and 0.2 - 0.7 μM in SKBR-3 cells, confirming the importance of bisnaphthalimidopropyl functionality. BNIPs were also found to increase intracellular ROS levels after 8 hours treatment and induce a significant increase in DNA strand breaks compared to endogenous levels, after 24 hour treatment in both cell lines. After cell synchronisation, cell cycle distribution was studied, revealing that trans,trans-BNIPDaCHM induces sub-G1 cell population arrest in MDA-MB-231 and SKBR-3 cells, after 24 hours treatment. In addition, BNIPs induced apoptotic phosphatidylserine exposure, after 0.5 hours treatment, inhibited Caspase-3 activity and increased autophagy, after 24 hour treatment in MDA-MB-231 and SKBR-3 cells. Moreover, BNIPs inhibited histone deacetylases (HDAC) activity after 24 hours treatment in MDA-MB-231 and SKBR-3 cells and BNIPDaCHM was identified as a potential SIRT2 inhibitor, in SKBR-3 cells. According to Proteome Profiler Arrays, BNIPDaCHM and BNIPPiEth altered the expression of cell stress-related proteins in a cell dependent manner and bioinformatic analysis revealed two novel, putative pathways for BNIP-induced oxidative stress-mediated cell death in MDA-MB-231 and SKBR-3 cells. The above findings indicate that BNIPs represent promising candidates for future breast cancer studies and cancer treatment.

蒲金氏細胞(Purkinje cell)是小腦中的一種主要神經元，其主要作用在於協調身體活動及平衡。蒲金氏細胞之早期分化需要兩個密切相關的LIM同源盒結構域基因Lhx1及Lhx5。在胚胎小腦發育期間，這兩個基因的失活化會導致蒲金氏細胞數量大量減少。但有趣的是，就算在蒲金氏細胞完成分化之後，Lhx1/5之表達依然維持在高水平。這顯示Lhx1/5在產後小腦發育過程中可能有更多作用。為了研究這些可能作用，我把條件性Lhx1/5雙基因剔除小鼠和Pcp2-IRES-Cre轉基因小鼠交配，從而令Lhx1/5在產後第二天的蒲金氏細胞失活化。結果顯示Lhx1/5雙突變體老鼠在出生後兩星期即有顯著但程度不太大的運動失調。但在八星期，牠們出現嚴重的運動協調及身體平衡能力缺失。可是，擁有一個正常的Lhx1或Lhx5等位基因的控制小鼠並沒有這些不正常行為出現。在出生後的三個星期內，缺乏Lhx1/5會導致蒲金氏細胞樹突不正常發展，但小腦的整體細胞結構和分層卻維持正常。另外，這兩個基因對維持蒲金氏細胞已發展的樹突並不起作用，而且在六個月大的成年突變小鼠並沒有蒲金氏細胞退化。利用微陣列及逆轉錄聚合酶鏈式反應，我們在成年突變小鼠的小腦中確定了數個參與在麩胺酸及鈣訊息的突觸基因表達量下降。而這些突觸基因也在其他運動失調小鼠有下降的表達量。研究結果說明了Lhx1及Lhx5對蒲金氏細胞樹突發展有著重要、但功能重疊的作用。 / 在探究Lhx1/5如何控制蒲金氏細胞樹突發展時，我們發現Lhx1/5與Foxp4有蛋白質交互作用。Foxp4屬forkhead家族成員轉錄因子，它表達在小腦原基、遷移中及成熟的蒲金氏細胞。為了初步瞭解Foxp4在蒲金氏細胞發展中的作用，我在產後第十天小腦薄片組織培養中，利用siRNA降低Foxp4基因的表達量。結果發現蒲金氏細胞樹突及關聯的伯格曼膠質細胞支架出現結構性受損。這顯示Foxp4對維持蒲金氏細胞樹突有重要作用。 / 為了進一步研究Foxp4在活體蒲金氏細胞及小腦發育的作用，我把條件性Foxp4基因剔除小鼠和不同的Cre轉基因小鼠交配，從而令Foxp4在不同的發育過程階段中失活化。但是有趣地，我只能在同質結合突變小鼠 (Foxp4Δ/Δ)，即Foxp4在生殖細胞時期已經被剔除的情況下，觀察到小腦發育遲緩。當Foxp4在其他發育過程階段中失活化，我並沒有觀察到任何缺陷表型。這個結果顯示了在活體中發生了功能性的彌補，但在小腦薄片組織培養中卻沒有發生。另外，條件性Lhx1/5雙基因剔除小鼠和條件性Foxp4基因剔除小鼠的不同表型意味著在控制蒲金氏細胞及小腦發育過程中，有其他蛋白質可能參與在Lhx1/5及Foxp4的轉錄複合子中。我們需要更多的研究去明白Foxp和 LIM同源盒結構域蛋白質在功能上的聯系及它們在中樞神經系統發育中的作用。 / Purkinje cells (PCs) are one of the principal neurons in the cerebellum that is essential for the coordination of fine-tuning body movement and balancing. Early differentiation of PCs requires two closely related LIM-homeodomain genes Lhx1 and Lhx5, as inactivation of both genes results in significant reduction of PC number in embryonic cerebellum. Interestingly, high levels of Lhx1/5 expressions persist even after PC differentiation in the postnatal cerebellum. Hence, there may be additional roles for these two genes during postnatal PC development. To address this question, conditional inactivation approach was used to inactivate both Lhx1/5 in postnatal PCs specifically beginning at postnatal day 2 (P2). Lhx1/5 double conditional knockout (DKO) mutants were generated by crossing Lhx1/5 conditional null mutant mice with Pcp2-IRES-Cre mice. The mutants initially showed modest but noticeable ataxic locomotion at around two weeks after birth. However at 8 weeks old, the mutants displayed severe deficits in motor coordination and body balance. The control animals with one functional copy of either Lhx1 or Lhx5 did not show any abnormality. Deficiencies of both genes could lead to abnormal PC dendritogenesis during the first three weeks of life although the general cytoarchitectural lamination of cerebellar cortex was maintained. However, the two genes were dispensable for the maintenance of developed dendrites in adult mouse and no PC degeneration was observed in the 6 month-old double mutant mouse. Further microarray and semi-quantitative RT-PCR analysis identified down-regulation of several synaptic genes that involved in glutamate and/or calcium signaling in our Lhx1/5 DKO mutant and such disturbance had also been found in other ataxic mouse models. Overall, our findings suggest that Lhx1/5 are required but functionally redundant in dendritogenesis of PCs. / During investigation on how Lhx1/5 control the dendritogenesis of PCs, Lhx1/5 proteins were found to physically interact with Foxp4. Foxp4 belongs to the forkhead transcription factor family that is expressed in developing cerebellum primordium, migrating and mature PCs. To initially examine the function of Foxp4 in PC development, Foxp4 was knocked down by siRNA in organotypic cerebellar slice culture at P10. Impaired organization of PC dendritic arbors and associated Bergmann glial scaffold were resulted, suggesting that Foxp4 is essential for the maintenance of PC dendritic arborization. / To further investigate the function of Foxp4 during the cerebellum and PCs development in vivo, a Foxp4 CKO mouse line was generated and crossed with different lines of Cre-deleter mice, including Zp3-Cre, Pax2-Cre, En1-Cre and Pcp2-IRES-Cre, to inactivate Foxp4 at different developmental stages. Intriguingly, although developmental delay of cerebellum was found in germline deletion of Foxp4 homozygous recombined null mutant, no defective phenotype was observed when Foxp4 was inactivated at other stages. Hence, functional compensation might take place in vivo but not in the cerebellar slice culture. The phenotypic difference between Lhx1/5 DKO and Foxp4 CKO mice imply potential involvement of other proteins in the transcription complex between Lhx1/5 and Foxp4 in regulating the cerebellum and/or PCs development. Thus further investigation is required to understand the functional association between Foxp and LIM-homeodomain protein families during the development of central nervous system. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tam, Wing Yip. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 187-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.1 / 摘要 --- p.4 / Acknowledgements --- p.6 / Abbreviations --- p.8 / Figure list --- p.12 / Table list --- p.15 / Chapter Chapter 1 --- General Introduction --- p.16 / Chapter 1.1 --- An overview of cerebellum functions and anatomy --- p.16 / Chapter 1.2 --- Purkinje cell development in the mouse --- p.19 / Chapter 1.2.1 --- Embryonic development of mouse cerebellum --- p.19 / Chapter 1.2.2 --- Postnatal development of mouse cerebellum --- p.21 / Chapter 1.3 --- Degeneration of Purkinje cell leads to spinocerebellar ataxia --- p.22 / Chapter 1.4 --- LIM-homeodomain genes Lhx1 and Lhx5 --- p.24 / Chapter 1.4.1 --- LIM-homeodomain --- p.24 / Chapter 1.4.2 --- Lhx1 and Lhx5 are crucial to Purkinje cell differentiation --- p.25 / Chapter 1.5 --- Hypothesis, aim and strategy of the study --- p.26 / Chapter Chapter 2 --- Generation of Lhx5 conditional knockout allele in the mouse --- p.31 / Chapter 2.1 --- Chapter summary --- p.31 / Chapter 2.2 --- Introduction --- p.32 / Chapter 2.3 --- Materials and methods --- p.35 / Chapter 2.3.1 --- Materials --- p.35 / Chapter 2.3.2 --- Construction of Lhx5-conditional targeting vector by recombineering --- p.39 / Chapter 2.3.3 --- Gene targeting in mouse embryonic stem cells --- p.53 / Chapter 2.3.4 --- Generation of Lhx5 CKO mouse --- p.62 / Chapter 2.3.5 --- Histological examination of Lhx5 CKO mouse brain --- p.63 / Chapter 2.4 --- Results --- p.64 / Chapter 2.4.1 --- Generation of Lhx5 conditional targeting construct --- p.64 / Chapter 2.4.2 --- Screening of targeted ES cell clones --- p.64 / Chapter 2.4.3 --- Karyotyping --- p.65 / Chapter 2.4.4 --- Generation of chimeric mice and maintenance of Lhx5 CKO mice --- p.66 / Chapter 2.4.5 --- Histological examination of Lhx5 recombined null mutant mouse --- p.67 / Chapter 2.4.6 --- Gross anatomical examination of Lhx5 recombined null mutant mouse --- p.69 / Chapter 2.5 --- Discussion --- p.71 / Chapter Chapter 3 --- Generation and characterization of Pcp2-CreER[superscript T]² transgenic mouse --- p.75 / Chapter 3.1 --- Chapter summary --- p.75 / Chapter 3.2 --- Introduction --- p.76 / Chapter 3.3 --- Materials and methods --- p.79 / Chapter 3.3.1 --- Materials --- p.79 / Chapter 3.3.2 --- Construction of pPcp2-IRES-CreER[superscript T]²-FRT-Kan-FRT --- p.81 / Chapter 3.3.3 --- Generation of BAC-Pcp2-IRES-CreER[superscript T]² transgene --- p.85 / Chapter 3.3.4 --- Generation of Pcp2-CreER[superscript T]² transgenic mice --- p.90 / Chapter 3.3.5 --- Characterization of Pcp2-CreER[superscript T]² transgenic mice --- p.91 / Chapter 3.4 --- Results --- p.93 / Chapter 3.4.1 --- Construction of BAC-Pcp2-IRES-CreER[superscript T]² --- p.93 / Chapter 3.4.2 --- Production of Pcp2-CreER[superscript T]² transgenic mice --- p.93 / Chapter 3.4.3 --- Expression of Cre recombinase in Pcp2-CreER[superscript T]² transgenic mice --- p.93 / Chapter 3.4.4 --- Histological examination of Pcp2-CreER[superscript T]² transgenic mice --- p.97 / Chapter 3.4.5 --- Behavioral test of Pcp2-CreER[superscript T]² transgenic mice by rotarod --- p.98 / Chapter 3.5 --- Discussion --- p.100 / Chapter Chapter 4 --- Characterization of Lhx1/5 double conditional knockout mouse --- p.103 / Chapter 4.1 --- Chapter summary --- p.103 / Chapter 4.2 --- Introduction --- p.104 / Chapter 4.3 --- Materials and methods --- p.106 / Chapter 4.3.1 --- Mouse strain --- p.106 / Chapter 4.3.2 --- Behavioral tests --- p.106 / Chapter 4.3.3 --- Histological examination of cerebellum --- p.107 / Chapter 4.3.4 --- CreER[superscript T]² induction by tamoxifen --- p.108 / Chapter 4.3.5 --- Gene expression profiling using microarray --- p.109 / Chapter 4.3.6 --- Transmission electron microscopy --- p.110 / Chapter 4.3.7 --- Statistical analysis --- p.111 / Chapter 4.4 --- Results --- p.112 / Chapter 4.4.1 --- Early postnatal developmental delay in female DKO mutant --- p.112 / Chapter 4.4.2 --- Lhx1/5 DKO mutants displayed significant motor deficit --- p.114 / Chapter 4.4.3 --- Abnormal Purkinje cell dendritic arborization in the adult Lhx1/5 DKO mutant mouse --- p.117 / Chapter 4.4.4 --- Reduction in the number of synaptic vesicles in the adult Lhx1/5 DKO mutant --- p.119 / Chapter 4.4.5 --- Abnormal Purkinje cell dendrite development in the Lhx1/5 DKO mutant mouse --- p.120 / Chapter 4.4.6 --- Lhx1/5 were not required for the maintenance of developed Purkinje cell dendrite --- p.122 / Chapter 4.4.7 --- Comparison of gene expression profiles in the Lhx1/5 DKO mutant and control --- p.128 / Chapter 4.5 --- Discussion --- p.130 / Chapter Chapter 5 --- Foxp4 - a potential interacting partner of Lhx1/5 --- p.137 / Chapter 5.1 --- Chapter summary --- p.137 / Chapter 5.2 --- Introduction --- p.138 / Chapter 5.3 --- Materials and methods --- p.139 / Chapter 5.3.1 --- Co-immunoprecipitation --- p.139 / Chapter 5.3.2 --- Foxp4 expression pattern and knockdown in cerebellar slice culture --- p.140 / Chapter 5.3.3 --- Generation of Foxp4 CKO mouse --- p.146 / Chapter 5.4 --- Results --- p.148 / Chapter 5.4.1 --- Lhx1 and Lhx5 physically interacted with Foxp4 --- p.148 / Chapter 5.4.2 --- Foxp4 expression during mouse cerebellum development --- p.150 / Chapter 5.4.3 --- Effective gene silencing by siRNA in cerebellar slice culture --- p.151 / Chapter 5.4.4 --- Silencing gene expression of Foxp4 at P5 exerted no observable effect on Purkinje cell survival or differentiation --- p.154 / Chapter 5.4.5 --- Developed Purkinje cell dendritic arbors and associated Bergmann glial fibers were impaired when Foxp4 was knockdown at P10 --- p.157 / Chapter 5.4.6 --- Generation of Foxp4 targeting construct and conditional knockout mouse --- p.159 / Chapter 5.4.7 --- Developmental delay of cerebellum in Foxp4 recombined homozygous mutants --- p.163 / Chapter 5.4.8 --- Normal cerebellum development in adult En1-Cre; Foxp4[superscript fx/fx] and Pax2-Cre; Foxp4[superscript fx/fx] mutants --- p.165 / Chapter 5.4.9 --- Purkinje cell-specific knockout of Foxp4 did not impair Purkinje cell maintenance, motor activity and learning --- p.167 / Chapter 5.5 --- Discussion --- p.171 / Chapter 5.6 --- Acknowledgements --- p.177 / Chapter Chapter 6 --- General discussion, future works and conclusion --- p.179 / Chapter 6.1 --- Evolutionary conserved function of Lhx1 and Lhx5 in neurons --- p.180 / Chapter 6.2 --- LHX1 and LHX5 in human diseases --- p.181 / Chapter 6.3 --- Transcription complex between LIM-homeodomain and forkhead domain proteins may be important in the cerebellum development --- p.182 / Chapter 6.4 --- Future works --- p.183 / Chapter 6.5 --- Conclusion --- p.186 / References --- p.187

"June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 162-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Rett syndrome (RTT) is a severe neurological disorder that affects approximately 1:10000 girls. Classical RTT is defined by a developmental regression phase and subsequent stabilisation of diagnostic criteria, which include partial or complete loss of spoken language, dyspraxic gait and stereotypic hand movements such as hand mouthing. RTT is a monogenic disorder, with the majority of cases being due to loss-of-function mutations in MeCP2 (methyl-CpG binding protein 2). Due to this clear genotype-phenotype link multiple RTT mouse models have been used to elucidate the molecular details, and consequent neuropathogenesis, of this complex neurological disease, as well as for the development of potential therapeutics for RTT. However, as the molecular details become clearer, the need for a simpler model system becomes evident. Human induced pluripotent stem cells (hiPSCs) generated from RTT patient fibroblasts are an option; however the handling of these cells is laborious, time-consuming and expensive and they often differentiate into a heterogeneous population of cells. To explore an alternative human model system I have been genetically engineering and experimenting with the human dopaminergic LUHMES cell line. LUHMES cells are an immortalised pre-neuronal cell line derived from an 8-week old, female foetus and can readily be differentiated into a homogeneous population of mature, electrically active neurons in just one week. In this thesis I have assessed the phenotypic properties of the wild-type cell line, demonstrated the ease of genetic manipulation of LUHMES cells by CRISPR/Cas9 approaches, generated seven mutant MECP2 LUHMES cell lines and explored the potential of protein therapy as a therapeutic approach for RTT. The LUHMES cell line proves to be extremely easy to handle and robust and has yielded novel molecular insights into the function of MeCP2 in human neurons. In particular, MeCP2-null cells show a striking relationship between the level of gene body methylation and the extent of transcriptional upregulation when compared to wild-type neurons. In contrast neurons that express a form of MeCP2 that can bind to DNA but cannot recruit a transcriptional corepressor complex (the R306C mutant) do not exhibit substantial gene expression alterations, yet do display a consistent decrease in total RNA amount. This decrease in total RNA is recapitulated in MeCP2-null LUHMES-derived neurons and in brain regions from MeCP2-R306C mice. The requirement for functional DNA binding for normal gene-body methylation dependent gene repression is demonstrated by assessing LUHMES cells that overexpress MeCP2-R111G, a protein that cannot bind to DNA. Furthermore, overexpression of the MeCP2-R306C protein highlights the importance of NCoR binding for normal gene repression, but also demonstrates that MeCP2-R306C protein retains some gene repression activity. Thinking more broadly, this cell line also has applications as a model system for a variety of other neurological disorders; as a simplified model system to elucidate molecular and neurological phenotypes, and as a relevant human system that can be cultured in a high-throughput manner for testing therapeutic strategies.