Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 143-162).

EGR1, one of the immediate-early response genes, plays an important role as a mediator for transmitting extracellular stimuli. EGR1 is down regulated in many cancers. Many studies show that it functions as a tumor suppressor gene in a variety of cancers. EGR1 also acts as an oncogene in number of cancers. We found that in rhabdomyosarcoma (RMS), which is a muscle derived pediatric cancer, EGR1 was expressed in both RMS subtypes, embryonal and alveolar, but with a much higher expression profile in embryonal RMS. This suggests different mechanisms of down regulation of EGR1 in these two subtypes. Molecular and cellular approaches were used to characterize the functional role of EGR1 in RMS. We found that over expression of EGR1 in alveolar RMS significantly decreased cell proliferation, mobility, and anchorage-independent growth. We showed that exogenous EGR1 up regulated the cell cycle regulator, p21, which is normally repressed in RMS. EGR1 also promoted differentiation in RMS cells by up regulating several genes involved in muscle differentiation including myosin heavy chain (MyHC), MyoD and myogenin. We found that EGR1 interacts with the oncogene TBX2 in RMS cells and that TBX2 inhibits EGR1 function. To understand how TBX2 inhibits EGR1, we depleted TBX2 in RMS and we found an up regulation of the EGR1 targeted tumor suppressor gene, PTEN, and the cysteine protease inhibitor gene, CST6. Also, we performed luciferase assays and found that TBX2 decreased the expression of luciferase constructs fused with the PTEN promoter when TBX2 was co-transfected with EGR1. Our novel findings on the EGR1 function in RMS highlights the significant role of EGR1 in muscle development and tumor growth. Significantly, our work also suggests the EGR1 could promote tumor regression in RMS through inducing programmed cell death, or apoptosis. We found that EGR1 induced apoptosis through triggering the intrinsic apoptosis pathway and activating caspase cascades involving caspase 3 and caspase 9, which are essential mitochondrial apoptotic factors. Also, we observed the activation of two pro-apoptotic factors, BAX and dephosphorylated BAD, which are both located upstream of the caspase cascades in the intrinsic pathway. Also, we found in our study that EGR1 is repressed by the catalytic subunit of PRC2 complex, EZH2, which mediates gene silencing through methylation of lysine 27 on histone 3 (H3K27me3). EGR1 also sensitized RMS cells to chemotherapeutic agents, which could be a future direction for improved therapeutic targeting. Therefore, this work provides a novel and powerful molecular therapeutic target for RMS cancer.

>Magister Scientiae - MSc / P53 is one of the most important tumour suppressor proteins in the body which protects the cell against the tumourigenic effects of DNA damage by initiating processes such as apoptosis, senescence and cell cycle arrest. Regulation of p53 is key — so that the abovementioned processes are not initiated inappropriately. The principle negative regulator of p53 is Murine Double Minute 2 (MDM2), a RING finger-containing protein which catalyses the attachment of lysine48-linked poly-ubiquitin chains, targeting it for degradation by the 26S proteasome. It has been found to work in conjunction with the MDM2 homologue MDMX. Retinoblastoma Binding Protein 6 (RBBP6) is a RING finger-containing protein known to play a role in mRNA 3’-end processing, as well as interacting with p53 and another crucial tumour suppressor, pRb. It has previously been shown to cooperate with MDM2 in the ubiquitination and degradation of p53 in vivo and acts as a scaffold. The objectives of this project are to investigate the proposed role of RBBP6 in the MDM2-catalysed ubiquitination of p53 using a fully in vitro ubiquitination system. Due to the difficulty of expressing full length RBBP6 in bacteria, a shortened version, dubbed "R3" was used which includes the RING finger domain but excludes the domain identified in earlier studies as the p53-binding domain. Proteins required to set up the fully in vitro p53 ubiquitination assays – including E1 and E2 enzymes, MDM2, R3, p53 and ubiquitin - were all successfully expressed in bacteria. The active 26S proteasome was successfully purified out of human cell lysates using antibodies targeting the α2-subunit. Cloning, expression and purification results showed that p53, MDM2 and R3 were not very stable proteins to work with — with degradation being initiated almost immediately after expression and purification which progressed during the downstream processing of the proteins. Although levels of intact protein were not always high, they were sufficient for in vitro assays. MDM2 and GST-R3 were both capable of poly-ubiquitinating p53 independently in "partially in vitro" assays using human cell lysate. The fully in vitro ubiquitination of p53 using MDM2 and R3 was established based on the well-known MDM2/MDMX system. When acting together R3 and MDM2 was shown to produce poly-ubiquitination which is lysine-48 linked and recognised by the 26S proteasome leading to degradation. When the proteasome inhibitor MG132 was added, the poly-ubiquitinated p53 was rescued from degradation. R3 was also shown to successfully poly-ubiquitinate p53 independently of MDM2 and also interact with p53 in vitro. These results suggest R3 to be of the same order of importance as that of MDM2 — which is known to be the most important regulator of p53. It would also rule out the proposed model of RBBP6 functioning as a scaffold as it is able to poly-ubiquitinate p53 independent of MDM2. These results allow us to better understand the mechanism in which p53 is down-regulated by E3s. / National Research Foundation (NRF)

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / In nature, just twenty canonical amino acids are responsible for the creation of nearly all proteins. Genetic code expansion (GCE), or the incorporation of noncanonical amino acids (ncAAs) into living cells, is a powerful tool that expands the studies we are capable of performing using proteins. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs, and one of the main limitations of GCE arises from the inefficiency of these suppressor tRNAs when expressed in a foreign host cell. To address this limitation, we have previously reported a strategy for the virus-assisted directed evolution of tRNAs (VADER) which is uniquely capable of addressing the specific needs of tRNA evolution. In order to advance the capabilities of VADER, we made a number of modifications to the VADER selection scheme. First, we designed and executed a modified VADER selection that enabled the evolution of a new class of tRNAs, and with this VADER selection, we were able to generate a first-generation E. coli tyrosyl tRNA (tRNATyr) variant that was three times as active as its wild-type equivalent. Next, we introduced a number of refinements to the VADER strategy to generate VADER 2.0, an improved workflow capable of screening larger libraries and libraries encoding more active variants. Using VADER 2.0, we created second-generation tRNAPyl and tRNATyr mutants that achieved incorporation efficiencies that were greater than five-fold higher than their wild-type equivalents across a wide variety of substrates, enabling exciting GCE experiments that would not be possible otherwise. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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全球人類健康的頭號殺手--腫瘤目前仍是難以攻克的醫學難題。腫瘤的發生是一個復雜的過程，主要由促癌基因的異常增多或激活及抑癌基因（TSG）的缺失或功能喪失的累積效果導致。近年來基於非基因序列改變所致基因表達水平變化的表觀遺傳學的研究進展表明，啟動子區CpG島甲基化所致的表觀遺傳沉默是抑癌基因轉錄失活的重要機制。Ras和Wnt信號轉導通路在癌病的發生和發展過程中均起到重要的作用，因此針對該兩種信號通路的拮抗因子的表觀遺傳調控及功能學研究將為我們提供有研究及應用前景的候選抑癌基因。 / 作為一種重要的原癌基因，Ras家族基因具有致癌活性的點突變及其導致的過度激活的Ras信號通路被發現廣泛存在於大約30%的人類腫瘤中。然而在一些缺乏Ras基因突變的腫瘤類型中，持續激活的Ras信號通路仍然普遍存在並具有重要作用，昭示著除了Ras基因點突變以外的信號轉導異常激活的機制。與GTP的結合可激活Ras，而RasGAP家族蛋白可通過水解GTP達到使Ras失活的作用。通過采用微陣列比較基因組雜交(aCGH)的實驗手段我們發現6p21.3染色體區具有半接合子缺失， 並於此區域發現了候選抑癌基因RASA5。在以往的研究報道中，RASA5被命名為SynGAP且其功能研究僅限於神經系統。我們的研究發現不同於RasGAP家族的其它基因RASA2-4，RASA5廣泛表達於人類正常器官組織中，並特異性地在腫瘤細胞，特別是鼻咽癌(NPC)，食管鱗狀上皮細胞癌(ESCC)和乳腺癌這些具有野生型Ras基因但Ras信號通路仍被過度激活的細胞中被表觀遺傳沉默。RASA5的異位表達可有效促進腫瘤細胞的雕亡，抑制腫瘤細胞的生長、遷移及“幹性（stemness）“。同時，使用siRNA敲除內源性RASA5可以激發細胞的克隆形成及上皮-間質(EMT)轉化。RASA5的抑癌功能是通過調低Ras-GTP水平並進而抑制其下遊信號通路的活性實現的。過量表達具有致癌活性的點突變的Ras或RasGAP結構域缺失均可部分逆轉這種抑癌作用。此項研究首次證明了RASA5的抑癌功能。 / Wnt/Dvl/β-catenin信號轉導通路在人類腫瘤中存在廣泛的異常激活。我們發現DACT (Dpr/Frodo)家族成員TUSC-T2的表觀遺傳沉默是一種普遍存在於人類腫瘤中的現象。TUSC-T2編碼一種胞質蛋白，外源性表達TUSC-T2可促進腫瘤細胞雕亡並導致腫瘤細胞的克隆形成能力下降。TUSC-T2可與Dvl蛋白結合並下調其活化水平，從而保護GSK-3β蛋白不被Dvl蛋白抑制。GSK-3β可與Axin及APC蛋白形成蛋白質復合物，該復合物可捕捉並降解細胞內信號分子β-catenin。TUSC-T2的過量表達可以抑制β-catenin的激活及其向細胞核內的富集，並進一步阻止β-catenin在細胞核內與Lef/Tcf轉錄因子家族的作用及下遊特定原癌基因，例如c-Myc, CCND1及Fibronectin的表達。因此TUSC-T2具有抑制腫瘤細胞增殖、遷移及上皮-間質(EMT)轉化的作用。 / 綜上所述，我們的研究結果表明RASA5及TUSC-T2是具有抑癌功能的Ras或Wnt/Dvl/β-catenin信號轉導通路抑制因子，其表觀遺傳沉默導致的轉錄失活對於腫瘤的發生發展具有重要意義。同時，針對這兩種抑癌基因的進一步研究將為我們提供富有應用前景的腫瘤標記物。值得註意的是，RASA5課題的研究開創性地闡明了Ras信號通路的拮抗因子的表觀遺傳沉默是一種Ras信號轉導通路於腫瘤細胞中異常激活的新機制。 / Cancer is the top killer of the world, as well as the medical problem difficult to overcome. The conversion of a normal cell to a cancer cell is usually caused by upregulation of oncogenes and downregulation of tumor suppressor genes (TSGs). Epigenetic silencing has been proved to be important in TSGs inactivation, often through methylation of CpG-rich promoter regions. Ras and Wnt signaling pathways are both important for the tumorigenesis, epigenetic and functional studies of antagonists to Ras and Wnt signaling would provide us with candidate TSGs. / Ras is a well-known oncogene. Aberrant mutations of Ras genes occur in approximately 30% of human tumors, causing constitutively activated Ras signaling. However, in certain types of tumors with wild type Ras genes, abnormally activated Ras signaling is still a common and critical event, suggesting alternative mechanisms for Ras signaling hyperactivation. Ras is active when it is bound to GTP, while the hydrolysis of bound GTP and inactivation of Ras is catalyzed by Ras GTPase activating proteins (RasGAPs). Using 1-Mb array CGH (aCGH), we refined a small hemizygous deletion at the 6p21.3 chromosome region that contains a RasGAP family member gene RASA5, which used to be named as SynGAP and studied only in the neuron systems. We demonstrated that RASA5, rather than other RasGAP family members RASA2-4, is broadly expressed in human normal tissues while frequently epigenetically silenced in multiple tumors, especially in certain tumor types such as nasopharyngeal (NPC), esophageal (ESCC) and breast carcinomas (BrCa) with wild-type Ras while Ras cascade is still constitutively active. Ectopic expression of RASA5 led to apoptosis, growth and migration inhibition, as well as ‘stemness’ repression of tumor cells. Meanwhile, knockdown of RASA5 by siRNA promoted the tumor cell colony formation as well as epithelial-mesenchymal transition (EMT). The tumor-suppressive function of RASA5 was exerted through downregulating Ras-GTP level and further inactivating Ras signaling. Such an inhibitory effect could be partially abrogated in the presence of mutated, activated Ras or by deletion of the RasGAP domain. For the first time, our study refined the role of RASA5 as a tumor suppressor. / Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers. We identified a DACT (Dpr/Frodo) family member TUSC-T2 as an epigenetically downregulated gene in human tumors. TUSC-T2 encodes a punctate cytoplasmic protein. Ectopic expression of TUSC-T2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. TUSC-T2 interacts and downregulates Dishevelled (Dvl) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by Wnt/Dvl and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of TUSC-T2 disrupted β-catenin activation and accumulation in nuclei, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of β-catenin target oncogenes such as c-Myc, CCND1 and Fibronectin as well as the inhibition of tumor cell proliferation and migration. We also observed that TUSC-T2 could inhibit tumor cell EMT. / Taken together, our data demonstrate that RASA5 and TUSC-T2 are functional tumor suppressors epigenetically silenced in multiple tumors through acting as negative regulators of the Ras or Wnt/Dvl/β-catenin cancer pathways, and could be developed as promising biomarkers for human tumors. Of note, our study reveals that epigenetic silencing of the Ras antagonist represents a new mechanism responsible for Ras aberrant activation in cancers with wild-type Ras. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fan, Yichao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 184-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii-iii / List of tables --- p.iv / List of Figures --- p.v-vii / List of Publications --- p.viii-ix / Abstract in English --- p.x-xii / Abstract in Chinese --- p.xiii-xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer epigenetics --- p.4 / Chapter 1.1.1 --- Epigenetic modifications --- p.5 / Chapter 1.1.1.1 --- DNA Methylation --- p.5 / Chapter 1.1.1.2 --- Histone modifications --- p.10 / Chapter 1.1.1.3 --- RNA interference --- p.14 / Chapter 1.1.1.4 --- Nucleosome positioning --- p.15 / Chapter 1.1.2 --- Epigenetic alteration induced Tumor suppressor genes (TSGs) silencing during carcinogenesis --- p.17 / Chapter 1.2 --- Epigenetic alterations in cancer pathways --- p.23 / Chapter 1.2.1 --- Brief introduction of cancer pathways --- p.23 / Chapter 1.2.2 --- Ras pathway --- p.25 / Chapter 1.2.2.1 --- Ras pathway and carcinogenesis --- p.25 / Chapter 1.2.2.2 --- Epigenetic regulation of RasGAP proteins in carcinogenesis --- p.28 / Chapter 1.2.2.3 --- Epigenetic silencing of other negative regulators of Ras signaling --- p.30 / RAS association domain family (RASSF) proteins --- p.30 / PTEN --- p.32 / Sprouty (SPRY) proteins --- p.33 / Chapter 1.2.2.4 --- Hypomethylation induced Ras oncogenes activation --- p.35 / Chapter 1.2.2.5 --- Ras mediates epigenetic regulation through feedback loop --- p.36 / Chapter 1.2.3 --- Wnt pathway --- p.43 / Chapter 1.2.3.1 --- Wnt signaling pathway and carcinogenesis --- p.43 / Chapter 1.2.3.2 --- Epigenetic silencing of negative regulators of Wnt signaling --- p.45 / Chapter 1.2.3.3 --- DACT family proteins and carcinogenesis --- p.48 / Chapter 1.3 --- Application of tumor specific epigenetic alterations as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.1 --- The potential and advantage of tumor specific epigenetic alterations used as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.2 --- Epigenetic-disrupted regulators of Ras signaling as tumor biomarkers and therapeutic targets --- p.50 / Chapter 1.3.3 --- Epigenetic-disrupted regulators of Wnt signaling as tumor biomarkers and therapeutic targets --- p.52 / Chapter Chapter 2 --- Aims of this study --- p.54 / Chapter 2.1 --- To identify epigenetically silenced candidate TSGs as antagonists to Ras or Wnt signaling --- p.55 / Chapter 2.2 --- To elucidate the functional of candidate TSGs --- p.56 / Chapter Chapter 3 --- Materials and Methods --- p.57 / Chapter 3.1 --- Cell lines, tumor samples and routine cell line maintenance --- p.57 / Chapter 3.2 --- Drug and stress treatments --- p.59 / Chapter 3.3 --- DNA and RNA extraction --- p.59 / Chapter 3.4 --- Semi-quantitative RT-PCR and Real time PCR --- p.60 / Chapter 3.5 --- Direct sequencing of PCR products --- p.67 / Chapter 3.6 --- CpG island analysis --- p.67 / Chapter 3.7 --- Bisulfite treatment --- p.67 / Chapter 3.8 --- Methylation-specific PCR (MSP) and bisulfite genomic sequencing --- p.68 / Chapter 3.9 --- Plasmid extraction --- p.69 / Chapter 3.9.1 --- Bacteria culture --- p.69 / Chapter 3.9.2 --- Mini-scale preparation of plasmid DNA --- p.70 / Chapter 3.9.3 --- Large-scale endotoxin-free plasmids extraction --- p.71 / Chapter 3.10 --- Construction of expression plasmids --- p.71 / Chapter 3.10.1 --- Gene cloning and plasmids construction of RASA5 --- p.71 / Chapter 3.10.2 --- Gene cloning and plasmids construction of TUSC-T2 --- p.74 / Chapter 3.11 --- Immunofluorescence Staining --- p.74 / Chapter 3.12 --- Colony formation assay --- p.76 / Chapter 3.13 --- Apoptosis assay --- p.77 / Chapter 3.14 --- Luciferase reporter assay --- p.78 / Chapter 3.15 --- Protein preparation and Western blot --- p.79 / Chapter 3.16 --- Ras Activity Assay --- p.80 / Chapter 3.17 --- Wound healing assay --- p.81 / Chapter 3.18 --- Matrigel invasion assay --- p.81 / Chapter 3.19 --- RNA Interference --- p.81 / Chapter 3.20 --- Statistical analysis --- p.82 / Chapter Chapter 4: --- Epigenetic disruption of Ras signaling through silencing of a Ras GTPase-activating protein RASA5 in human cancers --- p.83 / Chapter 4.1 --- Identification of RASA5 as a downregulated gene residing in the 6p21.3 deletion region --- p.86 / Chapter 4.2 --- RASA5 is widely expressed in human normal tissues but downregulated in tumor cell lines --- p.91 / Chapter 4.3 --- The tumor-specific downregulation pattern of RASA5 is unique in the RASA family genes --- p.95 / Chapter 4.4 --- RASA5 promoter CpG methylation resulted in its transcription inactivation --- p.96 / Chapter 4.5 --- Frequent methylation of RASA5 promoter in multiple primary tumors --- p.101 / Chapter 4.6 --- Cloning and characterization of human RASA5 --- p.104 / Chapter 4.7 --- RASA5 inhibits tumor cell clonogenicity through inducing apoptosis --- p.108 / Chapter 4.8 --- RasGAP domain is required for the tumor suppressive function of RASA5 --- p.111 / Chapter 4.9 --- Certain cancer types harbor wild type Ras but active Ras signaling, with RASA5 epigenetically silenced --- p.114 / Chapter 4.10 --- RASA5 antagonizes Ras signaling pathway --- p.117 / Chapter 4.10.1 --- RASA5 represses Ras signaling through downregulating Ras-GTP level --- p.117 / Chapter 4.10.2 --- Oncogenic mutant form of Ras abrogated colony formation inhibitory effect of RASA5 on tumor cells --- p.120 / Chapter 4.10.3 --- Knockdown of RASA5 promoted the tumor cell colony formation and Ras signaling activation --- p.122 / Chapter 4.10.4 --- RASA5 inhibits ERK1/2 nuclei translocation and activation --- p.123 / Chapter 4.10.5 --- RASA5 negatively regulates Ras target gene expression --- p.125 / Chapter 4.11 --- RASA5 inhibits tumor cell migration and invasion through the Ras/Rac/cofilin signaling --- p.127 / Chapter 4.12 --- RASA5 suppresses tumor cell epithelial-mesenchymal transition (EMT) and stemness --- p.133 / Chapter 4.13 --- RASA5 appears in the cellcell interaction region nanotubes --- p.139 / Chapter 4.14 --- Discussion --- p.141 / Chapter Chapter 5: --- The Wnt/Dvl signaling antagonist TUSC-T2 is a pro-apoptotic tumor suppressor epigenetically silenced in tumors and inhibits tumor cell proliferation and migration --- p.150 / Chapter 5.1 --- Expression of TUSC-T2 is downregulated in human tumors --- p.150 / Chapter 5.2 --- TUSC-T2 promoter methylation results in its transcriptional inactivation --- p.151 / Chapter 5.3 --- Cloning and characterization of TUSC-T2 --- p.155 / Chapter 5.4 --- TUSC-T2 inhibits tumor cell clonogenicity through inducing apoptosis --- p.157 / Chapter 5.5 --- TUSC-T2 inhibits Wnt/Dvl/β-catenin pathway --- p.161 / Chapter 5.6 --- TUSC-T2 suppresses cell migration and EMT through upregulating E-cadherin --- p.165 / Chapter 5.7 --- Discussion --- p.171 / Chapter Chapter 6: --- Conclusions --- p.176 / Chapter 6.1. --- RasGAP family member RASA5 is epigenetically silenced in human cancers, acting as a tumor suppressor through negatively regulating Ras signaling --- p.177 / Chapter 6.2. --- DACT family member TUSC-T2 functions as a candidate TSG silenced by promoter methylation and inhibits Wnt/Dvl/β-catenin pathway --- p.178 / Chapter Chapter 7: --- Future Studies --- p.181 / Chapter 7.1. --- Further functional study of RASA5 and TUSC-T2 --- p.181 / Chapter 7.2. --- Clinical application of epigenetic silenced candidate TSGs --- p.182 / Chapter 7.3. --- Further screening of candidate TSGs as antagonists to cancer pathways --- p.183 / Reference list --- p.184

Dab2是一個細胞內接頭蛋白和腫瘤抑制因子。在小鼠胚胎中，應用免疫熒光染色技術，從E8.5-E11.0 Dab2發現表達於肌節的生皮肌節中。從E8.5 E9.5，Dab2表達於生皮肌節的中部。在E10.5，Dab2表達於生皮肌節的腹外側唇部，與肌肉發育的早期標誌基因Pax3和 Myf5共定位。從E11.5-E14.5，Dab2表達於四肢與軀體的肌肉中,Dab2在出生後小鼠肌肉中的表達逐漸減弱。此外，因為肌肉正常發育需要很多細胞信號的調節並且Dab2已經發現調節MAPK, TGF-β和 Wnt信號轉導通路。這些發現預示了Dab2在肌肉發育和分化中可能具有重要作用。 / 為了進一步研究它在肌肉發育中的作用，非洲爪蟾的胚胎和C2C12 肌原細胞在此研究中分別被用作體內和體外的研究模型。原位雜交結果揭示非洲爪蟾的Dab2基因表達於其胚胎的肌節中，並與肌肉發育的標誌基因XPax3, XMyoD, XMef2c和 XMyos共定位於此。用morpholino敲低XDab2 在非洲爪蟾胚胎中的表達，下調了許多肌肉發育標誌基因的表達，例如：XPax3, XMyf5, XMef2c, XMyoS 和XAC100。與此同時，免疫熒光技術也檢測到MHC(MF20)和12/101在肌節中的表達下調。 / 來源於小鼠肌肉衛星細胞的C2C12肌原細胞系被用作體外模型來檢測Dab2基因在骨骼肌發育和分化中的作用。在C2C12肌原細胞被誘導分化形成肌管的過程中，Dab2基因在RNA和蛋白水平的表達被誘導性的升高。Dab2基因超表達能夠加速肌原細胞的融合，從而增加肌小管的形成。利用miRNA敲低Dab2基因的表達能夠減緩肌原細胞的融合，從而減少肌小管的形成。利用慢病毒shRNA技術我們得到了2個Dab2穩定敲低細胞系，命名為克隆5-2和克隆5-7。這兩個克隆具有減少或抑制減少或抑制肌小管形成的特點。蛋白免疫印跡實驗表明，磷酸化p38 MAPK的表達在這兩個克隆中被抑制。在克隆5-2中超表達Dab2基因能夠恢復肌小管的形成。這個研究表明Dab2基因在肌小管的形成過程中具有至關重要的作用。 / 利用Affymetrix微陣列技術，我們檢測並分析了在克隆5-2和對照細胞中差異表達的基因。235個探針（155個基因）的顯示出超過2倍的差異表達。在這155個基因中，127個基因下調表達，28個基因上調表達。熒光定量PCR結果顯示出與微陣列結果相一致的結果。這些差異表達基因的功能發現與肌肉系統的發育和功能具有顯著地聯系。它影響了與肌肉收縮，橫紋肌的收縮，肌前體細胞的分化和肌肉發育相關功能的基因。基因網絡分析結果揭示，在克隆5-2中Mef2c基因的下調表達可能是一個導致肌細胞分化抑制的原因。 Mef2c基因在克隆5-2中超表達能夠拯救肌細胞的分化。 / 總括來說，體內和體外實驗共同表明Dab2基因是一個肌肉發育和分化的正調控基因。 / Dab2 is an intracellular adaptor protein and a tumor suppressor. In mouse embryos, Dab2 was found to be expressed in the dermomyotome of somites from E8.5 to E11.0 using immunofluorescence staining, with expression first detected in the medial aspect of the dermomyotome at E8.5 and then co-localized with the early muscle markers Pax3 and Myf5 at the ventrolateral lip of the dermomyotome at E10.5. From E11.5 to E14.5, Dab2 was expressed in muscle masses of limb buds and the trunk. Dab2 expression in skeletal muscles was gradually decreased after birth. These observations suggested potential roles of Dab2 in the skeletal muscle myogenesis. In addition, since the normal development of skeletal muscles requires proper signal transduction, and Dab2 has been known to be involved in the MAPK, TGF-β and Wnt signaling pathways, Dab2 may therefore be important for the muscle development. / To determine the role of Dab2 in the skeletal muscle development, Xenopus laevis embryos and C2C12 myoblasts were employed as in vivo and in vitro models, respectively. In situ hybridization results showed that XDab2 was expressed in somites of Xenopus embryos and co-localized with the muscle markers XPax3, XMyoD, XMef2c and XMyos. Knockdown of XDab2 expression with antisense morpholinos down regulated the expression of several muscle markers in somites including XPax3, XMyf5, XMef2c, XMyoS and XAC100. Down-regulation of MHC and 12/101 were also observed in whole mount preparations and transverse sections of XDab2 morpholino-injected embryos after immunohistochemical staining. / The C2C12 cell line derived from mouse muscle satellite cells was then employed as an in vitro model to determine the role of Dab2 during early muscle development. When C2C12 myoblasts were induced to differentiate into myotubes, Dab2 expression was simultaneously increased at RNA and protein levels. Dab2 over-expression after transfection with Dab2 plasmids resulted in enhanced myoblast fusion and increased numbers of myotubes. Conversely, suppression of Dab2 expression with miRNAs resulted in reduced myoblast fusion and decreased numbers of myotubes. Lentiviral shRNA-mediated Dab2 stable knockdown reduced myotube formation in 2 representative stable clones, clone 5-2 and clone 5-7. Western blot analysis showed that expression of phospho-p38 MAPK was down-regulated in clone 5-2 and 5-7. Dab2 re-expression through plasmid-mediated transient transfection in clone 5-2 could partially restore the myotube formation. These observations therefore suggested that Dab2 plays essential roles in the formation of myotubes. / Comprehensive profiling of differentially expressed genes was performed with the Affymetrix microarray analysis between the Dab2-knockdown clone 5-2 and the C2C12 parental cell line. As compared to the parental cells, the clone 5-2 showed significant changes in the expression of 235 probe sets representing 155 genes (p<0.05) with 2 folds or greater changes. Among the 155 genes, 127 were down-regulated, while 28 up-regulated. qRT-PCR results were found to be consistent with the microarray results. Functions of the differentially expressed genes were found to be significantly associated with the development and functions of the muscular system. Knockdown of Dab2 affected the genes involved in muscle contraction, the contraction of striated muscle, differentiation of muscle precursor cells, and the development of skeletal muscle fibers. A network analysis and a gene expression study revealed that Mef2c down-regulation was related to the inhibition of myogenic differentiation in the clone 5-2. Furthermore, forced expression of Mef2c in the clone 5-2 could rescue the myogenic differentiation. / In conclusion, these results indicated that Dab2 is positive regulator of the skeletal muscle development and differentiation both in vivo and in vitro. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Na. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 211-227). / 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.i / 摘要 --- p.iv / Acknowledgements --- p.vi / Table of contents --- p.vii / Abbreviation --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Characterizations of the Dab2 gene --- p.1 / Chapter 1.2 --- The role of Dab2 in Wnt/ beta-catenin signaling --- p.2 / Chapter 1.3 --- The role of Dab2 in TGF beta signaling --- p.3 / Chapter 1.4 --- The role of Dab2 in Ras-MAPK signaling --- p.4 / Chapter 1.5 --- The role of Dab2 in protein trafficking and endocytosis --- p.5 / Chapter 1.6 --- Dab2 expression and its functions. --- p.7 / Chapter 1.7 --- Somite and skeletal muscle development --- p.8 / Chapter 1.8 --- The formation of the somite and its structure --- p.9 / Chapter 1.9 --- The formation of dermomyotome and its function --- p.10 / Chapter 1.10 --- The formation of myotome and its function --- p.11 / Chapter 1.11 --- The formation of muscle fibers and musculatures --- p.12 / Chapter 1.12 --- The formation of satellite cells and its function in skeletal muscle differentiation --- p.12 / Chapter 1.13 --- The gene expression during skeletal muscle development and differentiation --- p.13 / Chapter 1.14 --- Dab2 genetically modified mice --- p.16 / Chapter 1.15 --- Objectives of this research --- p.17 / Chapter Figures and legends --- p.21 / Chapter Chapter 2 --- Expression of Dab2 in the mouse somites and skeletal muscles --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Mouse embryos and tissue isolation --- p.34 / Chapter 2.2.2 --- Histological preparation of embryos and tissues --- p.34 / Chapter 2.2.3 --- Immunostaining using Tyramide signal amplification kits --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Dab2 expression in somites of the mouse embryos --- p.36 / Chapter 2.3.2 --- Dab2 expression in skeletal muscles of embryonic and postnatal mice --- p.36 / Chapter 2.3.3 --- Co-localization of Dab2 and Pax3 immunoreactivities with double immunofluorescence staining --- p.37 / Chapter 2.3.4 --- Co-localization of Dab2 and Myf5 immunoreactivities with double immunofluorescence staining --- p.38 / Chapter 2.3.5 --- Co-localization of Dab2 and Myogenin immunoreactivities with double immunofluorescence staining --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Summary --- p.42 / Chapter Table 2.1 --- p.44 / Chapter Figures and Legends --- p.45 / Chapter Chapter 3 --- Dab2 is a positive regulator of skeletal muscle development in Xenopus embryos --- p.58 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- RNA extraction --- p.61 / Chapter 3.2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.61 / Chapter 3.2.3 --- Gene cloning and sequencing analysis --- p.61 / Chapter 3.2.4 --- Transformation --- p.62 / Chapter 3.2.5 --- Plasmid mini and midi-preparation --- p.62 / Chapter 3.2.6 --- Frogs and embryos handling --- p.63 / Chapter 3.2.7 --- Synthesis of mRNA for microinjection --- p.64 / Chapter 3.2.8 --- Microinjection --- p.64 / Chapter 3.2.9 --- Synthesis of DIG-labeled anti-sense RNA probe --- p.65 / Chapter 3.2.10 --- Whole mount in situ hybridization (WMISH) and whole mount immunohistochemical localization --- p.65 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Cloning of Xenopus Dab2 long isoform and the sequence analysis --- p.67 / Chapter 3.3.2 --- Phylogenetic analysis --- p.67 / Chapter 3.3.3 --- RT-PCR analysis of Xenopus Dab2 (XDab2) expression --- p.68 / Chapter 3.3.4 --- Xenopus Dab2 spatial and temporal expression examined by WMISH analysis --- p.68 / Chapter 3.3.5 --- Dab2 expression in somites and its colocalization with myogenic transcription factors --- p.69 / Chapter 3.3.6 --- XDab2 knockdown led to down-regulation of myogenic transcription factors and muscle markers at the RNA level --- p.70 / Chapter 3.3.7 --- XDab2 knockdown led to down-regulation of muscle markers at the protein level --- p.70 / Chapter 3.3.8 --- XDab2 overexpression led to up-regulation of XPax3, XMyf5 and XMyoS --- p.71 / Chapter 3.4 --- Discussion --- p.72 / Chapter 3.5 --- Summary --- p.77 / Chapter Table 3.1 --- p.78 / Chapter Figures and Legends --- p.79 / Chapter Chapter 4 --- Potential roles of Dab2 in C2C12 myoblast differentiation --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Cell culture and differentiation in vitro --- p.101 / Chapter 4.2.2 --- Cell sample preparation --- p.102 / Chapter 4.2.3 --- Real-time PCR --- p.102 / Chapter 4.2.4 --- SDS-PAGE --- p.103 / Chapter 4.2.5 --- Western blotting and immunodetection --- p.104 / Chapter 4.2.6 --- Plasmids used for transient over-expression --- p.105 / Chapter 4.2.7 --- Generation of miRNAs targeting at Dab2 --- p.105 / Chapter 4.2.8 --- C2C12 differentiation after transfection --- p.106 / Chapter 4.2.9 --- Immunohistochemical staining for myotubes --- p.106 / Chapter 4.2.10 --- Lentiviral shRNA mediated Dab2 stable knockdown --- p.107 / Chapter 4.2.10.1 --- shRNA Lentiviral Transduction Particles and sequence information --- p.107 / Chapter 4.2.10.2 --- Optimization of puromycin treatment on C2C12 myoblasts --- p.107 / Chapter 4.2.10.3 --- Determination of the optimal MOI for C2C12 --- p.108 / Chapter 4.2.10.4 --- Lentivirus transduction method --- p.109 / Chapter 4.2.10.5 --- Stable cell line generation --- p.109 / Chapter 4.2.11 --- Rescue experiments --- p.109 / Chapter 4.2.12 --- Serum starvation and FGF treatment --- p.110 / Chapter 4.2.13 --- Microarray and data analysis --- p.110 / Chapter 4.3 --- Results --- p.113 / Chapter 4.3.1 --- Expression of Dab2 during myogenesis --- p.113 / Chapter 4.3.2 --- Generation of miRNAs targeting at Dab2 --- p.113 / Chapter 4.3.3 --- Improvement of the transfection efficiency --- p.114 / Chapter 4.3.4 --- Knockdown efficiencies of the 4 miRNAs --- p.114 / Chapter 4.3.5 --- Down-regulation of Dab2 expression by transient transfection inhibited C2C12 differentiation --- p.115 / Chapter 4.3.6 --- Up-regulation of Dab2 expression by transient transfection enhanced myogenic differentiation --- p.116 / Chapter 4.3.7 --- Lentivirus-mediated Dab2 stable knockdown inhibited myotube formation --- p.117 / Chapter 4.3.8 --- Re-expression of Dab2 partially restored myogenic differentiation in the clone 5-2 --- p.120 / Chapter 4.3.9 --- Dab2 knockdown affected the MAPK signaling pathway --- p.122 / Chapter 4.3.10 --- Transcriptome and network analysis revealed changes of gene expression patterns in the C2C12 cell line after Dab2 knockdown --- p.123 / Chapter 4.3.11 --- Mef2c down-regulation was related to the inhibition of the myotube formation in the clone 5-2 --- p.126 / Chapter 4.4 --- Discussion --- p.128 / Chapter 4.4.1 --- Dab2 expression was found to be induced upon differentiation and down-regulated after myotube formation --- p.128 / Chapter 4.4.2 --- Dab2 was found to be a positive regulator of C2C12 differentiation --- p.129 / Chapter 4.4.3 --- Dab2 knockdown affected the MAPK signaling pathway --- p.131 / Chapter 4.4.4 --- Potential roles of Dab2 in myogenic differentiation revealed by transcriptome and network analysis --- p.133 / Chapter 4.4.5 --- Mef2c down-regulation may be involved in the inhibition of myogenic differentiation after Dab2 knockdown --- p.135 / Chapter 4.5 --- Summary --- p.138 / Chapter Table 4.1 --- p.141 / Chapter Table 4.2 --- p.142 / Chapter Table 4.3 --- p.143 / Chapter Table 4.4 --- p.144 / Chapter Table 4.5 --- p.147 / Chapter Table 4.6 --- p.148 / Chapter Table 4.7 --- p.149 / Chapter Figures and Legends --- p.150 / Chapter Chapter 5 --- Conclusions and discussion --- p.192 / Chapter 5.1 --- Dab2 expression in somites and skeletal muscles of mouse embryos --- p.192 / Chapter 5.1 --- Dab2 as a positive regulator for skeletal muscle development in Xenopus embryos in vivo --- p.194 / Chapter 5.3 --- Dab2 as a positive regulator of skeletal muscle development in vitro --- p.196 / Chapter 5.3.1 --- Dab2 was found to be a positive regulator of C2C12 differentiation --- p.196 / Chapter 5.3.2 --- Dab2 knockdown affected the MAPK signaling pathway --- p.198 / Chapter 5.3.3 --- Potential functions of Dab2 revealed by transcriptomeand network analysis --- p.200 / Chapter 5.3.4 --- Mef2c down-regulation was closely related to the inhibition of myogenic differentiation upon Dab2 knockdown --- p.202 / Appendix I --- p.204 / Appendix II --- p.205 / References --- p.211

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Regulation of the immune response is mediated, in part, by the action of suppressor T cells (Ts). One intriguing aspect of these cells is the description of T cell suppressor factor (TsF): a soluble analog of the cell that shares many of its properties, such as the ability to bind free antigen (Ag) and suppress an Ag-specific immune response. The exact molecular nature of TsF and the relationship of TsF to Ts are unknown. The immune response to the small, bacterial protein, ferredoxin (Fd), was used as a model system to study TsF. A Fd-specific suppressor cell network has been described in mice that are genetically nonresponsive to this Ag. Previously, a soluble mediator, known as Fd11F, was found in the culture supernatant (SN) of the Ts hybridoma, Fd11. Fd11F possessed both Ag-binding activity and the ability to suppress the anti-Fd Ab response in mice. The TsF-specific monoclonal antibody, B16G, was used for both the recovery of Fd11F-enriched material from SN and its detection by the enzyme-linked immunosorbent assay. ' Further immunochemical, biological, and biochemical characterization of Fd11F was done with emphasis on describing the Ag-binding properties of Fd11F. It was found that Fd11F bound to solid- and liquid-phase Fd, and demonstrated preferential binding to the carrier determinant of the Ag. A spleen cell culture assay was devised which showed that Fd11F suppressed Ab production in a concentration-dependent manner. Additional experiments suggested that the suppressive effect was Ag-specific. The identification of the Ag-binding molecule was attempted by the fractionation of Fd11F-enriched material using high performance gel filtration or preparative SDS-PAGE (run under non-reducing conditions). Using SDS-PAGE, a unique, single polypeptide of about 30k relative molecular mass (Mr) was identified as the Ag-binding moiety of Fd11F. The possible relationship of this moiety to other identified materials is discussed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate