Metastasis is the primary cause of mortality in women with breast cancer. Recently, elevated serum levels of a glycoprotein known as chitinase-3 likeprotein- 1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in tumor progression have not yet been elucidated. In this document, we describe the role of CHI3L1 in tumor growth and metastasis and its relationship with inflammation. Using well-established models of breast cancer, we show that CHI3L1 is increased in the serum of tumor bearing mice. We found that CHI3L1 levels are increased at both the “pre-metastatic” and “metastatic stage” and that tumor cells, splenic, alveolar and interstitial macrophages; and myeloid derived population produce CHI3L1. Furthermore, we demonstrated that CHI3L1 has an inhibitory role on the expression of interferon-gamma (IFN γ) by T cells, while enhancing the production of pro-inflammatory mediators by macrophages such as Cchemokine ligand 2 (CCL2/MCP-1), Chemokine CX motif ligand 2 (CXCL2/IL-8) and matrix metalloproteinase-9 (MMP-9), all of which promote tumor growth and metastasis. We demonstrated that in vivo treatment of tumor-bearing mice with chitin microparticles, a TH1 adjuvant and a substrate for CHI3L1, promoted immune effector functions with increased production of IFN-γ but decreased CCL2/MCP-1, CXCL2/IL-8 and MMP-9 expression by splenic and pulmonary macrophages. Significantly, in vivo administration of chitin microparticles decreased tumor growth and pulmonary metastasis in mammary tumor bearing mice. These results suggest that CHI3L1 may play a role in tumor progression. Inflammation plays a pivotal role during tumor progression and metastasis by promoting the production of pro-inflammatory molecules such as CHI3L1. However, little is known about how CHI3L1 expression can affect secondary sites to enhance metastasis. In these studies, we demonstrated that CHI3L1 alters the cellular composition and inflammatory mediators that aid in the establishment of a metastatic niche for the support of infiltrating tumor cells leading to accelerated tumor progression. Since previous studies showed that CHI3L1 modulates inflammation, we determined the role of CHI3L1 in the context of pre-existing inflammation and metastasis. We found that CHI3L1 deficient mice with preexisting inflammation had decreased pro-inflammatory mediators, and significant reduction in tumor volume and metastasis compared to wild type controls. Preexisting inflammation and CHI3L1 may be driving the establishment of a premetastatic milieu in the lungs and aiding in the establishment of metastasis. Understanding the role of CHI3L1 in inflammation during tumor progression could result in the design of targeted therapies for breast cancer patients. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed. / by Kori Mulholland. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly. / by Kelli Robbins. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Colorectal cancer (CRC), with an estimated 1.2 million new cases annually, is the third leading cause of cancer incidence and death worldwide. Generally, the majority of CRC patients are diagnosed at the advanced stages with poor prognosis and unfavorable response to multiple therapeutic drugs. In spite of increasing knowledge of the molecular mechanism for the tumorigenesis in CRC patients, the translation from basic science into clinical therapy has been limited for quite a long time. In order to develop novel treatment strategies against CRC, intensive and extensive attempts have been made in the past decades. / The epithelial to mesenchymal transition (EMT) is a multi-step process characterized by the loss of cell polarity, decreased cell-cell adhesion as well as enhanced migration and invasion capacity. It is well documented that EMT is essential for a variety of cellular biological events ranging from embryogenesis to tumor progression. The field of lncRNA is developing rapidly and currently it is one of the most intensively studied fields in the biomedical sciences. Emerging evidence indicates that the majority of human genome encodes thousands of non-protein-coding RNA transcripts, nevertheless, the function of long non-coding RNAs (lncRNAs) in orchestrating EMT progression remains elusive. Historically, the lncRNA H19 was the first identified imprinted non-coding RNA transcript in human, and the H19/IGF2 locus acted as an ideal paradigm for the investigation of genomic imprinting genes. In recent years, the expression profiling and functional characterization of the H19 gene in a variety of human diseases has been extensively studied. / In our studies, H19 was characterized as a novel regulator of EMT in colon cancer. We first observed significant mesenchymal characteristics in the methotrexate-resistant HT-29 cells. Interestingly, significant upregulation of H19 was observed in mesenchymal-like MTX resistant HT-29 cells. We subsequently demonstrated that after treatment of TGF-β1, one of the most widely used EMT inducers, H19 presented dramatic increase during the EMT progression. To further investigate the functional role of H19 in EMT, we generated the stable cell lines overexpressing H19 in colon cancer cells using retroviral infection. Stable overexpression of H19 significantly promoted EMT progression in two epithelial colon cancer cell lines HT-29 and HCT-116. However, overexpression of H19 did not affect cell proliferation as well as cell cycle progression. Further proteomics studies screened out that ectopic expression of H19 upregulated the protein level of Vimentin, a vital biomarker for mesenchymal cells. By using the bioinformatics study in combination with luciferase reporter assays, we demonstrated that H19 potentiated the expression of several core marker genes essential for mesenchymal cells by serving as a competing endogenous RNA(ceRNA), which builds up the missing link between the regulatory miRNA network and EMT progression. According to the results from xenograft tumor model and soft agar assay, stable expression of H19 reinforced the in vitro and in vivo tumor growth. Moreover, the investigation of clinical specimens verified that H19 RNA level was significantly increased in colon cancer tissues compared with corresponding adjacent normal tissues. Taken together, the above observations imply that the lncRNA H19, by acting as a competing endogenous RNA, is an important regulator which tightly modulated the expression of multiple important genes involved in EMT and it could probably serve as a novel therapeutic target against colon cancer. / 大腸癌每年有一百二十萬新增個案，是世界第三大癌症殺手。通常情況下，大部分大腸癌病人發現時已經處於晚期，該時期的癌症病人對多種臨床治療藥物已無法治愈。盡管關於大腸癌發病的分子生物學機制已經不斷完善，但如何從基礎研究轉化為臨床治療手段在很長一段時間內不可實現。為了進一步研究新的抗擊大腸癌治療手段，廣泛且深入的研究已經不斷開展。 / 上皮間充質轉化是一個多步驟的過程，該過程的典型特徵為失去細胞的極性，細胞間粘連減弱以及細胞爬行遷移能力的不斷加強。目前科學家已經知道上皮間充質轉化對於從胚胎發育到腫瘤發展都起著重要的作用。近年來，長非編碼RNA的研究不斷快速發展，已然成為醫學研究中最激烈的領域之一。眾多證據表明人體基因組編碼數以千計不編碼蛋白質的RNA轉錄體。然而，這些RNA轉錄體在上皮間充質轉化中的功能依然所知甚少。長非編碼RNA H19是人體內第一個被鑒別出來參與到基因印記的非編碼RNA。資料表明H19/IGF2位點是一個非常理想的研究基因印記的位點。近年來，H19在眾多癌症中的表達以及功能學研究已不斷湧現，同時也不斷取得令人鼓舞的研究成果。 / 在我們的研究中，H19被鑒定為大腸癌裏上皮間充質轉化過程中一個重要的參與者。通過研究甲氨蝶呤耐藥大腸癌HT-29細胞株，我們發現該HT-29耐藥細胞株有著顯著的間充質細胞特性。有趣的是，H19在該細胞株中有著顯著升高。我們隨後用經典的上皮間充質轉化誘導劑TGF-β1處理兩株大腸癌細胞，處理後H19亦有著顯著升高。為了進一步研究H19在上皮間充質轉化，通過使用逆轉錄病毒，我們建立H19的穩定表達細胞株。穩定表達H19顯著地促進了HT-29以及SW620大腸癌細胞株的上皮間充質轉化。然後，高水平表達(過表達)H19並不影響細胞的生長以及細胞周期的進程。進一步的蛋白質組學研究表明，過表達H19能促進間充質細胞一個重要標記基因Vimentin的表達。通過生物信息學以及熒光素酶報告基因實驗，我們證明了H19通過其競爭內源性RNA的作用，能夠促進間充質細胞所需的幾個重要基因的表達。該發現建立起了miRNA網絡以及上皮間充質轉化進程的交流網絡。通過異位移植以及軟瓊脂實驗，我們發現過表達H19能夠促進腫瘤細胞的生長。而在臨床大腸癌病人組織中，我們更發現H19在大腸癌病人組織中高表達。綜上所述，我們的結果證明H19這一長非編碼RNA，能夠通過其競爭內源性RNA的作用機制，從而調控上皮間充質轉化過程中的關鍵基因。同時H19亦有可能成為治療大腸癌的臨床新靶點。 / Liang, Weicheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 95-124). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Liang, Weicheng.

腸神經系統(enteric nervous system, ENS)是由大量神經元和神經膠質細胞聚集而成的最複雜的周圍神經系統。這些腸道的神經元和神經膠質細胞來源于迷走神經脊和骶神經脊細胞，在胚胎發育過程中，這些神經脊細胞沿著腸道移動最終占滿整個腸道。儘管神經脊細胞的遷移對於腸道神經系統的形成及功能的正常發揮起到很重要的作用，然而影響神經脊細胞遷移的分子機制的研究卻相對較少。因此找出參與調控神經脊細胞遷移的基因對於更好的瞭解腸道神經脊系統的發育起到非常重要的作用，並且為治療腸道神經系統紊亂所導致的相關疾病提供治療靶點。 / 本研究論文是由兩部份實驗課題所組成來研究影響腸和調控道神經脊細胞遷移及腸道神經系統發育的相關基因。第一部份課題主要研究的是Semaphorin3A (Sema3A)對於骶神經脊細胞遷移的影響。本論文的研究發現Sema3A不僅被腸道內的上皮細胞所表達，腸道兩側的盆神經節周圍的間質細胞也表達Sema3A。同時Sema3A的受體neuropilin-1被骶神經脊細胞所表達。體外培養的實驗表明Sema3A能夠抑制骶神經脊細胞的遷移。另外，當表達Sema3A的腸道末端與骶神經脊細胞共同培養時，骶神經脊細胞的遷移同樣也受到抑制。這些研究結果表明由腸道末端的上皮細胞和腸道外圍的間質細胞所表達的Sema3A共同作用來調控骶神經脊細胞在停滯時期的遷移活動。 / 第二部份的研究課題主要研究的是轉錄因子Sox10以及其靶基因對於迷走神經脊細胞遷移的影響。Dominant megacolon (Dom)是一種攜帶有Sox10突變的巨結腸癥小鼠模型。本研究利用這種小鼠模型來發現突變鼠中可能影響迷走神經脊細胞遷移的基因。從迷走神經脊細胞體外培養發現： 由於Sox10突變，迷走神經脊細胞在體外培養24小時后，細胞遷移延遲，細胞的分化能力被改變，並且細胞死亡增加。利用基因芯片的方法比較了純和變異鼠迷走神經脊細胞和正常鼠迷走神經脊細胞的基因表達的差異。螢光素酶報告基因分析顯示，Sox10可以結合Lama4, Itga4和Gfra2的啟動子并激活它們的表達。 Sox10能與Gfra2啟動子上-116bp到-58bp之間序列的結合誘導Gfra2的表達。在純和變異鼠迷走神經脊細胞中，通過上調Gfra2信使RNA的表達，細胞死亡的數目大大下降，表明Gfra2作為Sox10的靶基因，對迷走神經脊細胞的存亡有著重要作用。 / 綜上所述，我們發現在骶神經脊細胞未進入腸道末端的這段停滯期內，Sema3A對於骶神經脊細胞的遷移起到抑制作用，Sema3A通過其表達在這段停滯期內的時空改變來調控骶神經脊細胞進入腸道。另外我們發現由於Sox10的突變，迷走神經脊細胞表現出非正常的遷移和基因表達的變化。作為Sox10的靶基因，Gfra2對於迷走神經脊細胞的死亡有重要的作用。 / The enteric nervous system (ENS) is the most complex part of the peripheral nervous system which is composed of a vast number of neurons and glial cells. The enteric neurons and glial cells arise from vagal and sacral neural crest cells (NCCs) which migrate along the gastrointestinal tract to colonize the whole gut during the embryonic development. The molecular mechanisms regulating the NCC migration are poorly characterized despite the importance of this migration process in the ENS formation. Therefore, identification and characterization of molecules involved in the modulation of NCC migration are essential to understand the ENS development and could provide potential therapeutic targets for the treatment of human ENS disorders. / The present study was aimed to identify and characterize the molecules involved in modulating the NCC migration during the ENS development, and was divided into two parts. The first part focused on semaphorin3A (Sema3A) signaling, Sema3A was found to be expressed in the hindgut epithelium and also the adjacent regions of pelvic ganglia, while its receptor, neuropilin-1, was expressed by sacral NCCs before sacral NCCs entered the hindgut. Sacral NCC migration and neuronal fiber extension in vitro were retarded in the culture medium containing Sema3A. When a hindgut segment expressing Sema3A was co-cultured with sacral NCCs, sacral NCC migration and neuronal fiber extension were also suppressed by the hindgut segment. These findings provide evidence for the repulsive activity of Sema3A before the entry of sacral NCCs to the hindgut. / The second part focused on the potential target genes of the transcription factor Sox10 which is expressed by migrating NCCs. A naturally occurring mouse mutant Dominant megacolon (Sox10Dom) which expresses a mutant Sox10 was used to identify candidate molecules which may possibly affect the NCC migration. After 24 hours in culture, vagal NCCs from Sox10Dom/Dom embryos showed retarded migration, abnormal cell differentiation and excessive cell death in vitro when compared to Sox10⁺/⁺ vagal NCCs. Results of microarray analyses revealed differentially expressed genes in Sox10Dom/Dom as compared to Sox10⁺/⁺ vagal NCCs after 24 hours in culture. Among these genes, Sox10 was able to bind to the promoter of Itga4, Lama4, and Gfra2 to induce their expression. Sox10 activated Gfra2 promoter by direct binding to the critical region located between -116bp and -58bp upstream of the Gfra2 transcription start site. Finally, re-expression of Gfra2 in Sox10Dom/Dom vagal NCCs resulted in decreased cell death, suggesting that down-regulation of Gfra2 in the mutant mice played an important role in early cell death of vagal NCCs. / In conclusion, before sacral NCCs entered into the hindgut, Sema3A inhibited the sacral NCC migration, and the spatiotemporal change of the Sema3A distribution regulated the entry of sacral NCCs into hindgut. Furthermore, retarded cell migration, abnormal cell differentiation, increased cell death and differential gene expression were found in Sox10Dom/Dom vagal NCCs as compared with those from Sox10⁺/⁺ embryos in vitro. The expression of Gfra2, a potential target gene of Sox10, promoted the cell viability of vagal NCCs. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Cuifang. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 180-196). / Abstracts also in Chinese. / Wang, Cuifang.

Despite the tremendous advances of the last century, the cause of Alzheimer disease (AD) remains unclear. Genetic analysis of families with Alzheimer disease has revealed a disease-associated variant of the APOE gene, which encodes apolipoprotein E, a transporter of lipids in the blood and central nervous system. The effect of the AD-associated isotype, termed ApoE-E4, on disease risk has been validated, though it is unclear by what mechanism apoE-E4 confers AD risk. Mitochondria have long been implicated in AD pathogenesis, as the canonical histopathological findings of amyloid plaques and tau tangles occur in the setting of mitochondrial dysfunction. The disrupted processes include calcium homeostasis, cholesterol metabolism, phospholipid synthesis, and mitochondrial dynamics, and are all regulated by a subcompartment of the ER that is in physical contact with mitochondria. This compartment, called the mitochondria-associated ER membrane, or MAM, has been found to be overactive in AD patient cell lines and cell models of AD. Given that MAM is dysfunctional in AD and that ApoE-ε4 is the most important risk factor for AD, this dissertation examines if ApoE4 contributes to the MAM dysfunction seen in AD. The MAM dysfunction seen in AD patients and in cell models of AD has been best characterized in the context of familial AD, and it is the purpose of this study to extend those findings to the more common, sporadic, form of the disease. Familial AD is the result of autosomal dominant mutations in one of three genes, amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2). APP is the protein from which amyloid-beta, the component of amyloid plaques, is cleaved. The presenilins constitute the enzymatic core of the γ-secretase complex, which cleaves amyloid-beta from a precursor APP molecule. Both PSEN1 (PS1) and PSEN2 (PS2) localize at the MAM, and their action is speculated to influence MAM activity. Fibroblasts from familial AD patients, which contained mutations in APP, PSEN1 or PSEN2, showed a marked increase in MAM activity when compared to that of age-matched controls. In mouse embryonic fibroblasts, one can recapitulate this increased MAM activity by knocking out presenilins 1 and 2. In these Psen1/2 double knockout (DKO) cells, the typical measures of MAM function, i.e. increased cholesteryl ester and phosphatidylethanolamine synthesis, calcium transport from ER to mitochondria, and co-localization of ER and mitochondria by confocal and electron microscopy, mimicked the same phenotype found in fibroblasts obtained from familial AD patients, which suggests that the presenilins are negative regulators of ER-mitochondrial apposition. The precise mechanism by which they regulate the ER-mitochondria interface, whether directly as part of a tethering complex, or indirectly though the metabolism of APP-derived substrates, is unclear. While the effect of familial AD mutations on MAM has been characterized, the mechanism of mitochondrial dysfunction seen in the more common sporadic form of the disease remains obscure. Sporadic AD patients harbor no mutations in APP, PSEN1, or PSEN2, but rather inherit mutations in other genes which do not guarantee the development of the disease, but are instead considered risk factors. The most important of these risk factors, in terms of both amount of AD risk conferred and prevalence in the population, is ApoE. Embedded in the phospholipid monolayer of lipoproteins, ApoE is involved in the transport of phospholipids, cholesterol, and cholesteryl esters in plasma and the central nervous system (CNS). In the CNS, it is the most abundant apolipoprotein, and is secreted primarily by astrocytes and taken up by neurons. Once endocytosed, ApoE can follow three different pathways: degradation by the lysosome, intracellular retention in early endosomes, or re-lipidation and re-secretion out of the cell. Our approach takes advantage of the physiological role of ApoE as part of a high densitylike lipoprotein particle (HDL). Using astrocytes from ApoE targeted gene replacement mice in which murine APOE has been replaced by either human APOE-E3 or human APOE-E4, cultured media containing ApoE3 and ApoE4-lipoproteins can be produced and applied to target cells that do not express ApoE, such as neurons or fibroblasts. These target cells can then be analyzed for MAM activity. To examine the contribution of ApoE towards MAM dysfunction, target cells, either neurons or fibroblasts, were grown in the presence of astrocyte conditioned media (ACM) from ApoE targeted gene replacement mice. Several measures of phospholipid and cholesteryl ester synthesis were performed to analyzed MAM function. To confirm that the alterations in phospholipid synthesis were the result of altered MAM activity, the same assay was performed in cells in which a protein tethers that bind mitochondria and ER were genetically ablated. Finally, to confirm that the effects seen were the result of the HDL particles and not the result of other components of the ACM, lipoproteins were extracted from ACM by density ultracentrifugation and applied to fibroblasts. In all of the assays performed, ApoE4 conditioned media or ApoE4 isolated lipoproteins were able to induce a significant increase in MAM activity, whereas ApoE4 from recombinant sources did not. These data suggest a contribution of ApoE4 towards MAM dysfunction seen in AD. The mechanism of these ApoE4 induced MAM alterations remains to be deduced. One may speculate that given the role of ApoE in cholesterol transport outside of the cell, its intracellular retention may impact the distribution of cholesterol within the cell. MAM is a cholesterol rich subdomain with lipid raft-like properties, and any change in the cholesterol content or lipid nature of this membrane may alter its activity. To test this hypothesis, MAM was biochemically extracted from ApoE3 and ApoE4 treated cells and analyzed for cholesterol and lipidomic content. The results described in this thesis demonstrate an AD-like effect in wildtype cells when treated with ApoE-E4, and that the mechanism for these alterations may be due to disturbances in cholesterol distribution in the MAM.

Le séquençage du génome humain et l'émergence de nouvelles technologies de génomique à haut débit, ont initié de nouveaux modèles d'investigation pour l'analyse systématique des maladies humaines. Actuellement, nous pouvons tenter de comprendre les maladies tel que le cancer avec une perspective plus globale, en identifiant des gènes responsables des cancers et en étudiant la manière dont leurs produits protéiques fonctionnent dans un réseau d’interactions moléculaires. Dans ce contexte, nous avons collecté les gènes spécifiquement liés à la Leucémie Lymphoblastique Aiguë (LLA), et identifié de nouveaux partenaires d'interaction qui relient ces gènes clés associés à la LLA tels que NOTCH1, FBW7, KRAS et PTPN11, dans un réseau d’interactions. Nous avons également tenté de prédire l’impact fonctionnel des variations génomiques tel que des fusions de gènes impliquées dans LLA. En utilisant comme modèles trois différentes translocations chromosomiques ETV6-RUNX1 (TEL-AML1), BCR-ABL1, et E2A-PBX1 (TCF3-PBX1) fréquemment identifiées dans des cellules B LLA, nous avons adapté une approche de prédiction d’oncogènes afin de prédire des perturbations moléculaires dans la LLA. Nous avons montré que les circuits transcriptomiques dépendant de Myc et JunD sont spécifiquement dérégulés suite aux fusions de gènes TEL-AML1 et TCF3-PBX1, respectivement. Nous avons également identifié le mécanisme de transport des ARNm dépendant du facteur NXF1 comme une cible directe de la protéine de fusion TCF3-PBX1. Grâce à cette approche combinant les données interactomiques et les analyses d'expression génique, nous avons fourni un nouvel aperçu à la compréhension moléculaire de la Leucémie Lymphoblastique Aiguë. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Law Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 214-225). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES --- p.X / LIST OF TABLES A --- p.XII / APPENDIX --- p.XIII / Chapter CHAPTER ONE --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Chinese herbs --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- Leigongteng --- p.2 / Chapter 1.1.2.1 --- Leigongteng and its importance --- p.2 / Chapter 1.1.2.2 --- Chemical components and pharmacological effects of Leigongteng --- p.4 / Chapter 1.1.3 --- Problems in authentication of Leigongteng --- p.5 / Chapter 1.1.3.1 --- Taxonomic problems of Tripterygium --- p.5 / Chapter 1.1.3.2 --- Confusion caused by other species --- p.7 / Chapter 1.1..3.3 --- Adulterants --- p.9 / Chapter 1.2 --- Celastraceae --- p.10 / Chapter 1.2.1 --- Introduction --- p.10 / Chapter 1.2.2 --- Controversial taxonomic issue --- p.12 / Chapter 1.2.1.1 --- Subfamilies of Celastraceae --- p.12 / Chapter 1.2.1.2 --- Subfamily Tripterygioideae --- p.13 / Chapter 1.3 --- Molecular authentication --- p.14 / Chapter 1.4 --- Molecular systematics --- p.18 / Chapter 1.4.1 --- DNA sequence markers --- p.19 / Chapter 1.4.2 --- Molecular phylogeny --- p.25 / Chapter 1.4.2.1 --- Tree-building method --- p.25 / Chapter 1.4.2.2. --- Measures of support --- p.28 / Chapter 1.5 --- Objectives --- p.29 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.31 / Chapter 2.1 --- Plant and herb samples --- p.31 / Chapter 2.2 --- DNA extraction --- p.41 / Chapter 2.2.1 --- Modified CTAB extraction --- p.41 / Chapter 2.2.2 --- Commercial kit extraction --- p.42 / Chapter 2.3 --- Polymerase chain reaction (PCR) condition --- p.43 / Chapter 2.4 --- DNA gel electrophoresis --- p.44 / Chapter 2.5 --- PCR product purification --- p.45 / Chapter 2.5.1 --- GEL-M´ёØ gel extraction system --- p.45 / Chapter 2.6 --- Ligation and transformation --- p.46 / Chapter 2.6.1 --- Ligation and transformation --- p.46 / Chapter 2.6.2 --- Cell cultivation --- p.47 / Chapter 2.6.3 --- Plasmid extraction --- p.47 / Chapter 2.7 --- Determination of DNA concentration --- p.49 / Chapter 2.8 --- Cycle sequencing --- p.49 / Chapter 2.9 --- Sequence analysis --- p.50 / Chapter 2.10 --- Materials preparation --- p.51 / Chapter CHAPTER THREE --- MOLECULAR AUTHENTICATION OF LEIGONGTENG --- p.54 / Chapter 3.1. --- Authentication based on internal transcribed spacer (ITS) region --- p.54 / Chapter 3.1.1 --- Sequence alignment --- p.54 / Chapter 3.1.2 --- ITS region nucleotide differences significant in authentication of Leigongteng --- p.55 / Chapter 3.1.3 --- Relationship of samples --- p.70 / Chapter 3.1.4 --- Comparison of sequences --- p.75 / Chapter 3.2 --- Authentication based on 5s-rDNA region --- p.78 / Chapter 3.2.1 --- Sequence alignment --- p.78 / Chapter 3.2.2 --- 5s-rDNA nucleotide differences significant in authentication of Leigongteng --- p.78 / Chapter 3.2.3 --- Relationship of samples --- p.88 / Chapter 3.2.4 --- Comparison of sequences --- p.90 / Chapter 3.3 --- Authentication based on psbA-trnH region --- p.93 / Chapter 3.3.1 --- Sequence alignment --- p.93 / Chapter 3.3.2 --- psbA-trnH nucleotide differences significant in authentication of Leigongteng --- p.101 / Chapter 3.3.3 --- Relationship of samples --- p.113 / Chapter 3.3.4 --- Comparison of sequences --- p.115 / Chapter 3.4 --- Authentication based on trnL-F region --- p.118 / Chapter 3.4.1 --- Sequence alignment --- p.118 / Chapter 3.4.2 --- trnL-F region nucleotide differences significant in authentication of Leigongteng --- p.121 / Chapter 3.4.3 --- Relationship of samples --- p.139 / Chapter 3.4.4 --- Comparison of sequences --- p.141 / Chapter 3.5 --- Discussion --- p.144 / Chapter 3.5.1 --- Molecular markers --- p.144 / Chapter CHAPTER FOUR --- PHYLOGENETIC STUDIES ON TRIPTERYGIUM --- p.151 / Chapter 4.1 --- Combine loci of ITS and 5s-rDNA regions --- p.152 / Chapter 4.1.1 --- Homogenity test --- p.152 / Chapter 4.1.2 --- Sequence alignment --- p.152 / Chapter 4.1.3 --- Phylogenetic study --- p.173 / Chapter 4.2 --- psbA-trnH region --- p.174 / Chapter 4.2.1 --- Sequence alignment --- p.174 / Chapter 4.3 --- Discussion --- p.177 / Chapter CHAPTER FIVE --- PHYLOGENETIC STUDIES ON TRIPTERYGIOIDEAE AND CELASTRACEAE --- p.191 / Chapter 5.1 --- ITS regions --- p.191 / Chapter 5.1.1 --- Sequence alignment --- p.191 / Chapter 5.1.2 --- Phylogenetic analysis --- p.205 / Chapter 5.2 --- Discussion --- p.206 / Chapter 5.2.1 --- Subfamily Tripterygioideae --- p.206 / Chapter 5.2.2 --- Subfamilies of Celastraceae --- p.210 / Chapter CHAPTER SIX --- CONCLUSION --- p.212 / BILBIOGRAPHY --- p.214

Made available in DSpace on 2014-07-29T15:26:23Z (GMT). No. of bitstreams: 1 Tese Marcia Alves Dias Matos.pdf: 1405580 bytes, checksum: cff6253aff8dbc7e48a7a6c687cebfa0 (MD5) Previous issue date: 2007-12-18 / Hepatitis B virus (HBV) infection occurs throughout the world. In Africa, this infection is highly endemic, with the majority of individuals becoming infected during childhood. Although Brazil has been globally considered a country of HBV intermediate endemicity, variable rates have been found in all five Brazilian regions and even inside the same region. This study aimed to investigate the epidemiological and molecular profile of the HBV infection among the Kalunga population in Goiás, Central Brazil, which is considered the largest Afro-Brazilian isolated community. A total of 878 individuals were interviewed about sociodemographic characteristics, risk factors and HBV vaccination. Blood samples were collected from all participants and serum samples were screened by enzyme-linked immunosorbent assay for the presence of HBsAg, anti-HBc and anti-HBs serological markers. HBsAg-positive samples were submitted to HBeAg and anti-HBe detection. HBsAg and anti-HBc positive samples were tested for HBV DNA detection by polymerase chain reaction and genotyping by subsequent restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of preS/S region. The overall prevalence of HBV infection was 35.4% (95% CI: 32.3-38.7). HBsAg carrier rate was 1.8% (95% CI: 1.1- 3.0). Multivariate analysis of risk factors showed that increased age, male gender, illiteracy and history of multiple sexual partners were associated with this infection. Isolated anti-HBs was found in 301 (34.3%) individuals who were immune for hepatitis B. HBV DNA was detected in 75% (12/16) of the HBsAg positive samples, in 100% (2/2) of the HBeAg and in 83.3% (10/12) of the anti-HBe positive samples. An occult HBV infection rate of 1.7% (5/295) was found among anti-HBc positive individuals. All genotyped isolates belonged to genotype A by RFLP analysis. Nucleotide sequencing of preS/S region confirmed the circulation of genotype A (subgenotype Aa) in this community. The epidemiological findings indicate that preventive measures, such as additional health education and HBV vaccination programs, are needed to control HBV infection in this population. In addition, the molecular results suggest the introduction of genotype A, subgenotype Aa in Brazil from Africa during the slave trade. / infecção pelo vírus da hepatite B (HBV) apresenta distribuição mundial. Na África, é altamente endêmica, sendo a maioria dos indivíduos infectada durante a infância. Embora o Brasil seja considerado um país de endemicidade intermediária, taxas variadas de prevalência têm sido encontradas nas cinco regiões geográficas e mesmo dentro de uma mesma região. Este estudo teve como objetivo investigar o perfil epidemiológico e molecular da infecção pelo HBV na população Kalunga em Goiás, Brasil Central, que é considerada a maior comunidade afro-descendente isolada no Brasil. Um total de 878 indivíduos foi entrevistado sobre características sócio-demográficas, fatores de risco e vacinação contra hepatite B. Amostras sanguíneas foram coletadas de todos os participantes e os soros triados para detecção dos marcadores HBsAg, anti-HBc total e anti-HBs por ensaio imunoenzimático. As amostras HBsAg positivas foram submetidos à detecção dos marcadores HBeAg e anti-HBe. O DNA viral foi detectado nas amostras HBsAg e anti-HBc reagentes pela reação da polimerase em cadeia, sendo as amostras HBV DNA positivas genotipadas pela análise do polimorfismo de comprimento dos fragmentos de restrição (RFLP) e sequenciamento da região Pré-S/S. A prevalência global da infecção pelo HBV foi de 35,4% (IC 95% 32,3-38,7), sendo de 1,8% (IC 95% 1,1-3,0) para o HBsAg. A análise multivariada mostrou que aumento da idade, gênero masculino, analfabetismo e história de múltiplos parceiros sexuais foram fatores associados a esta infecção. Em 301 (34,3%) indivíduos, verificou-se positividade isolada ao marcador anti-HBs, sugerindo imunidade ao HBV. O HBV DNA foi detectado em 75% (12/16) das amostras HBsAg reagentes, em 100% (2/2) das HBeAg e 83,3% (10/12) das anti-HBe positivas. Um índice de 1,7% (5/295) para infecção oculta pelo HBV foi encontrado nos indivíduos anti-HBc reagentes. Todas as amostras genotipadas por RFLP foram do genótipo A. O sequenciamento da região Pré-S/S confirmou a circulação do genótipo A (subgenótipo Aa) nesta comunidade. Os achados epidemiológicos indicam que medidas preventivas, como programas de educação em saúde e de vacinação contra hepatite B, são necessárias para o controle da infecção pelo HBV nesta população. Além disso, os resultados moleculares sugerem que o genótipo A, subgenótipo Aa foi introduzido no Brasil durante o tráfico de escravos da África.