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Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-28Bitstream added on 2014-06-13T19:33:15Z : No. of bitstreams: 1 silveira_sm_me_botib.pdf: 772404 bytes, checksum: 2fd06918cebfdbcf5a739b4c7f827bc0 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Angiofibroma Nasofaríngeo Juvenil (ANJ) é um tumor vascular e localmente invasivo e, por ocorrer predominantemente em adolescentes do sexo masculino, sugere-se que é uma neoplasia hormônio dependente. Embora seja um tumor bastante raro e de crescimento lento, o ANJ pode progredir para lesões avançadas, tendo sérias seqüelas para seu portador. Por estas razões, este tumor pouco relatado em literatura é um modelo interessante para análise de alterações genômicas e moleculares. Neste estudo foi utilizada a metodologia de hibridação genômica comparativa de alta resolução (HR-CGH) para a triagem de perdas e ganhos cromossômicos em 18 amostras de ANJs. Em nove destes casos, as células endoteliais e os fibroblastos foram isolados por microdissecção a laser e analisados separadamente por HRCGH. As regiões genômicas consistentemente alteradas foram investigadas em bancos de dados com o objetivo de identificar genes candidatos que pudessem estar relacionados com a etiologia destes tumores. Baseando-se na função e localização, dois genes foram selecionados e validados pela análise de expressão quantitativa em tempo real (qRT-PCR) em 18 amostras de ANJs. Entre os 18 ANJs, foram detectadas 281 regiões genômicas preferencialmente alteradas. A análise dos componentes celulares isolados revelou aproximadamente 90 regiões cromossômicas significativamente alteradas. Os ganhos genômicos foram mais prevalentes nas células endoteliais do que nas células estromais. Regiões consistentes de perdas e ganhos comuns entre as mesmas amostras microdissecadas (dois componentes isolados) e não microdissecadas revelaram concordância de 58%. A análise individualizada dos dois componentes celulares presentes no tumor mostrou diversas regiões significativamente alteradas incluindo 3p, 4q, 8q, 16q e X. Ganhos em 8q21-q22, 4q25 e Xq11.2-q12 foram detectados nos dois componentes... / Juvenile Nasopharyngeal Angiofibroma (JNA) is a locally invasive vascular tumor that occurs predominantly in male adolescents, suggesting that the tumor may be hormonedependent. Although this tumor is rare and presents a slow growth, JNA may evolve to advanced lesions, resulting in severe consequences for the patient. For these reasons, this tumor, which is scarcely reported on literature, is an interesting subject to research for genomic and molecular alterations. In this study, the high-resolution comparative genomic hybridization (HR-CGH) methodology was performed to screening of genomic gains and losses in 18 JNA samples. In nine out of 18 cases, the endothelial and stromal components were isolated by laser microdissection and they were analyzed by HR-CGH separately. The consensus abnormal genomic regions were investigated using databases to identify potential candidate genes which could be associated to JNA etiology. According to the function data, two potential genes were selected and validated in a second group of 18 JNA samples by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) method. In 18 JNA cases, were observed 281 significant altered genomic regions. The differential analysis of the endothelial and stromal components identified about ninety significant altered chromosomal regions. The genomic gains were more prevalent in endothelial than fibroblasts cells. The comparison between non-microdissected and microdissected sample HR-CGH data showed concordance in 58% genomic regions. The individual analysis of each cellular component identified many chromosome regions significantly altered, including 3p, 4q, 8q, 16q e X. Gains in 8q21-q22, 4q25 and Xq11.2-q12, were detected in both endothelial and fibroblastic components. Genomic losses on 16q22.1 were frequently observed in stromal cells (8/9 cases) and 3p21 gain in endothelial cells (4/9 cases)... (Complete abstract click electronic access below)

Orientador: Silvia Regina Rogatto / Banca: Jair Cortez Montovani / Banca: Carla Rosenberg / Resumo: O Angiofibroma Nasofaríngeo Juvenil (ANJ) é um tumor vascular e localmente invasivo e, por ocorrer predominantemente em adolescentes do sexo masculino, sugere-se que é uma neoplasia hormônio dependente. Embora seja um tumor bastante raro e de crescimento lento, o ANJ pode progredir para lesões avançadas, tendo sérias seqüelas para seu portador. Por estas razões, este tumor pouco relatado em literatura é um modelo interessante para análise de alterações genômicas e moleculares. Neste estudo foi utilizada a metodologia de hibridação genômica comparativa de alta resolução (HR-CGH) para a triagem de perdas e ganhos cromossômicos em 18 amostras de ANJs. Em nove destes casos, as células endoteliais e os fibroblastos foram isolados por microdissecção a laser e analisados separadamente por HRCGH. As regiões genômicas consistentemente alteradas foram investigadas em bancos de dados com o objetivo de identificar genes candidatos que pudessem estar relacionados com a etiologia destes tumores. Baseando-se na função e localização, dois genes foram selecionados e validados pela análise de expressão quantitativa em tempo real (qRT-PCR) em 18 amostras de ANJs. Entre os 18 ANJs, foram detectadas 281 regiões genômicas preferencialmente alteradas. A análise dos componentes celulares isolados revelou aproximadamente 90 regiões cromossômicas significativamente alteradas. Os ganhos genômicos foram mais prevalentes nas células endoteliais do que nas células estromais. Regiões consistentes de perdas e ganhos comuns entre as mesmas amostras microdissecadas (dois componentes isolados) e não microdissecadas revelaram concordância de 58%. A análise individualizada dos dois componentes celulares presentes no tumor mostrou diversas regiões significativamente alteradas incluindo 3p, 4q, 8q, 16q e X. Ganhos em 8q21-q22, 4q25 e Xq11.2-q12 foram detectados nos dois componentes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Juvenile Nasopharyngeal Angiofibroma (JNA) is a locally invasive vascular tumor that occurs predominantly in male adolescents, suggesting that the tumor may be hormonedependent. Although this tumor is rare and presents a slow growth, JNA may evolve to advanced lesions, resulting in severe consequences for the patient. For these reasons, this tumor, which is scarcely reported on literature, is an interesting subject to research for genomic and molecular alterations. In this study, the high-resolution comparative genomic hybridization (HR-CGH) methodology was performed to screening of genomic gains and losses in 18 JNA samples. In nine out of 18 cases, the endothelial and stromal components were isolated by laser microdissection and they were analyzed by HR-CGH separately. The consensus abnormal genomic regions were investigated using databases to identify potential candidate genes which could be associated to JNA etiology. According to the function data, two potential genes were selected and validated in a second group of 18 JNA samples by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) method. In 18 JNA cases, were observed 281 significant altered genomic regions. The differential analysis of the endothelial and stromal components identified about ninety significant altered chromosomal regions. The genomic gains were more prevalent in endothelial than fibroblasts cells. The comparison between non-microdissected and microdissected sample HR-CGH data showed concordance in 58% genomic regions. The individual analysis of each cellular component identified many chromosome regions significantly altered, including 3p, 4q, 8q, 16q e X. Gains in 8q21-q22, 4q25 and Xq11.2-q12, were detected in both endothelial and fibroblastic components. Genomic losses on 16q22.1 were frequently observed in stromal cells (8/9 cases) and 3p21 gain in endothelial cells (4/9 cases)... (Complete abstract click electronic access below) / Mestre

[Truncated abstract] Introduction: Breast cancer is a heterogeneous disease, resulting in very different outcomes for women with apparently similar tumour characteristics. In order for patients to have optimal treatment, a better understanding of the molecular nature of their disease is required. Aims: The aims of this thesis were: 1) To determine whether methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes are associated with phenotypic features of breast cancer and the prognostic significance of methylation of these genes. 2) To investigate for possible associations between the frequency of methylation at RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST genes and the presence of germ-line variants in the TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes, as well as for possible correlations between these polymorphisms and clincopathological features of breast cancer including patient outcome. 3) To determine whether PIK3CA mutations determined clinical phenotype and the prognostic significance of PIK3CA mutations in a large and well characterized cohort of breast cancer patients. Methods: A large and well characterized series of primary breast tumours were selected for methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes using MSP, and for polymorphisms in TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes using PCR, PCR-RFLP and PCR-SSCP. Mutations to PIK3CA were detected using F-SSCP. Results and Conclusions: Methylation frequencies ranged from 11% for CCND2 to 84% for ER. More frequent hypermethylation was observed in tumours with poor histological differentiation compared to those with well/moderate differentiation, as well as trends for association with larger tumour size and mutant TP53. Tumours with ER and CDH1 methylation were associated with significantly lower hormone receptor levels, younger age at diagnosis and the presence of mutant p53. TWIST methylation is firstly reported to be associated with significantly older patient age at diagnosis and larger tumour size. Our data suggests that gene methylation may be linked to various pathological features of breast cancer. However, there appears to be little support for a distinctive CpG island methylator phenotype in breast cancer.

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of monotherapies. A major challenge facing tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. In contrast, certain strains of bacteria are gaining recognition as a new class of antigen-agnostic cell therapy due to their selective growth within the immunosuppressive niche of the solid tumor microenvironment (TME). In response, this dissertation aims to pair the cytotoxicity of CAR-T cells with the antigen-independent specificity of tumor-colonizing bacteria to create a new strategy for solid tumor recognition. Here, we reprogram the probiotic strain of E. coli Nissle 1917 to release synthetic CAR targets and human chemokines directly within the solid tumor core. To enable universal targeting, we design synthetic targets to bind ubiquitous components of the TME and broadly tag tumor tissue for CAR-mediated lysis. We demonstrate that these targets robustly coat the surface of cancer cell lines and lead to effective killing by CAR-T cells across various cancer types. We additionally show that injected probiotics selectively grow within the tumor core and maintain target production ¬ in situ – leading to therapeutic efficacy across multiple genetically distinct tumor models. Within this dissertation, we also reveal that intratumoral bacteria provide natural adjuvant effects that serve to activate and increase the effector functions of CAR-T cells in vivo. However, we discover that this can lead to early T cell exhaustion and terminal effector differentiation. To mitigate the counterproductive effects of overstimulation, we generate a new probiotic strain with reduced inflammatory properties that significantly improves CAR-T cell phenotype – leading to enhanced therapeutic benefit in a human model of leukemia. We conclude by discussing the numerous avenues available to optimize cross-Kingdom signaling and to ultimately leverage the full therapeutic benefit of combined cell therapies for future translation. Altogether, this dissertation highlights the potential of the probiotic-guided CAR-T cell (ProCAR) platform to address the critical roadblock of identifying suitable CAR targets by providing an antigen in situ that is orthogonal to both healthy tissue and tumor genetics – and, in turn, aims to establish the foundation for engineered communities of living medicines.

Medulloblastoma (MB) and supratentorial primitive neuroectodermal tumor (stPNET) are pediatric embryonic brain tumors, which arise in a brain that is in the process of growth and development. They differ significantly from adult lesions and may involve unique genetic and epigenetic factors. However, the pathogenesis of these tumors is still elusive. My project consisted of four parts, investigating major genetic and epigenetic alterations of these tumors. / Multiple genetic studies have shown high frequency of loss (30--60%) on chromosome 8p in MBs. Microcell-mediated transfer of chromosome 8 suppressed tumorigenesis or the proliferation of colon and breast cancer cell, indicating that chromosome 8p is likely to include several TSGs in human cancers. In previous studies from our laboratory, results showed the frequency of loss on chromosome 8p is also rather high (66.7%). An overlapping HD region was identified in a 1.8cM interval on 8p22-23.1, between markers D8S520 and D8S1130, in two MBs (Yin et al., 2002), indicating that several candidate TSGs are located within or near this region. PinX1 on 8p23.1, a potential inhibitor of telomerase, is most likely the candidate TSG in MBs due to its location and function. To evaluate the genetic alterations of PinX1 and to investigate its role in MBs, the first part of my study is to perform mutation analysis in a series of 52 primary MBs, 3 MB cell lines and 4 primary stPNETs. Transcript expression of PinX1 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected tumors and normal cerebellum. Using the telomeric repeat amplification protocol (TRAP) assay, 19 MBs, 2 stPNETs and all 3 MB cell lines were analyzed for telomerase activity. No somatic point mutations and loss of expression of PinX1 were detected in our series, suggesting that PinX1 is not the target gene on 8p23.1 in MBs. Although we did not find a significant association between PinX1 expression and telomerase activity, the presence of telomerase activity in 16 of 22 MBs and 1 of 2 stPNETs indicate that telomerase activation is associated with the development of this malignant disease. Our study represents the largest series of MB examined by telomerase repeat amplification protocol (TRAP) assay. (Abstract shortened by UMI.) / Chang Qing. / "April 2005." / Adviser: Ho-Keung Ng. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0191. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 201-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Indiana University-Purdue University Indianapolis (IUPUI)

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 1 is a genetic disease that results from either heritable or spontaneous autosomal dominant mutations in the NF1 gene, which encodes a protein serving, at least in part, to accelerate the intrinsic hydrolysis of active Ras-GTP to inactive Ras-GDP. A second-hit NF1 mutation precedes predominant NF1 neoplasms, including juvenile myelomoncytic leukemia (JMML) and plexiform neurofibroma formation, potentially fatal conditions with no medical therapy. While NF1 loss of heterozygosity (LOH) in myeloid progenitor cells sufficiently engenders leukemogenesis, plexiform neurofibroma formation depends on LOH in Schwann cells and Nf1 heterozygosity in the hematopoietic system. Specifically, recruited Nf1+/- mast cells accelerate tumorigenesis through secreted cytokines and growth factors. Nf1+/- mast cells depend upon deregulated signaling in c-kit pathways, a receptor system conserved in hematopoietic stem cells (HSCs). Accordingly, Nf1-/- myeloid progenitor cells, which can induce a JMML-like disease in mice, also demonstrate deregulated c-kit receptor signaling. C-kit-activated Nf1+/- mast cells and Nf1-/- myeloid progenitors both show increased latency and potency of active Erk1 and Erk2, the principal cytosolic-to-nuclear effectors of canonical Ras-Raf-Mek signaling. Thus, Erk represents a potential regulator of leukemogenesis and tumor-associated inflammation. However, single and combined Erk1 and Erk2 roles in HSC function, myelopoiesis, and mature mast cell physiology remain unknown, and recent hematopoietic studies relying on chemical Mek-Erk inhibitors have produced conflicting results. Here, we show that hematopoietic stability, myelopoiesis, and mast cell generation require Erk1 or Erk2, but individual isoforms are largely dispensable. Principally, Erk-disrupted hematopoietic stem cells incorporate BrdU but are incapable of dividing, a novel and cell type-specific Erk function. Similarly, mast cell proliferation requires Erk but cytokine production proceeds through other pathways, elucidating molecule-specific functions within the c-kit cascade. Based on these findings, we have reduced tumor mast cell infiltration by treating genetically-engineered tumor model mice with PD0325901, a preclinical Mek-Erk inhibitor. Moreover, we have devised a quadruple transgenic HSC transplantation model to examine dual Erk disruption in the context of Nf1 nullizygosity, testing whether diseased hematopoiesis requires Erk. These insights illuminate cell-specific Erk functions in normal and Nf1-deficient hematopoiesis, informing the feasibility of targeting Mek-Erk in NF1-associated disease.

Poon, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-124). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.6 / Chapter Chapter 2 --- Literature Reviews --- p.8 / Chapter 2.1 --- Cancer genetics --- p.8 / Chapter 2.1.1 --- Oncogenes and TSGs --- p.8 / Chapter 2.1.2 --- Kundsońةs two-hit event of cancer gene --- p.9 / Chapter 2.2 --- Cancer Epigenetics --- p.9 / Chapter 2.2.1 --- Types of Epigenetic regulation --- p.10 / Chapter 2.2.2 --- DNA methylation in TSGs --- p.10 / Chapter 2.2.2.1 --- Promoter CpG island in DNA methylation --- p.10 / Chapter 2.2.2.2 --- Protection system in DNA methylation --- p.11 / Chapter 2.2.2.3 --- Transcriptional silencing by DNA methylation --- p.11 / Chapter 2.2.2.4 --- DNA methylation of TSG silencing in cancers --- p.13 / Chapter 2.2.3 --- Hypomethylation of the cancer genome --- p.14 / Chapter 2.2.4 --- Clinical relevance of cancer epigenetic --- p.14 / Chapter 2.3 --- EGFR/STAT/SOCS pathway --- p.15 / Chapter 2.3.1 --- General Introduction of the EGFR pathway --- p.15 / Chapter 2.3.2 --- EGFR survival signaling pathways --- p.16 / Chapter 2.3.3 --- EGFR/STAT/SOCS signaling --- p.17 / Chapter 2.3.4 --- EGFR/STAT/SOCS signaling and cancers --- p.18 / Chapter 2.3.4.1 --- EGF and cancers --- p.18 / Chapter 2.3.4.2 --- EGFR/STAT/SOCS pathway and cancers --- p.18 / Chapter 2.3.4.3 --- EGF survival signaling as a target for cancer therapy --- p.19 / Chapter 2.4 --- TSGs in the EGFR/STAT/SOCS pathway --- p.20 / Chapter 2.4.1 --- Suppressors of cytokine signaling (SOCS) family --- p.20 / Chapter 2.4.2 --- Signal transducers and activators of transcription (STATs) family --- p.22 / Chapter 2.4.3 --- Sprouty (SPRY) family --- p.23 / Chapter 2.4.4 --- Protein Inhibitor of Activated STAT (PIASs) family --- p.25 / Chapter 2.4.5 --- Ras and Rab Interactor (RIN) family --- p.26 / Chapter 2.4.6 --- Ras-association domain family (RASSF) --- p.26 / Chapter 2.4.7 --- Glycine N-methyltransferase (GNMT) --- p.28 / Chapter 2.5 --- Nasopharyngeal carcinoma (NPC) --- p.30 / Chapter 2.5.1 --- Epidemiology of NPC --- p.30 / Chapter 2.5.2 --- Histopathology of NPC --- p.30 / Chapter 2.5.3 --- Genetic and epigenetic alteration in NPC --- p.31 / Chapter 2.5.4 --- EGFR signaling in NPC --- p.32 / Chapter 2.6 --- Esophageal squamous cell carcinoma (ESCC) --- p.33 / Chapter 2.6.1 --- Epidemiology of ESCC --- p.34 / Chapter 2.6.2 --- Histopathology of ESCC --- p.34 / Chapter 2.6.3 --- Genetic and epigenetic alteration in ESCC --- p.35 / Chapter 2.6.4 --- EGFR signaling in ESCC --- p.36 / Chapter Chapter 3 --- Materials and Methods --- p.38 / Chapter 3.1 --- General Materials --- p.38 / Chapter 3.1.1 --- "Cell lines, tumor and normal tissue samples" --- p.38 / Chapter 3.1.2 --- Maintenance of cell lines --- p.38 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.39 / Chapter 3.1.4 --- Total RNA extraction --- p.39 / Chapter 3.1.5 --- Genomic DNA extraction --- p.40 / Chapter 3.2 --- General techniques --- p.40 / Chapter 3.2.1 --- Agarose gel electrophoresis of DNA --- p.40 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.40 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.41 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.41 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.41 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.42 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.42 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.42 / Chapter 3.3 --- Preparation of reagents and medium --- p.43 / Chapter 3.4 --- Semi-quatitative Reverse-Transcription (RT) PCR expression analysis --- p.44 / Chapter 3.4.1 --- Reverse transcriptin reaction --- p.44 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.44 / Chapter 3.4.2.1 --- Primers design --- p.44 / Chapter 3.4.2.2 --- PCR reaction --- p.46 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.47 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.47 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.48 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.48 / Chapter 3.5.2.2 --- Primers design --- p.48 / Chapter 3.5.2.3 --- PCR reaction --- p.49 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.50 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.51 / Chapter 3.6.1 --- Sub-cloning of expression vector of candidate genes --- p.51 / Chapter 3.6.1.1 --- Mouse Socsl expression vector --- p.51 / Chapter 3.6.1.2 --- SPRY1 expression vector --- p.51 / Chapter 3.6.1.3 --- GNMT expression vector --- p.52 / Chapter 3.6.2 --- Restriction digestion of cloning vectors and expression --- p.52 / Chapter 3.6.3 --- Ligation of cloning fragments --- p.53 / Chapter 3.6.4 --- Colony formation assay on monolayer culture --- p.53 / Chapter 3.6.5 --- Statistical analysis --- p.54 / Chapter Chapter 4 --- Screening of candidate TSGs in EGFR pathway --- p.55 / Chapter 5.3.3 --- Restoration of GNMT expression by pharmacological demethylation --- p.89 / Chapter 5.3.4 --- Confirmation of the methylation status of GNMT promoter by BGS --- p.90 / Chapter 5.3.5 --- Methylation status of GNMT in ESCC and NPC primary tumors --- p.90 / Chapter 5.3.6 --- GNMT inhibited the growth of tumor cells in-vitro --- p.90 / Chapter 5.3.7 --- Discussion --- p.95 / Chapter Chapter 6 --- General Discussion --- p.100 / Chapter Chapter 7 --- Summary --- p.105 / Chapter Chapter 8 --- Future Study --- p.107 / Reference --- p.109