No description available.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described. / text

As células pluripotentes (CPs), em teoria, são capazes de dar origem a todos os mais de 200 tipos de células do organismo. Na natureza, há três tipos de células pluripotentes: células-tronco embrionárias, células germinais embrionárias e células de carcinoma embrionário. As características das CPs têm permitido um importante avanço para a pesquisa básica e apontam uma grande aplicabilidade na medicina regenerativa. No núcleo das CPs existem fatores atuantes responsáveis pela manutenção da identidade pluripotente; dentre eles destacam-se OCT4, NANOG, SOX2, KLF4 e MYC. Muito já se sabe sobre os mecanismos que estes fatores atuam para promover a manutenção da pluripotência celular. Baseados nestes estudos foi possível gerar células de pluripotência induzida (iPSCs). Porém, os mecanismos moleculares que direcionam a indução da pluripotência ainda não estão muito bem esclarecidos. Alguns estudos revelaram que componentes chaves da via NFB estão envolvidos na regulação da pluripotência, bem como na diferenciação e destino celular das células-tronco. Neste estudo, analisamos a participação de componentes da via canônica (RelA e NFB1) e não-canônica (RelB e NFB2) de NFB nos processos de diferenciação e destino celular ou manutenção da pluripotência. Para isto usamos técnicas de PCR quantitativa em Tempo Real (qPCR) e Imunoprecipitação de Cromatina (ChIP) investigando os papéis das vias canônica e não-canônica de NFB na manutenção da pluripotência e diferenciação de CPs, em um modelo de indução de diferenciação celular mediado por ácido trans-retinóico (atRA) em células de carcinoma embrionário NTera-2. Foram avaliadas as ligações dos fatores de transcrição RelA e RelB nas regiões promotoras dos genes OCT4, SOX2, MYC, KLF4 e GFAP e a regulação transcricional associada. Nossos resultados identificaram que as células não tratadas com atRA apresentaram níveis baixos na expressão dos componentes da via canônica de NFB, RelA e NFB1, e GFAP e quando induzidas à diferenciação por atRA durante 4 dias esses níveis se elevaram. Uma situação oposta foi vista nos componentes da via não-canônica de NFB, RelB e NFB2, e na expressão dos fatores de pluripotência OCT4, NANOG, SOX2 e KLF4, que apresentaram níveis de expressão elevados nas células não tratadas com atRA e sofreram redução com a indução da diferenciação celular. O ensaio de ChIP revelou que RelA liga-se nas regiões de regulação dos genes OCT4, SOX2, KLF4, MYC e GFAP apenas quando a célula está em processo de diferenciação, enquanto RelB se apresentou ligado às mesmas regiões tanto nas células indiferenciadas quanto naquelas induzidas à diferenciação por 4 dias. Com estes dados sugerimos que a via canônica de NFB pode estar relacionada com o processo de diferenciação e destino celular através da regulação negativa executada por RelA e NFB1 nos genes responsáveis pela identidade pluripotente das células aqui estudadas enquanto a via não-canônica de NFB, representada pela ativação de RelB e NFKB2, pode participar na manutenção da pluripotência através da regulação positiva destes mesmos fatores. / Human pluripotent stem cells (hPSCs) are able to give rise to all the 200 cell types of the adult organism. In nature, there are three types of hPSCs: embryonic stem cells, germ line stem cells and embryonal carcinoma cells. hPSCs characteristics have allowed a major advance in basic research, and are thought to have great applicability in regenerative medicine. In the nucleus of hPSCs there are transcription factors responsible for the maintenance of their pluripotent identity. OCT4, NANOG, SOX2, KLF4 and MYC are considered the core pluripotency factors in hPSCs. A great deal of knowledge about the mechanisms that promote and maintain pluripotency has been generated. Based on these studies it was possible to generate induced pluripotent stem cells (iPSCs). However, the molecular mechanisms that drive the induction of pluripotency are not fully understood. Some studies have recently indicated that key components of the NFkB may be involved in regulating pluripotency as well as cell differentiation and cell fate. In this study we analyzed the involvement of components of the canonical (RelA and NFB1) and the non-canonical NFB pathways (RelB and NFB2) in the maintenance of pluripotency, differentiation and cell fate processes. The techniques of quantitative real-time PCR (qPCR) and chromatin immunoprecipitation (ChIP) were used to interrogate the roles of the canonical and non-canonical NFB pathways in maintenance of pluripotency and differentiation in a model of cell differentiation induced by all trans-retinoic acid (atRA) on embryonal carcinoma cells NTera-2. The transcription factors RelA and RelB occupancy in the promoter regions of OCT4, SOX2, KLF4, MYC and GFAP, and the transcriptional regulation associated were evaluated. Our results showed that undifferentiated cells exhibited low expression levels of canonical NFB pathway components, RelA and NFB1, while cells induced to differentiate for 4 days exhibited downregulated expression of these factors. In the other hand, the non-canonical NFB pathway components, RelB and NFB2, and the pluripotency factors OCT4, NANOG, SOX2 and KLF4 were expressed in higher levels in undifferentiated cells, and were downregulated upon the differentiation process. ChIP assay revealed that RelA binds to the regulatory regions of OCT4, SOX2, KLF4, MYC, and GFAP only when cells are induced to differentiate, while RelB was found bound to the same regions in both undifferentiated and differentiated cells. This data suggests that the canonical NFB pathway may be associated to differentiation and cell fate processes by downregulation of genes responsible for the pluripotent identity, and that the non-canonical NFB pathway may act in the maintenance of pluripotency through the upregulation of the same factors.

It was previously demonstrated that miR-199a was down-regulated in testicular germ cell tumor (TGCT) partly caused by hypermethylation of its promoter. More detailed analyses showed that miR-199a-5p, one of its two derivatives, suppressed TGCT invasiveness and proliferation via directing targeting PODXL and MAFB. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this project we identified DNMT3A, the de novo methyltransferase, as a direct target of miR-199a-3p using a 3’-UTR reporter assay. In NT2 (NTera 2) and HT (Hs 1.Tes) cells, miR-199a-3p regulated the expression of endogenous DNMT3A (both DNMT3A1 and DNMT3A2 isoforms), especially DNMT3A2 isoform. In clinical samples, the expression of DNMT3A2 and miR-199a-3p were reciprocally regulated. However, DNMT3A did not regulate miR-199a expression. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. MiR-199a-3p could restore the expression of APC and MGMT via de-methylation in their promoters. Our studies demonstrated the dysregulation of miR-199a-3p in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potential therapeutic use of synthetic miR-199a-3p oligonucleotides as effective demethylation agent in the treatment of TGCT. However, since DNMT3A expression did not regulate miR-199a expression, the mechanism of promoter DNA hypermethylation of miR-199a in TGCT needs further investigation. / MiR-199a is encoded by two loci in the human genome, namely, miR-199a-1 on chromosome 19 and miR-199a-2 on chromosome 1. Another microRNA, miR-214, also locates on chromosome 1. Previous study revealed that it is co-transcribed with miR-199a-2, which is directed by miR-199a-2 promoter. However, the biological significance of the co-expression of miR-199a and miR-214 remains largely unknown. In this project, it was determined that miR-199a and miR-214 were concordantly expressed in TGCT. Silencing of DNMT1 increased the expression of miR-199a and miR-214, accompanied by de-methylation in the promoters of miR-199a-1/2. Overexpression of TP53 down-regulated the expression of DNMT1 and increased the expression of mature miR-199-3p/5p and miR-214. In addition, silencing of PSMD10 up-regulated the expression of TP53, while miR-214 over-expression resulted in PSMD10 down-regulation and TP53 up-regulation. Collectively, our findings highlighted a miR-199a/miR-214/PSMD10/TP53/DNMT1 self-regulatory network, which caused the down-regulation of miR-199a, miR-214 and TP53, as well as the up-regulation of DNMT1 and PSMD10 in TGCT. These observations partly explain the mechanism of promoter DNA hypermethylation in miR-199a in TGCT. They also suggest a potential therapeutic approach by targeting the miR-199a/miR-214/PSMD10/TP53/DNMT1 regulatory network in the treatment of TGCT. / 先前的研究證實miR-199a在睾丸生殖細胞腫瘤 (簡稱睾丸癌) 中是低表達的，部分歸因於其啟動子區域過度甲基化。對其功能研究發現miR-199a能抑制睾丸癌細胞的生長，侵襲和轉移，且miR-199a的抑癌屬性應歸功於它的兩個衍生物之一miR-199a-5p。然而，miR-199a的另一個衍生物miR-199a-3p在睾丸癌中的生物學功能仍然在很大程度上是未知的。此研究中，DNMT3A被鑒定為miR-199a-3p的直接靶定目標。在NT2和HT細胞中，miR-199a-3p能調控內源性DNMT3A(DNMT3A1和DNMT3A2)的表達水準，尤其是DNMT3A2。在臨床樣本中，DNMT3A2的表達水準與miR-199a-3p的表達水準呈負相關。但DNMT3A並不能調控miR-199a的表達水準。進一步研究顯示過表達miR-199a-3p能減少APC和MGMT啟動子區域甲基化而恢復其表達水準。研究證實異常表達的miR-199-3p可能在睾丸癌的癌變過程中發揮作用，並提出一個潛在的治療方案，即使用miR-199a -3p作為有效的去甲基化藥劑治療睾丸癌。然而睾丸癌中導致miR-199a啟動子區域過度甲基化的機制有待進一步研究。 / 在人類基因組中，miR-199a-1(位於19號染色體)和miR-199a-2(位於1號染色體)都編碼miR-199a。同時miR -214也位於1號染色體，研究表明miR-214與miR-199a-2由miR-199a-2啟動子介導共同轉錄，但miR-199a和miR- 214共同表達的生物學意義仍未知。此研究中，miR-199a和miR-214在睾丸癌中的表達呈現一致性。沉默DNMT1後miR-199a和miR-214的表達水準顯著提高，並伴隨著miR-199a-1/2啟動子區域的DNA去甲基化。在NT2細胞中。過表達TP53能下調DNMT1的表達水準，同時上調miR-199-3p/5p和miR- 214的表達水準。此外，過表達miR -214能導致PSMD10表達水準的下調以及TP53表達水準的上調。綜上所述，我們提出一個miR-199a/miR-214/PSMD10/TP53/DNMT1自我調控網路，此調控通路能引起睾丸癌中miR-199a，miR-214和TP53表達水準的下調，以及DNMT1和PSMD10表達水準的上調，且部分解釋睾丸癌中miR-199a啟動子區域過度甲基化的機制，同時該調控網路可作為治療睾丸癌的一個潛在靶點。 / Chen, Bifeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 103-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 20, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

No description available.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (Leaves 188-223).

As células pluripotentes (CPs), em teoria, são capazes de dar origem a todos os mais de 200 tipos de células do organismo. Na natureza, há três tipos de células pluripotentes: células-tronco embrionárias, células germinais embrionárias e células de carcinoma embrionário. As características das CPs têm permitido um importante avanço para a pesquisa básica e apontam uma grande aplicabilidade na medicina regenerativa. No núcleo das CPs existem fatores atuantes responsáveis pela manutenção da identidade pluripotente; dentre eles destacam-se OCT4, NANOG, SOX2, KLF4 e MYC. Muito já se sabe sobre os mecanismos que estes fatores atuam para promover a manutenção da pluripotência celular. Baseados nestes estudos foi possível gerar células de pluripotência induzida (iPSCs). Porém, os mecanismos moleculares que direcionam a indução da pluripotência ainda não estão muito bem esclarecidos. Alguns estudos revelaram que componentes chaves da via NFB estão envolvidos na regulação da pluripotência, bem como na diferenciação e destino celular das células-tronco. Neste estudo, analisamos a participação de componentes da via canônica (RelA e NFB1) e não-canônica (RelB e NFB2) de NFB nos processos de diferenciação e destino celular ou manutenção da pluripotência. Para isto usamos técnicas de PCR quantitativa em Tempo Real (qPCR) e Imunoprecipitação de Cromatina (ChIP) investigando os papéis das vias canônica e não-canônica de NFB na manutenção da pluripotência e diferenciação de CPs, em um modelo de indução de diferenciação celular mediado por ácido trans-retinóico (atRA) em células de carcinoma embrionário NTera-2. Foram avaliadas as ligações dos fatores de transcrição RelA e RelB nas regiões promotoras dos genes OCT4, SOX2, MYC, KLF4 e GFAP e a regulação transcricional associada. Nossos resultados identificaram que as células não tratadas com atRA apresentaram níveis baixos na expressão dos componentes da via canônica de NFB, RelA e NFB1, e GFAP e quando induzidas à diferenciação por atRA durante 4 dias esses níveis se elevaram. Uma situação oposta foi vista nos componentes da via não-canônica de NFB, RelB e NFB2, e na expressão dos fatores de pluripotência OCT4, NANOG, SOX2 e KLF4, que apresentaram níveis de expressão elevados nas células não tratadas com atRA e sofreram redução com a indução da diferenciação celular. O ensaio de ChIP revelou que RelA liga-se nas regiões de regulação dos genes OCT4, SOX2, KLF4, MYC e GFAP apenas quando a célula está em processo de diferenciação, enquanto RelB se apresentou ligado às mesmas regiões tanto nas células indiferenciadas quanto naquelas induzidas à diferenciação por 4 dias. Com estes dados sugerimos que a via canônica de NFB pode estar relacionada com o processo de diferenciação e destino celular através da regulação negativa executada por RelA e NFB1 nos genes responsáveis pela identidade pluripotente das células aqui estudadas enquanto a via não-canônica de NFB, representada pela ativação de RelB e NFKB2, pode participar na manutenção da pluripotência através da regulação positiva destes mesmos fatores. / Human pluripotent stem cells (hPSCs) are able to give rise to all the 200 cell types of the adult organism. In nature, there are three types of hPSCs: embryonic stem cells, germ line stem cells and embryonal carcinoma cells. hPSCs characteristics have allowed a major advance in basic research, and are thought to have great applicability in regenerative medicine. In the nucleus of hPSCs there are transcription factors responsible for the maintenance of their pluripotent identity. OCT4, NANOG, SOX2, KLF4 and MYC are considered the core pluripotency factors in hPSCs. A great deal of knowledge about the mechanisms that promote and maintain pluripotency has been generated. Based on these studies it was possible to generate induced pluripotent stem cells (iPSCs). However, the molecular mechanisms that drive the induction of pluripotency are not fully understood. Some studies have recently indicated that key components of the NFkB may be involved in regulating pluripotency as well as cell differentiation and cell fate. In this study we analyzed the involvement of components of the canonical (RelA and NFB1) and the non-canonical NFB pathways (RelB and NFB2) in the maintenance of pluripotency, differentiation and cell fate processes. The techniques of quantitative real-time PCR (qPCR) and chromatin immunoprecipitation (ChIP) were used to interrogate the roles of the canonical and non-canonical NFB pathways in maintenance of pluripotency and differentiation in a model of cell differentiation induced by all trans-retinoic acid (atRA) on embryonal carcinoma cells NTera-2. The transcription factors RelA and RelB occupancy in the promoter regions of OCT4, SOX2, KLF4, MYC and GFAP, and the transcriptional regulation associated were evaluated. Our results showed that undifferentiated cells exhibited low expression levels of canonical NFB pathway components, RelA and NFB1, while cells induced to differentiate for 4 days exhibited downregulated expression of these factors. In the other hand, the non-canonical NFB pathway components, RelB and NFB2, and the pluripotency factors OCT4, NANOG, SOX2 and KLF4 were expressed in higher levels in undifferentiated cells, and were downregulated upon the differentiation process. ChIP assay revealed that RelA binds to the regulatory regions of OCT4, SOX2, KLF4, MYC, and GFAP only when cells are induced to differentiate, while RelB was found bound to the same regions in both undifferentiated and differentiated cells. This data suggests that the canonical NFB pathway may be associated to differentiation and cell fate processes by downregulation of genes responsible for the pluripotent identity, and that the non-canonical NFB pathway may act in the maintenance of pluripotency through the upregulation of the same factors.

The Hedgehog (HH) signalling pathway and its primary transducer, GLI2, regulate cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells. To further assess the role of HH signalling during mouse embryonic stem (mES) cell differentiation, we studied the effects of GLI2 overexpression during mES cell differentiation. GLI2 overexpression resulted in temporal enhancement of cardiac progenitor genes, Mef2c and Nkx2-5, along with enhancement of Tbx5, Myhc6, and Myhc7 in day 6 differentiating mES cells. Mass spectrometric analysis of proteins that immunoprecipitate with GLI2 determined that GLI2 forms a complex with BRG1 during mES cell differentiation. Furthermore, modulation of HH signalling during P19 EC cell differentiation followed by chromatin immunoprecipitation with an anti-BRG1 antibody determined that HH signalling regulates BRG1 enrichment on Mef2c. Therefore, HH signalling accelerates cardiac progenitor gene expression during mES cell differentiation potentially by recruiting a chromatin remodelling factor to at least one cardiac progenitor gene.

Magister Chirurgiae Dentium - MChD / This is a retrospective study of malignant tumours of the maxillofacial and oral region in children that presented over a 20 year period (1973 to 1993) at the Red Cross War Memorial Children's Hospital and Groote Schuur Hospital, Cape Town. Of the 352 children that were treated for a malignant tumour arising from various anatomic sites in the head and neck region, 30 were found to have had maxillofacial and oral involvement. This represented an incidence of 8,5%. Histologically, the majority of the tumours were non-odontogenic and mesenchymal in origin. The rhabdomyosarcoma was found to be the most common neoplasm, followed by the Burkitt's lymphoma. The age range was 6 months to 13.8 years (mean age 5.7 years). Males were more commonly affected than females, with a ratio of 1.3:1. There were 26 (86,7%) black patients and 4 (13,3%) white patients, representing a ratio of 6.5:1. Fifty percent of the cases were from the Eastern Cape. The mandible and the maxilla were the most common sites to be involved, followed by the soft tissues of the face. The most common presenting symptom was a painless swelling (73,3%) of the face. Twenty percent of the patients had "floating" or loose teeth. Radiographic features in the jaws were poorly circumscribed destructive lytic lesions with displacement of teeth. Histologic type was found to be the most significant variable affecting the outcome, with the Burkitt's lymphomas having the best prognosis and the rhabdomysarcomas the worst. The most common cause of death was metastases to the lungs. It is concluded that although malignant tumours of the maxillofacial and oral region in children are rare, their prognosis is poor. Therefore, any child presenting with a facial swelling should be viewed with suspicion.