There is a strong association between estrogen exposure and breast cancer risk. Estrogen can activate estrogen receptor alpha (ERalpha) to increase cell proliferation. Estrogen can also be metabolized into genotoxic compounds to induce DNA damage and mutations. Activation of the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid-2 like 2 (NFE2L2; NRF2) can alter the production of genotoxic estrogen. The present thesis investigated the signalling mechanisms of ERalpha, AHR, and NRF2 and how their interaction might modulate breast cancer risk. In Chapter 2, genome-wide, but promoter-focused analysis of ERalpha binding sites in T-47D breast cancer cells identified potential cell line specific differences in estrogen signalling between T-47D and the commonly used MCF-7 breast cancer cells. CYP2B6 was identified to be an ERalpha target gene in T-47D cells but not MCF-7 cells, supporting cell line dependent effect in estrogen signalling. In Chapter 3 and 4, genome-wide analyses of AHR binding sites were performed to investigate the molecular criteria governing genomic AHR transactivation in vivo in mouse and in vitro in MCF-7 breast cancer cells. Our analysis identified 1) the previously established aryl hydrocarbon response element to be an important, but not an absolute requirement in AHR transactivation and 2) key epigenetic modifications that modulate AHR-dependent gene regulation. Lastly, in Chapter 5, interaction among ERalpha, AHR, and NRF2 was presented at the regulatory region of two NRF2 target genes, NADPH Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase 1 (HMOX1). ERalpha repressed, whereas AHR enhanced NRF2-dependent NQO1 and HMOX1 mRNA expression through altered p300 recruitment and Histone H3 Lysine 9 acetylation. Collectively, this thesis examined novel molecular mechanisms that might alter breast cancer development/progression by modulating ER, AHR, and NRF2 activity.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborg University, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves: 147-162). Also available on the Internet.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 125 pages. Includes Vita. Includes bibliographical references.

There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type. A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method. Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method. I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T19:57:00Z : No. of bitstreams: 1 sanches_oc_me_botfmvz.pdf: 854358 bytes, checksum: 39b64088c65063724feff29da0e935b5 (MD5) / Universidade Estadual Paulista (UNESP) / A insuficiência renal crônica é a principal afecção renal dos cães e a anemia é a principal seqüela desta infecção. Os rins são os principais responsáveis pela produção do hormônio estimulador e regulador da eritropoiese - eritropoetina. O presente estudo teve por objetivos investigar e descrever as alterações histopatológicas dos rins e da linhagem eritróide na medula óssea de cães portadores de insuficiência renal crônica (IRC), identificando e quantificando as células precursoras das hemácias na medula óssea. Foram utilizados 15 animais controle e 15 animais portadores de insuficiência renal crônica. As amostras foram analisadas em microscopia ótica, tendo como auxílio à coloração de Giemsa, que foi utilizada para diferenciar os precursores eritróides. Os animais do grupo controle apesar de apresentarem alterações morfológicas nos rins, estas não foram suficientes para promover alterações nos parâmetros hematológicos, bem como na bioquímica sérica. Já nos animais do grupo com IRC as alterações morfológicas se caracterizaram por glomerulonefrite crônica, seguida da glomerulonefrite proliferativa mesangial, glomerulonefrite membranoproliferativa e da glomerulonefrite membranosa com 46,66%, 26,66%, 13,33% e 13,33% dos animais respectivamente. Vale ressaltar ainda que 73,33% dos animais exibiam nefrite intersticial crônica e 20% apresentavam nefrite intersticial focal. Dos quinze animais do grupo com IRC, 80% exibiam quadro de anemia, sendo 46,66% do tipo normocítica normocrômica, 20% macrocítica hipocrômica, 6,66% com anemia microcítica normocrômica e 6,66% do tipo macrocítica hipocrômica. As alterações morfológicas túbulo-intersticiais apresentaram maior correlação com as concentrações de uréia e creatinina e com a contagem dos precursores eritróides. / The chronic renal failure, the main renal afection of the dogs and the anemia is the main sequel of this afection. The kidneys are the main responsible for the production of the hormone stimulator and regulator of the erythropoiesis - erythropoietin. The present work had as objectives to investigate and to describe the histopathologics alterations of the kidneys and of the erythroid lineage in the bone marrow of dogs bearers of chronic renal failure (CRF), identifying and quantifying the precursory cells of the erythrocytes in the bone marrow. 15 control animals and 15 animals bearers of chronic renal failure were used. The samples were analyzed in optic microscopy, with the use of the Giemsa coloration, used to differentiate the erythroid precursors. The animals of the control group inspite of the morphologic alterations presented in the kidneys, were not enough to promote alterations in the hematological parameters, as well as in the serum biochemistry. In the animals of the group with CRF the morphologic alterations are characterized by chronic glomerulonephritis, following by proliferative mesangial glomerulonephritis, membranousproliferative glomerulonephritis and the membranous glomerulonephritis with 46,66%, 26,66%, 13,33% and 13,33% of the animals respectively. It is worth to stand out that 73,33% of the animals exhibited chronic interstitial nephritis and 20% presented focal interstitial nephritis. Of the 15 animals of the group with CRF, 80% exhibited anemia, being 46,66% of the type normocytic normochromic, 20% macrocytic hypochromic, 6,66% with anemia microcytic normochromic and 6,66% of the type macrocytic hypochromic. The morphologic tubulo-interstitial alterations presented larger correlation with the urea concentrations and creatinine and with the counting of the erythroid precursors.

Orientador: Julio Lopes Siqueira / Resumo: A insuficiência renal crônica é a principal afecção renal dos cães e a anemia é a principal seqüela desta infecção. Os rins são os principais responsáveis pela produção do hormônio estimulador e regulador da eritropoiese - eritropoetina. O presente estudo teve por objetivos investigar e descrever as alterações histopatológicas dos rins e da linhagem eritróide na medula óssea de cães portadores de insuficiência renal crônica (IRC), identificando e quantificando as células precursoras das hemácias na medula óssea. Foram utilizados 15 animais controle e 15 animais portadores de insuficiência renal crônica. As amostras foram analisadas em microscopia ótica, tendo como auxílio à coloração de Giemsa, que foi utilizada para diferenciar os precursores eritróides. Os animais do grupo controle apesar de apresentarem alterações morfológicas nos rins, estas não foram suficientes para promover alterações nos parâmetros hematológicos, bem como na bioquímica sérica. Já nos animais do grupo com IRC as alterações morfológicas se caracterizaram por glomerulonefrite crônica, seguida da glomerulonefrite proliferativa mesangial, glomerulonefrite membranoproliferativa e da glomerulonefrite membranosa com 46,66%, 26,66%, 13,33% e 13,33% dos animais respectivamente. Vale ressaltar ainda que 73,33% dos animais exibiam nefrite intersticial crônica e 20% apresentavam nefrite intersticial focal. Dos quinze animais do grupo com IRC, 80% exibiam quadro de anemia, sendo 46,66% do tipo normocítica normocrômica, 20% macrocítica hipocrômica, 6,66% com anemia microcítica normocrômica e 6,66% do tipo macrocítica hipocrômica. As alterações morfológicas túbulo-intersticiais apresentaram maior correlação com as concentrações de uréia e creatinina e com a contagem dos precursores eritróides. / Abstract: The chronic renal failure, the main renal afection of the dogs and the anemia is the main sequel of this afection. The kidneys are the main responsible for the production of the hormone stimulator and regulator of the erythropoiesis - erythropoietin. The present work had as objectives to investigate and to describe the histopathologics alterations of the kidneys and of the erythroid lineage in the bone marrow of dogs bearers of chronic renal failure (CRF), identifying and quantifying the precursory cells of the erythrocytes in the bone marrow. 15 control animals and 15 animals bearers of chronic renal failure were used. The samples were analyzed in optic microscopy, with the use of the Giemsa coloration, used to differentiate the erythroid precursors. The animals of the control group inspite of the morphologic alterations presented in the kidneys, were not enough to promote alterations in the hematological parameters, as well as in the serum biochemistry. In the animals of the group with CRF the morphologic alterations are characterized by chronic glomerulonephritis, following by proliferative mesangial glomerulonephritis, membranousproliferative glomerulonephritis and the membranous glomerulonephritis with 46,66%, 26,66%, 13,33% and 13,33% of the animals respectively. It is worth to stand out that 73,33% of the animals exhibited chronic interstitial nephritis and 20% presented focal interstitial nephritis. Of the 15 animals of the group with CRF, 80% exhibited anemia, being 46,66% of the type normocytic normochromic, 20% macrocytic hypochromic, 6,66% with anemia microcytic normochromic and 6,66% of the type macrocytic hypochromic. The morphologic tubulo-interstitial alterations presented larger correlation with the urea concentrations and creatinine and with the counting of the erythroid precursors. / Mestre

Un être humain adulte produit environ deux millions d’érythrocytes par seconde, à travers un processus connu sous le nom d’érythropoïèse. L’érythropoïèse est contrôlée par une balance entre prolifération et différenciation finement régulée. L’expression des gènes impliqués dans ces deux processus distincts, est régulée extrinsèquement (cytokines) et intrinsèquement (microenvirennement métabolique, facteurs de transcription). Les facteurs de transcription, fonctionnent sous forme de complexes multiprotéiques et contrôlent l’activité transcriptionnelle des cellules. Parmi eux, le complexe LDB1 joue un rôle clé dans la régulation de la balance prolifération/différenciation pendant l’érythropoïèse, puisqu’il contrôle l’expression des gènes impliquées dans ces deux processus. Au cours de mon doctorat, nous avons d’abord caractérisé les mécanismes moléculaires de la “pré-activation” des gènes de différenciation, également nommés marqueurs erythroides, dans les progéniteurs erythroides immatures. La pré-activation, est un état dans lequel, les gènes sont exprimés à un niveau basal très bas, permissif pour une activation significative pendant la différenciation. Nous avons ainsi montré que les répresseurs : ETO2, IRF2BP2 et NCOR1, interagissent avec le complexe LDB1, et lient ensemble les gènes des marqueurs erythroides et les répriment. Au cours de l’érythropoïèse, ces corépresseurs sont déstabilisés et LDB1 agit alors comme un activateur. En ce qui concerne les gènes de prolifération, nous avons observé que le complexe LDB1 est déstabilisé au niveau de ces loci pendant l’érythropoïèse. Afin d’étudier les mécanismes moléculaires de la répression génique des gènes de prolifération au cours de l’érythropoïèse, nous avons choisi d’étudier Myb, une cible du complexe LDB1, étudié auparavant dans le laboratoire. Nous avons testé trois facteurs : ZEB1, OGT et RNF12, en tant que candidats dans la répression de Myb. Nous avons montré que RNF12 est le seul facteur intervenant dans la transcription de Myb. RNF12 régule Myb probablement par une modification de complexes épigénétiques. / Every second about 2 million erythrocytes are produced in the adult human body, through a process called erythropoiesis. Erythropoiesis is controlled by a highly regulated balance between proliferation and differentiation. Expression of genes responsible for cell proliferation and differentiation is controlled external (such as cytokines) and internal (such as metabolic microenvironment and transcription factors). Transcription factors bind DNA and recruit co-factors generating transcriptional complexes. The LDB1 complex has a key role in the balance between erythroid proliferation vs. differentiation, since it regulates genes involved in both processes. During my Ph.D., we investigated the molecular mechanisms that LDB1 employs to regulate genes with divergent function. We first showed that in erythroid progenitors, differentiating genes, also known as erythroid markers, are primed. Gene priming consists of genes expressed in low basal but significant levels in progenitors, which can rapidly be activated during differentiation. We showed that in progenitors, ETO2, IRF2BP2 and NCOR1, bind the LDB1 complex therefore generating a priming complex. During differentiation, binding of the repressive (ETO2-IRF2BP2-NCOR1) co-factors to the LDB1 complex, is destabilized and genes become active. In genes involved in erythroid proliferation, we observed that LDB1 is destabilized, a feature leading to gene silencing. We used Myb, as a model of gene silencing in the context of regulation by the LDB1 complex. We tested three transcription factors: ZEB1, OGT and RNF12, as candidates in gene silencing. Among these factors, only RNF12 regulates Myb expression, probably through modifications of epigenetic silencers (Polycomb/MLL).