A ribonucleoproteína heterogênea nuclear K (hnRNP K) e o inibidor endógeno da fosfatase 2A (SET) são superexpressos e propostos como marcadores prognósticos em leucemia mieloide aguda e crônica. O objetivo do estudo foi caracterizar a participação das proteínas hnRNP K e SET na leucemogênese da leucemia promielocítica aguda (LPA), assim como na diferenciação celular induzida pelo ácido all-trans retinóico (ATRA). Os resultados iniciais de qRT-PCR demonstram que os níveis de RNAm de HNRNPK e SET estão aumentados em pacientes ao diagnóstico de LPA em comparação com amostras de indivíduos saudáveis e diminuem após indução e durante a manutenção do tratamento. Os resultados foram validados por Western blot, sugerindo hnRNP K e SET como marcadores diagnóstico e de resposta ao tratamento. O knockdown de hnRNP K e SET foi realizado em células de LPA sensível, NB4, e resistente ao ATRA, NB4-R2, utilizando RNA de interferência. Ambas as proteínas também foram testadas como alvo terapêutico com a utilização de inibidores de hnRNP K (U0126) e SET (OP449 e FTY720). A diminuição de hnRNP K nas células levou ao aumento da diferenciação celular granulocítica em ambas as células, principalmente na presença de ATRA, e portanto, foi capaz de reverter o fenótipo de resistência ao ATRA das células NB4-R2. O knockdown de hnRNP K, assim como o tratamento com U0126, levou a perda de viabilidade dessas células por indução de apoptose acompanhada da clivagem da proteína SET. O knockdown de SET em células LPA confirmou que a indução de apoptose em células com knockdown de hnRNP K ocorreu por clivagem e não pela diminuição da proteína SET nas células. Além disso, demonstrou também que SET prejudica a atuação do ATRA no processo de diferenciação celular. O modelo in vivo utilizando transplante de NB4-R2 em camundongos nude confirmou que o trióxido de arsênico (ATO) combinado a U0126 tem um maior potencial contra a progressão tumoral quando comparado ao tratamento isolado com ATO. FTY720 e OP449 tiveram efeito anti-leucêmico significativo com redução da viabilidade celular. Quando em associação, FTY720 e OP449, apresentaram efeito sinérgico significativo em NB4-R2 (CID<0,7). Concluímos que a superexpressão de hnRNP K e SET contribui para o bloqueio da diferenciação celular em promielócitos e prejudicam a atuação do ATRA no tratamento da LPA e, portanto, hnRNPK em associação com a proteína SET representam alvo terapêutico em potencial para terapia anti-leucêmica da LPA, principalmente para pacientes resistentes ao ATRA / Heterogeneous nuclear ribonucleoprotein K (hnRNP K) and endogenous inhibitor of phosphatase 2A (SET) are overexpressed and proposed as prognostic markers in acute and chronic myeloid leukemia. The study aim was to characterize the hnRNP K and SET proteins involvement in acute promyelocytic leukemia leukemia (APL) leukemogenesis as well as all-trans retinoic acid (ATRA) induced cell differentiation. Initial qRT-PCR results demonstrate that HNRNPK and SET mRNA levels are increased in patients diagnosed with APL compare to samples from healthy donors and decrease after induction and during maintenance of treatment. The results were validated by Western blot, suggesting hnRNP K and SET as diagnostic and response to treatment markers. The knockdown of hnRNP K and SET was performed on sensitive, NB4, and ATRA-resistant, NB4-R2, LPA cells using interfering RNA. Both proteins were also tested as a therapeutic target with a use of hnRNP K (U0126) and SET inhibitors (OP449 and FTY720). The decrease of hnRNP K in cells led to increased granulocyte cell differentiation in both cells, especially in the presence of ATRA, and thus was able to reverse the NB4-R2 cells resistance to ATRA phenotype. The hnRNP K knockdown, as well as the treatment with U0126, had a loss of cell viability by induction of apoptosis accompanied by cleavage of the SET protein. The SET knockdown in APL cells confirmed that an induction of apoptosis in cells with hnRNP K knockdown occurred by cleavage and not by the SET protein decrease in the cells. Furthermore, it has also shown that SET impairs the ATRA\'s performance in the cellular differentiation process. The in vivo model using NB4-R2 transplant in nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has a greater potential against tumor progression compared to the treatment isolated with ATO. FTY720 and OP449 have significant anti-leukemic effect reducing cell viability. When in combination, FTY720 and OP449, they had a significant synergistic effect on NB4-R2 (CDI <0.7). We conclude that overexpression of hnRNP K and SET contributes to block cell differentiation in promyelocytes and impair the performance of ATRA in the treatment of APL and therefore hnRNPK in association with a SET protein represent a potential therapeutic target for anti-leukemic therapy of APL, mainly for patients resistant to ATRA

La LMMC est une hémopathie est caractérisée par une monocytose persistante (>1.10^9/L) ainsi que par la présence anormale de granulocytes immatures dans le sang des patients. Actuellement, il n’existe aucun traitement ciblé, il est donc essentiel de mieux caractériser et de comprendre les mécanismes qui causent cette monocytose afin d’établir de nouvelles stratégies thérapeutiques et de proposer des traitements ciblés. A partir de monocytes primaires humain stimulés par du CSF-1, nous avons observé que l’autophagie, est induite et qu’elle est dépendante des protéines Beclin1, ATG5, ATG7 et ULK1. Cette autophagie est également nécessaire à la différenciation macrophagique. Nous avons déterminé que l’AMP kinase (AMPK), est nécessaire à l’induction de l’autophagie et par conséquent à la différenciation macrophagique. Nous avons également découvert que le récepteur purinergique P2RY6 via la stimulation du CSF-1R active l’axe PLCβ3-CaMKKβ-AMPK-ULK1 conduisant à l’induction de l’autophagie nécessaire à la différenciation macrophagique. Enfin, dans le cadre de la LMMC, où la différenciation macrophagique est altérée, nous avons confirmé que la présence de granulocytes immatures est responsable de l’inhibition de l’axe P2RY6-AMPK. Dans ce contexte, nous avons observé que l’ajout d’agonistes du P2RY6 est capable d’une part de réinduire l’expression de l’AMPK et d’autre part de restaurer la différenciation macrophagique. L’ensemble de ces résultats souligne l’importance de l’induction du processus autophagique au cours de la différenciation macrophagique des monocytes mais également désigne l’axe P2RY6-AMPK comme cible thérapeutique potentielle dans le traitement de la LMMC. / CMML is a hematologic malignancy characterized by a persistent monocytosis (> 1.10^9/ L) and an abnormal presence of immature granulocytes in the blood of patients. Currently, there is no targeted therapy for this disease, so there is a real need to better understand the mechanisms leading to monocytosis in order to establish new therapeutic strategies and propose targeted treatments for CMML patients. From primary human monocytes, stimulated by CSF-1 to induce their differentiation into macrophages, we found that autophagy is induced during this process and is required for proper macrophagic differentiation. Then we were able to show that AMPK kinase (AMPK) is required for the induction of autophagy and therefore macrophage differentiation. We also found that the P2RY6 purinergic receptor via the CSF-1R stimulation, activate the PLCβ3-CaMKKβ-AMPK-ULK1 axis leading to the induction of autophagy, which is necessary for the macrophagic differentiation. Finally, in CMML context, where the macrophagic differentiation is impaired, we confirmed that the presence of immature granulocytes is responsible for the inhibition of AMPK P2RY6-axis. In this context, we observed that the addition of agonists P2RY6 is first capable of re-inducing the expression of AMPK and then restoring the macrophagic differentiation. All of these results emphasize the importance of autophagy induction during macrophage differentiation of monocytes and highlight the P2RY6 AMPK-axis as a potential therapeutic target in the treatment of CMML.

Na mucosa gástrica, cinco tipos compõe o epitélio gástrico: mucosas superficiais, parietais (CP), mucosas do colo (CMC), endócrinas e zimogênicas (CZ). As CMC originam as células CZ por transdiferenciação e a população de CP é importante para que o processo ocorra.Durante a transdiferenciação observamos células com características de CMCs e CZs, denominadas células em transição (CT) que ocupam o segmento entre o colo e a base da glândula. Essa transição entre células e a presença de CP são essenciais para a homeostase da base da glândula. Porém, estudos mostram que alterações no padrão alimentar influenciam a organização do epitélio gástrico de ratos, e o desmame precoce (DP) modifica a dinâmica de proliferação, migração e maturação das células. Nosso objetivo foi avaliar os efeitos imediatos do DP sobre as populações de CMC, CZ, CT e CP, e investigar se tais efeitos são mantidos até a vida adulta. Para isso, ratos Wistar (CEUA ICB USP 18/2015) foram divididos em dois grupos: amamentado controle (A) (permaneceram com a mãe até 21 dias), e DP (separados das mães aos 15 dias). O estômago foi coletado aos 18, 30 e 60 dias de vida pósnatal. Em cortes histológicos, verificamos que o índice de CP não foi alterado pelo DP, porém a distribuição dessa célula na glândula foi modificada aos 18 e 30 dias. Sob microscopia de fluorescência e confocal, observamos que o DP aumentou a população de CMC (GSIIFITC+) aos 18 dias (A vs DP; P<0,01), e de CZ (Mist1-Cy3+) aos 18 e 60 dias (A vs DP; P<0,05). O número de CT/campo (GSII-FITC+Mist1-Cy3) não se alterou após o DP, evidenciando que o segmento de transição entre o colo e a base da glândula representa um importante controle de tamanho populacional. Para avaliar o comportamento de CT em relação ao seu destino (CZ) e sua origem (CMC), analisamos o índice de CT sobre essas populações. Sobre o destino, devido à ausência de resposta no número de CT/campo e ao aumento de CZ nos animais DP, o índice CT/CZs variou aos 18 e 30 dias. No entanto, o índice de CT/CMC (origem) não foi alterado pelos tratamentos nas diferentes idades. Sob microscopia eletrônica de transmissão, avaliamos as características ultraestruturais das CMC, CT e CZ aos 18 dias, e observamos principalmente as organelas envolvidas na reorganização das células durante a transdiferenciação. Nosso estudo mostrou que o desmame precoce acelera a diferenciação celular e muda a distribuição de células mucosas do colo e zimogênicas na glândula gástrica, porém esse processo ocorre com preservação do tamanho compartimento de transição e do número de células parietais. Assim, podemos sugerir que o desmame precoce aumenta a população de células zimogênicas por meio de uma passagem mais rápida entre a região do colo e da base, na presença de células parietais, e esse mecanismo seria acionado logo após a transição da dieta, podendo manter-se ativo até a vida adulta. / Five epithelial cell types are found in the gastric mucosa: surface mucous, parietal (PC), mucous neck (MNC), endocrine, and zymogenic (ZC). The MNC originates ZC through transdifferentiation, and the presence of PC is important for the process. During transdifferentiation, we observe cells that present characteristics from MNC and ZC that are identified as transition cells (TC). They occupy the area between the neck and base of the gastric gland. The transition between these cells and the presence PC are essential for homeostasis at the base of the gland. However, studies show that changes in dietary pattern influence the organization of rat gastric epithelium, and early weaning (EW) modifies the proliferation, migration and maturation dynamics of these cells. Our aim was to evaluate the immediate effects of EW on the populations of MNC, ZC, TC and PC, and to investigate if such effects are maintained until adult life. To that, Wistar rats (CEUA ICB USP 18/2015) were divided into two groups: suckling control (S) (pups remained with the mother until 21 days), and EW (pups were separated from mother at 15 days). The stomach was collected at 18, 30 and 60 postnatal days. After analyses of histological sections, we verified that PC indices were not altered by EW, but the distribution of this cell was modified at the 18 and 30 days. By fluorescence and confocal microscopy, we determined that EW increased MNC population at 18 days (A vs PD, P <0.01), and ZC at 18 and 60 day (A vs DP; P <0.05). TC number/field did not change after EW, indicating that the transition between neck and gland base is under an important population control. In order to evaluate the behavior of TC regarding its final differentiation (ZC) and origin (MNC), we calculated TC index on these populations. In relation to ZC, we found a variation in TC index at 18 and 30 days, specially because TC number did not change, whereas ZC population increased. However, the TC index on GSII + cells (origin) was not altered by treatments throughout growth and ageing. Finally, under electron microscopy, we studied the ultra structure of MNC, TC and ZC and observed the reorganization of secretory apparatus during transdifferentiation at 18 days. Thus, we can suggest that EW increases ZC population through a rapid traffic through transition compartment between neck and base, in the presence of parietal cells, and such mechanism would be triggered soon after dietary transition and be kept activated until adult life.

Includes bibliographical references (leaves 142-168) Aim was the purification and identification of the early primitive ectoderm-like (EPL) cell induction signals within the medium conditioned by the human hepatocellular carcinoma cell line HepG2 and the localisation of the signals that induce EPL cell and primitive ectoderm formation.

Uroplakin 1B (UPKIB) is an integral structural protein interacting with uroplakins 1A, 2 and 3 to form hexameric plaques along the bladder lumen in the asymmetric unit membrane of urothelial umbrella cells in humans and other mammals. UPKIB mRNA expression is deregulated in transitional cell carcinomas (TCCs), however the mechanisms of regulation of UPKIB have not been established. Using genome databases, a Xenopus UPKIB homologue was identified. Maximum Parsimony and BAMBE (Bayesian Analysis in Molecular Biology and Evolution) data support a close evolutionary relationship between mammalian and amphibian UPKIB mRNA. Using Unigene, UPKIB human expressed sequence tags were identified in tissues including brain, skeletal muscle and liver, suggesting the relatively widespread distribution of this membrane protein. The UPKIB genomic structure was also deduced using genome databases. Contig AC083800, identified in a high throughput genomic sequence database, spanned UPKIB and 9 exons and 8 introns were defined. A 67bp 5' untranslated region was identified using 5' rapid amplification of cDNA ends. This product was sequenced and a putative UPKIB promoter and transcription start site was deduced. Contig AC083800 spanned the transcription start site and putative promoter. Transcription factor binding motif prediction programs detected no TATA box, but did predict a CCAAT box and several binding motifs including 4 Sp-1 sites and a NFKB site. A weak CpG island was identified within a 0.5kb region including the putative promoter, exon 1 and intron 1, which was 54% GC rich with CpG:GpC ratio of 0.46, containing 15 CpG dinucleotides. Seven TCC cell lines and five peripheral blood lymphocyte samples were analysed for UPKIB expression using RT-PCR and two cell lines expressed UPKIB transcripts. Eleven CpG sites in the putative promoter were investigated for methylation using bisulfite modification analysis in normal PBL, TCC cell lines and patient TCC samples. An inverse correlation was established in TCC cell lines between UPKIB mRNA expression and degree of methylation. 5-Aza-2'deoxycytidine induced UPKIB mRNA expression in T24 cells, previously observed not to express UPKIB. Sequence analysis of patient samples revealed more complex CpG methylation patterns, reflecting tumour heterogeneity. In summary, the uroplakin 1B gene has been characterised and one mechanism of regulation of gene expression involves methylation. / Thesis (Ph.D.)--Dept of Surgery, 2003.

Includes bibliographical references (leaves 142-168) / x, 168 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aim was the purification and identification of the early primitive ectoderm-like (EPL) cell induction signals within the medium conditioned by the human hepatocellular carcinoma cell line HepG2 and the localisation of the signals that induce EPL cell and primitive ectoderm formation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences (Biochemistry), 2001

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 34-36).

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

La línea celular HT-29 M6 es una línea tumoral humana derivada de adenocarcinoma de colon, con capacidad de diferenciación in vitro hacia un fenotipo mucosecretor, obtenida por selección con 10-7 M y 10-6 M de metotrexato, en tratamientos sucesivos, de la línea parental indiferenciada HT-29 (Lesuffleur T, et al, 1990). Nosotros utilizamos esta línea celular como modelo para estudiar el proceso de diferenciación in vitro que ocurre de manera espontánea durante el crecimiento hacia confluencia. Los resultados que presentamos en este trabajo aportan evidencias sólidas acerca del papel del calcio extracelular como modulador no sólo de la epitelialización, sino también del ciclo celular y de la expresión génica durante la diferenciación in vitro. Aunque puede existir más de un mecanismo por el que el calcio extracelular sea responsable de los efectos observados, los resultados obtenidos son consistentes con el requerimiento de contactos de adhesión para la diferenciación epitelial. Además, los resultados sugieren que reguladores del ciclo celular, concretamente ciclina D1 y p27, desempeñan un papel determinante en el control del programa de expresión génica durante la diferenciación. Este resultado es muy relevante ya que: (1) su función parece tener lugar de manera general sobre el programa de expresión génica, y (2) al estar estas proteínas implicadas en la progresión tumoral, su mecanismo de acción emerge como una diana para manipular el fenotipo de diferenciación celular y con ello la tumorigenicidad. Por otro lado, hemos descubierto que parte del proceso de diferenciación in vitro es independiente de la formación de contactos de adhesión y de la epitelialización, concretamente niveles basales de expresión génica asociada a diferenciación y la producción de vesículas de moco. Asimismo, en condiciones de no adherencia celular, las células no adquieren capacidad invasiva, lo cual nos está indicando que la desdiferenciación celular observada en tumores y la adquisición de capacidad invasiva podría tener lugar por vías separadas. Finalmente, hemos obtenido evidencias preliminares acerca de la existencia de una vía de regulación de APC sobre p21CIP1 cuya inactivación podría estar relacionada con la reversibilidad del programa de diferenciación in vitro y con su incapacidad para llevar a término un programa de diferenciación terminal.

In pathological states such as osteoarthritis, the complex metabolic balance of cartilage is disrupted, leading to a loss in the integrity and biomechanical function of cartilage. Osteoarthritis affects more than 20 million Americans, costing the United States economy over $60 billion yearly. Risk factors for osteoarthritis include age, excessive joint loading, and joint injury. Tissue engineering offers a potential solution for the replacement of diseased and/or damaged cartilage. Unfortunately, plentiful donor cell populations are difficult to assemble, as chondrocytes have a well characterized lack of expansion potential. Mesenchymal progenitor cells offer an alternative with a high expansion potential capable of supplying large quantities of cells. Using an immature bovine model, the chondrogenic differentiation of articular chondrocytes and bone marrow stromal cells was found to be scaffold, media and mechanical stimulation dependent. TGF-beta signaling participated in the response of articular chondrocytes to dynamic compressive loading, as well as enhanced the chondrogenesis of bovine BMSCs, through interactions between loading and TGF-beta/Smad signaling. Also, dynamic loading altered gene expression, matrix synthesis rates and intracellular phosphorylation for bovine BMSCs. However the response of the cells to dynamic loading depends on both media supplementation and the duration of unloaded culture. These studies establish signaling through the TGF-beta pathway as a mechanotransduction pathway for chondrocytes and chondroprogenitors in 3D culture.