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1.
Nature ; 478(7369): 349-55, 2011 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-21993628

RESUMO

Determining the signalling pathways that direct tissue expansion is a principal goal of regenerative biology. Vigorous pancreatic ß-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing ß-cell expansion, a long-sought goal for diabetes therapy. Here we show that platelet-derived growth factor receptor (Pdgfr) signalling controls age-dependent ß-cell proliferation in mouse and human pancreatic islets. With age, declining ß-cell Pdgfr levels were accompanied by reductions in ß-cell enhancer of zeste homologue 2 (Ezh2) levels and ß-cell replication. Conditional inactivation of the Pdgfra gene in ß-cells accelerated these changes, preventing mouse neonatal ß-cell expansion and adult ß-cell regeneration. Targeted human PDGFR-α activation in mouse ß-cells stimulated Erk1/2 phosphorylation, leading to Ezh2-dependent expansion of adult ß-cells. Adult human islets lack PDGF signalling competence, but exposure of juvenile human islets to PDGF-AA stimulated ß-cell proliferation. The discovery of a conserved pathway controlling age-dependent ß-cell proliferation indicates new strategies for ß-cell expansion.


Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Fatores Etários , Animais , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Fatores de Transcrição E2F/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Histona-Lisina N-Metiltransferase/genética , Humanos , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexo Repressor Polycomb 2 , Proteína do Retinoblastoma/metabolismo
2.
J Undergrad Neurosci Educ ; 13(1): A29-40, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25565917

RESUMO

Learning and practicing scientific inquiry is an essential component of a STEM education, but it is often difficult to teach to novices or those outside of a laboratory setting. To promote scientific thinking in a freshmen introductory neuroscience course without a lab component, we developed a series of learning activities and assignments designed to foster scientific thinking through the use of scientific grant proposals. Students wrote three short grant proposals on topics ranging from molecular to cognitive neuroscience during a 10-week class (one quarter). We made this challenging and advanced task feasible for novice learners through extensive instructional scaffolding, opportunity for practice, and frequent peer and instructor feedback. Student and instructor reports indicate that the assignments were highly intellectually engaging and that they promoted critical thinking, a deeper understanding of neuroscience material, and effective written communication skills. Here we outline the mechanics of the assignment, student and instructor impressions of learning outcomes, and the advantages and disadvantages of implementing this approach.

3.
PLoS Genet ; 6(6): e1001003, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20585628

RESUMO

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells.


Assuntos
Ciclo Celular , Fatores de Transcrição E2F/metabolismo , Regiões Promotoras Genéticas , Proteína p107 Retinoblastoma-Like/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ciclo Celular , Células Cultivadas , Sequência Conservada , Humanos , Camundongos , Dados de Sequência Molecular , Proteína p107 Retinoblastoma-Like/química , Proteína p107 Retinoblastoma-Like/genética , Alinhamento de Sequência , Transcrição Gênica
4.
PLoS One ; 9(11): e112652, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25409432

RESUMO

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse "GS" (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.


Assuntos
Células-Tronco Adultas/metabolismo , Endonucleases/metabolismo , Engenharia Genética/métodos , Fenótipo , Células-Tronco Adultas/transplante , Animais , Linhagem Celular , Masculino , Camundongos , Espermatozoides/citologia , Transplante de Células-Tronco , Testículo/citologia
5.
Curr Opin Immunol ; 24(5): 609-16, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22981684

RESUMO

Genome engineering is an emerging strategy to treat monogenic diseases that relies on the use of engineered nucleases to correct mutations at the nucleotide level. Zinc finger nucleases can be designed to stimulate homologous recombination-mediated gene targeting at a variety of loci, including genes known to cause the primary immunodeficiencies (PIDs). Recently, these nucleases have been used to correct disease-causing mutations in human cells, as well as to create new animal models for human disease. Although a number of hurdles remain before they can be used clinically, engineered nucleases hold increasing promise as a therapeutic tool, particularly for the PIDs.


Assuntos
Enzimas de Restrição do DNA/fisiologia , Marcação de Genes/métodos , Engenharia Genética/métodos , Genoma Humano , Animais , Animais Geneticamente Modificados , Células Cultivadas , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Marcação de Genes/tendências , Engenharia Genética/tendências , Humanos , Dedos de Zinco/genética
6.
Cell Div ; 5: 9, 2010 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-20359370

RESUMO

p107 and its related family members Rb and p130 are critical regulators of cellular proliferation and tumorigenesis. Due to the extent of functional overlap within the Rb family, it has been difficult to assess which functions are exclusive to individual members and which are shared. Like its family members, p107 can bind a variety of cellular proteins to affect the expression of many target genes during cell cycle progression. Unlike Rb and p130, p107 is most highly expressed during the G1 to S phase transition of the cell cycle in actively dividing cells and accumulating evidence suggests a role for p107 during DNA replication. The specific roles for p107 during differentiation and development are less clear, although emerging studies suggest that it can cooperate with other Rb family members to control differentiation in multiple cell lineages. As a tumor suppressor, p107 is not as potent as Rb, yet studies in knockout mice have revealed some tumor suppressor functions in mice, depending on the context. In this review, we identify the unique and overlapping functions of p107 during the cell cycle, differentiation, and tumorigenesis.

7.
J Cell Biol ; 191(4): 809-25, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21059851

RESUMO

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.


Assuntos
Diferenciação Celular/fisiologia , Fase G1/fisiologia , Proteína do Retinoblastoma/metabolismo , Animais , Padronização Corporal/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/fisiologia , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismo , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/metabolismo
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