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1.
J Microsc ; 2023 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-37534621

RESUMO

The low reproducibility of scientific data published in articles has recently become a cause of concern in many scientific fields. Data involving light microscopy is no exception. The low awareness of researchers of the technologies they use in their research has been identified as one of the main causes of the problem. Potential solutions have hinted at the need to improve technological and methodological education within research. Despite the pivotal role of microscopy core facilities in the education of researchers being well documented, facility staff (FS) often learn their trade on the job, without receiving themselves any structured education about the technology they teach others to use. Additionally, despite endorsing an important role at the highest level of education, most FS never receive any training in pedagogy, the field of research on teaching and learning methods. In this article, we argue that the low level of awareness that researchers have of microscopy stems from a knowledge gap formed between them and microscopy FS during training routines. On the one hand, FS consider that their teaching task is to explain what is needed to produce reliable data. On the other, despite understanding what is being taught, researchers fail to learn the most challenging aspects of microscopy, those involving their judgement and reasoning. We suggest that the misunderstanding between FS and researchers is due to FS not being educated in pedagogy and thus often confusing understanding and learning. To bridge this knowledge gap and improve the quality of the microscopy education available to researchers, we propose a paradigm shift where training staff at technological core facilities be acknowledged as full-fledged teachers and offered structured education not only in the technology they teach but also in pedagogy. We then suggest that training routines at facilities be upgraded to follow the principles of the Constructive Alignment pedagogical method. We give an example of how this can be applied to existing microscopy training routines. We also describe a model to define where the responsibility of FS in training researchers begins and ends. This involves a major structural change where university staff involved in teaching research technologies themselves receive appropriate education. For this to be achieved, we advocate that funding agencies, universities, microscopy and core facility organisations mobilise resources of time and funding. Such changes may involve funding the creation and development of 'Train-the-trainer' type of courses and giving incentives for FS to upgrade their technological and pedagogical knowledge, for example by including them in career paths. We believe that this paradigm shift is necessary to improve the level of microscopy education and ultimately the reproducibility of published data.

2.
Sci Rep ; 7(1): 14571, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-29109414

RESUMO

Evading cell death is a major driving force for tumor progression that is one of the main problems in current cancer research. Mitotic catastrophe (MC) represents attractive platform compromising tumor resistance to current therapeutic modalities. MC appeared as onco-suppressive mechanism and is defined as a stage driving the cell to an irreversible destiny, i.e. cell death via apoptosis or necrosis. Our study highlights that MC induction in colorectal carcinoma cell lines ultimately leads to the autophagy followed by apoptosis. We show that autophagy suppression in Atg 13 knockout non-small cell lung carcinoma cells lead to the dramatic decrease of MC rate. Furthermore, mitochondria-linked anti-apoptotic proteins Mcl-1 and Bcl-xL play a crucial role in the duration of MC and a cross-talk between autophagy and apoptosis. Thus, the suppression of apoptosis by overexpression of Mcl-1 or Bcl-xL affected MC and lead to a significant induction of autophagy in HCT116 wt and HCT116 14-3-3σ-/- cells. Our data demonstrate that MC induction is a critical stage, in which a cell decides how to die, while mitochondria are responsible for the maintaining the balance between MC - autophagy - apoptosis.


Assuntos
Autofagia/fisiologia , Mitose , Apoptose , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/fisiopatologia , Células HCT116 , Humanos , Neoplasias Pulmonares/fisiopatologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/metabolismo
4.
Biochim Biophys Acta Biomembr ; 1859(2): 238-244, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27913278

RESUMO

Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.


Assuntos
Glutationa Transferase/metabolismo , Microssomos Hepáticos/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Estresse Oxidativo/fisiologia
5.
Int J Biochem Cell Biol ; 81(Pt A): 133-136, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27840153

RESUMO

Mitotic catastrophe (MC) is a sequence of events resulting from premature or inappropriate entry of cells into mitosis that can be caused by chemical or physical stresses. There are several observations permitting to define MC as an oncosuppressive mechanism. MC can end up in apoptosis, necrosis or senescence. Here we show that the anticancer drug doxorubicin triggers DNA damage and MC independently of ROS production. In contrast, doxorubicin-induced apoptosis was found to be ROS-dependent. Antioxidants NAC or Trolox suppressed apoptosis, but facilitated MC development. Our data demonstrate that evasion of apoptosis and subsequent stimulation of MC can contribute to tumor cell elimination improving anticancer therapy.


Assuntos
Apoptose , Mitose , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Células HCT116 , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitose/efeitos dos fármacos
7.
Genes Dev ; 28(24): 2726-38, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25512560

RESUMO

The WD40 domain-containing protein WRAP53ß (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53ß as an essential regulator of DNA double-strand break (DSB) repair. WRAP53ß rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53ß targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53ß facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53ß controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53ß impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53ß as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.


Assuntos
Reparo do DNA/fisiologia , Telomerase/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Telomerase/genética , Transativadores/metabolismo , Ubiquitina-Proteína Ligases
8.
J Cell Sci ; 124(Pt 17): 2951-63, 2011 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-21878502

RESUMO

Although the cause and outcome of mitotic catastrophe (MC) has been thoroughly investigated, precisely how the ensuing lethality is regulated during or following this process and what signals are involved remain unknown. Moreover, the mechanism of the decision of cell death modalities following MC is still not well characterised. We demonstrate here a crucial role of the γH2AX-ATM-p53 pathway in the regulation of the apoptotic outcome of MC resulting from cells entering mitosis with damaged DNA. In addition to p53 deficiency, the depletion of ATM (ataxia telangiectasia mutated), but not ATR (ataxia telangiectasia and Rad3-related protein), protected against apoptosis and shifted cell death towards necrosis. Activation of this pathway is triggered by the augmented chromosomal damage acquired during anaphase in doxorubicin-treated cells lacking 14-3-3σ (also known as epithelial cell marker protein-1 or stratifin). Moreover, cells that enter mitosis with damaged DNA encounter segregation problems because of their abnormal chromosomes, leading to defects in mitotic exit, and they therefore accumulate in G1 phase. These multi- or micronucleated cells are prevented from cycling again in a p53- and p21-dependent manner, and subsequently die. Because increased chromosomal damage resulting in extensive H2AX phosphorylation appears to be a direct cause of catastrophic mitosis, our results describe a mechanism that involves generation of additional DNA damage during MC to eliminate chromosomally unstable cells.


Assuntos
Apoptose/genética , Proteínas de Ciclo Celular/metabolismo , Quebra Cromossômica , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Mitose/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/genética , Proteínas Mutadas de Ataxia Telangiectasia , Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/genética , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Cromátides/efeitos dos fármacos , Cromátides/genética , Aberrações Cromossômicas/induzido quimicamente , Dano ao DNA , Proteínas de Ligação a DNA/genética , Doxorrubicina/farmacologia , Exonucleases/deficiência , Exonucleases/genética , Exorribonucleases , Fase G1/genética , Técnicas de Inativação de Genes , Instabilidade Genômica , Células HCT116 , Histonas/genética , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fase S/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
9.
Proc Natl Acad Sci U S A ; 102(47): 17020-5, 2005 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-16286652

RESUMO

Transthyretin (TTR) is a transport protein for thyroxine and, in association with retinol-binding protein, for retinol, mainly existing as a tetramer in vivo. We now demonstrate that TTR tetramer has a positive role in pancreatic beta-cell stimulus-secretion coupling. TTR promoted glucose-induced increases in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release. This resulted from a direct effect on glucose-induced electrical activity and voltage-gated Ca(2+) channels. TTR also protected against beta-cell apoptosis. The concentration of TTR tetramer was decreased, whereas that of a monomeric form was increased in sera from patients with type 1 diabetes. The monomer was without effect on glucose-induced insulin release and apoptosis. Thus, TTR tetramer constitutes a component in normal beta-cell function. Conversion of TTR tetramer to monomer may be involved in the development of beta-cell failure/destruction in type 1 diabetes.


Assuntos
Células Secretoras de Insulina/fisiologia , Pré-Albumina/fisiologia , Animais , Cálcio/fisiologia , Glucose/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Obesos , Técnicas de Patch-Clamp , Cloreto de Potássio
10.
Proc Natl Acad Sci U S A ; 101(27): 10090-4, 2004 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-15210953

RESUMO

In type 1 diabetes (T1D), there is a specific destruction of the insulin secreting pancreatic beta cell. Although the exact molecular mechanisms underlying beta cell destruction are not known, sera from T1D patients have been shown to promote Ca(2+)-induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular Ca(2+) concentration ([Ca(2+)](i)) and beta cell death. The apoCIII-induced increase in [Ca(2+)](i) reflects an activation of the voltage-gated L-type Ca(2+) channel. Both the effects of T1D sera and apoCIII on the beta cell are abolished in the presence of antibody against apoCIII. Increased serum levels of apoCIII can thus account for the increase in beta cell [Ca(2+)](i) and thereby beta cell apoptosis associated with T1D.


Assuntos
Apolipoproteínas C/fisiologia , Apoptose , Cálcio/fisiologia , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/patologia , Adulto , Animais , Apolipoproteína C-III , Canais de Cálcio Tipo L/fisiologia , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Masculino , Camundongos
11.
Diabetes ; 52(12): 2943-50, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14633855

RESUMO

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.


Assuntos
Apoptose , Insulina/metabolismo , Internet , Ilhotas Pancreáticas/metabolismo , Monitorização Fisiológica , Obesidade/fisiopatologia , Animais , Proteínas de Bactérias , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Precursores Enzimáticos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fluorometria , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Secreção de Insulina , Proteínas Luminescentes , Camundongos , Microscopia Confocal , Obesidade/genética , Obesidade/metabolismo , Fótons
12.
Psychol Sci ; 13(4): 342-9, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12137137

RESUMO

A concert pianist recorded her practice as she learned the third movement, Presto, of J.S. Bach's Italian Concerto. She also described the formal structure of the piece and reported her decisions about basic features (e.g., fingering), interpretive features (e.g., phrasing), and cues to attend to during performance (performance cues). These descriptions were used to identify which locations, features, and cues she practiced most, which caused hesitations when she first played from memory, and which affected her recall 2 years later. Effects of the formal structure and performance cues on all three activities indicated that the pianist used the formal structure as a retrieval scheme and performance cues as retrieval cues. Like expert memorists in other domains, she engaged in extended retrieval practice, going to great lengths to ensure that retrieval was as rapid and automatic from conceptual (declarative) memory as from motor and auditory memory.


Assuntos
Memória , Desempenho Psicomotor , Humanos , Fatores de Tempo , Gravação de Videoteipe
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