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1.
PLoS Comput Biol ; 16(2): e1007322, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059013

RESUMO

We present a multi-disciplinary image-based blood flow perfusion modeling of a whole organ vascular network for analyzing both its structural and functional properties. We show how the use of Light-Sheet Fluorescence Microscopy (LSFM) permits whole-organ micro-vascular imaging, analysis and modelling. By using adapted image post-treatment workflow, we could segment, vectorize and reconstruct the entire micro-vascular network composed of 1.7 million vessels, from the tissue-scale, inside a ∼ 25 × 5 × 1 = 125mm3 volume of the mouse fat pad, hundreds of times larger than previous studies, down to the cellular scale at micron resolution, with the entire blood perfusion modeled. Adapted network analysis revealed the structural and functional organization of meso-scale tissue as strongly connected communities of vessels. These communities share a distinct heterogeneous core region and a more homogeneous peripheral region, consistently with known biological functions of fat tissue. Graph clustering analysis also revealed two distinct robust meso-scale typical sizes (from 10 to several hundred times the cellular size), revealing, for the first time, strongly connected functional vascular communities. These community networks support heterogeneous micro-environments. This work provides the proof of concept that in-silico all-tissue perfusion modeling can reveal new structural and functional exchanges between micro-regions in tissues, found from community clusters in the vascular graph.


Assuntos
Circulação Sanguínea , Modelos Biológicos , Animais , Simulação por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL
2.
J Microsc ; 251(2): 128-32, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23691992

RESUMO

Single Plane Illumination Microscopy is an emerging and powerful technology for live imaging of whole living organisms. However, sample handling that relies on specimen embedding in agarose or gel is often a key limitation, especially for time-lapse monitoring. To address this issue, we developed a new concept for a holder device allowing us to prepare a sample container made of hydrogel. The production process of this holder is based on 3D printing of both a frame and casting devices. The simplicity of production and the advantages of this versatile new sample holder are shown with time-lapse recording of multicellular tumour spheroid growth. More importantly, we also show that cell division is not impaired in contrast to what is observed with gel embedding. The benefit of this new holder for other sample types, applications and experiments remains to be evaluated, but this innovative concept of fully customizable sample holder preparation potentially represents a major step forward to facilitate the large diffusion of single plane illumination microscopy technology.


Assuntos
Imageamento Tridimensional/instrumentação , Iluminação/instrumentação , Microscopia/instrumentação , Imagem com Lapso de Tempo/instrumentação , Linhagem Celular Tumoral , Humanos , Imageamento Tridimensional/métodos , Iluminação/métodos , Microscopia/métodos , Imagem com Lapso de Tempo/métodos
3.
Int J Legal Med ; 127(1): 169-76, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22358423

RESUMO

Characterization of sharp-force trauma on human bones can be extremely useful in providing information regarding the nature and context of death. Nevertheless, in the identification of weapons used to cause sharp-force trauma and analysis of bone wounds, challenging tasks still remain. Current analysis attempting to dissect bone wound characteristics varied quite a lot and mixed different criteria, thus leading sometimes to conflicting results. In this context, the aim of our study is to clarify qualitative aspects of cut marks induced by sharp weapons on human bones. For that purpose, we analyzed bone samples via an original approach based on bone autofluorescence with an epifluorescence macroscope and compared it to previous existing methods. In this study, we used bone sections from human clavicles on which three different kinds of lesions were manually implemented, using different weapons. The bone wounds were analyzed by three different methodologies, light microscopy, scanning electron microscopy (SEM), and micro-computed tomography, and were compared with epifluorescence macroscopy. We paid attention more significantly to the aspect of walls and floor of the kerf, so as to conclude on the nature and distinguish between weapons used. Among all technologies used in this study, the most precise and efficient methods were epifluorescence macroscopy and SEM. Nonetheless, epifluorescence macroscopy is faster, cheaper, and more accessible than SEM. More significantly, this technique, which has the potential to accurately document the nature of the damage, is nondestructive, and could thus be highly useful in forensic science as anthropology.


Assuntos
Clavícula/lesões , Clavícula/patologia , Ferimentos Penetrantes/patologia , Adulto , Idoso , Patologia Legal , Humanos , Microscopia , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Armas , Microtomografia por Raio-X
4.
Chromosome Res ; 19(7): 883-99, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21987186

RESUMO

Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.


Assuntos
Cromossomos/efeitos da radiação , Dano ao DNA/efeitos da radiação , Inativação Gênica/efeitos da radiação , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Feminino , Imunofluorescência , Raios gama/efeitos adversos , Histonas/genética , Humanos , Metilação/efeitos da radiação , Microscopia Confocal , Osteossarcoma/genética , Osteossarcoma/patologia , Processamento de Proteína Pós-Traducional/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
5.
Front Physiol ; 13: 899626, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35910575

RESUMO

Traditional thin sectioning microscopy of large bone and dental tissue samples using demineralization may disrupt structure morphologies and even damage soft tissues, thus compromising the histopathological investigation. Here, we developed a synergistic and original framework on thick sections based on wide-field multi-fluorescence imaging and spectral Principal Component Analysis (sPCA) as an alternative, fast, versatile, and reliable solution, suitable for highly mineralized tissue structure sustain and visualization. Periodontal 2-mm thick sections were stained with a solution containing five fluorescent dyes chosen for their ability to discriminate close tissues, and acquisitions were performed with a multi-zoom macroscope for blue, green, red, and NIR (near-infrared) emissions. Eigen-images derived from both standard scaler (Std) and Contrast Limited Adaptive Histogram Equalization (Clahe) pre-preprocessing significantly enhanced tissue contrasts, highly suitable for histopathological investigation with an in-depth detail for sub-tissue structure discrimination. Using this method, it is possible to preserve and delineate accurately the different anatomical/morphological features of the periodontium, a complex tooth-supporting multi-tissue. Indeed, we achieve characterization of gingiva, alveolar bone, cementum, and periodontal ligament tissues. The ease and adaptability of this approach make it an effective method for providing high-contrast features that are not usually available in standard staining histology. Beyond periodontal investigations, this first proof of concept of an sPCA solution for optical microscopy of complex structures, especially including mineralized tissues opens new perspectives to deal with other chronic diseases involving complex tissue and organ defects. Overall, such an imaging framework appears to be a novel and convenient strategy for optical microscopy investigation.

6.
Exp Cell Res ; 316(10): 1662-80, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20302859

RESUMO

Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored.


Assuntos
Núcleo Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/fisiologia , Antioxidantes/farmacologia , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Histonas/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Piperazinas/metabolismo , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Transcrição Gênica , Inativação do Cromossomo X
7.
Cancers (Basel) ; 13(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804934

RESUMO

Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20-30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

8.
Chromosome Res ; 17(6): 801-10, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19731052

RESUMO

The nuclear architecture is considered an important contributor to genome function. Although the fine structural features of the cell nucleus have been investigated extensively by means of ultrastructural cytochemistry, mainly on ultrathin sections in two dimensions (2D), there was a of lack routine methods for a rapid reconstruction of three-dimensional (3D) distribution of different structural constituents throughout the nuclear volume. We have now filled this gap by the application of a novel approach associating a pre-embedding selective visualization of nuclear components with a method making use of ultramicrotomy combined with scanning electron microscopy (microtome serial block face scanning electron microscopy--'3View'). We have been able to apply this method to the study of DNA distribution within the nuclear volume and reconstruction of 3D chromatin arrangement in nuclei of rat hepatocytes and endothelial cells. Our observations demonstrate that while chromatin appears to occupy the interior of nuclei rather sparsely on 2D images, once reconstructed in 3D from a series of sequential 2D images it gives the impression of considerably filling the nuclear volume. However, quantitative evaluation of the nuclear volume occupied by DNA in the above two types of nuclei leaves a significant part to the interchromatin space (66.2% for hepatic cells and 41.7% for endothelial cells, including nuclear space occupied by nucleoli). Detailed analysis of the reconstructed nuclei reveals a high degree of superposition of chromatin domains, giving rise to a false impression that they fill a much larger part of the nuclear volume than they really do. Our results show the importance of the contribution of such reconstruction techniques to our understanding of the nuclear architecture.


Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Células Endoteliais/ultraestrutura , Hepatócitos/ultraestrutura , Microscopia Eletrônica de Transmissão , Animais , Ratos
9.
Cell Death Differ ; 27(6): 1907-1923, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31819159

RESUMO

Chronic remodeling postmyocardial infarction consists in various maladaptive changes including interstitial fibrosis, cardiomyocyte death and mitochondrial dysfunction that lead to heart failure (HF). Reactive aldehydes such as 4-hydroxynonenal (4-HNE) are critical mediators of mitochondrial dysfunction but the sources of mitochondrial 4-HNE in cardiac diseases together with its mechanisms of action remain poorly understood. Here, we evaluated whether the mitochondrial enzyme monoamine oxidase-A (MAO-A), which generates H2O2 as a by-product of catecholamine metabolism, is a source of deleterious 4-HNE in HF. We found that MAO-A activation increased mitochondrial ROS and promoted local 4-HNE production inside the mitochondria through cardiolipin peroxidation in primary cardiomyocytes. Deleterious effects of MAO-A/4-HNE on cardiac dysfunction were prevented by activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2), the main enzyme for 4-HNE metabolism. Mechanistically, MAO-A-derived 4-HNE bound to newly identified targets VDAC and MCU to promote ER-mitochondria contact sites and MCU higher-order complex formation. The resulting mitochondrial Ca2+ accumulation participated in mitochondrial respiratory dysfunction and loss of membrane potential, as shown with the protective effects of the MCU inhibitor, RU360. Most interestingly, these findings were recapitulated in a chronic model of ischemic remodeling where pharmacological or genetic inhibition of MAO-A protected the mice from 4-HNE accumulation, MCU oligomer formation and Ca2+ overload, thus mitigating ventricular dysfunction. To our knowledge, these are the first evidences linking MAO-A activation to mitoCa2+ mishandling through local 4-HNE production, contributing to energetic failure and postischemic remodeling.


Assuntos
Aldeídos/metabolismo , Insuficiência Cardíaca/metabolismo , Mitocôndrias Cardíacas/metabolismo , Monoaminoxidase/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Remodelação Ventricular
10.
Sci Rep ; 9(1): 6684, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040317

RESUMO

Adipose tissue, as the main energy storage organ and through its endocrine activity, is interconnected with all physiological functions. It plays a fundamental role in energy homeostasis and in the development of metabolic disorders. Up to now, this tissue has been analysed as a pool of different cell types with very little attention paid to the organization and putative partitioning of cells. Considering the absence of a complete picture of the intimate architecture of this large soft tissue, we developed a method that combines tissue clearing, acquisition of autofluorescence or lectin signals by confocal microscopy, segmentation procedures based on contrast enhancement, and a new semi-automatic image analysis process, allowing accurate and quantitative characterization of the whole 3D fat pad organization. This approach revealed the unexpected anatomic complexity of the murine subcutaneous fat pad. Although the classical picture of adipose tissue corresponds to a superposition of simple and small ellipsoidal lobules of adipose cells separated by mesenchymal spans, our results show that segmented lobules display complex 3D poly-lobular shapes. Despite differences in shape and size, the number of these poly-lobular subunits is similar from one fat pad to another. Finally, investigation of the relationships of these subunits between each other revealed a never-described organization in two clusters with distinct molecular signatures and specific vascular and sympathetic nerve densities correlating with different browning abilities. This innovative procedure reveals that subcutaneous adipose tissue exhibits a subtle functional heterogeneity with partitioned areas, and opens new perspectives towards understanding its functioning and plasticity.


Assuntos
Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Gordura Subcutânea/citologia , Gordura Subcutânea/diagnóstico por imagem , Adipócitos/metabolismo , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Metabolismo dos Lipídeos , Microscopia Confocal , Gordura Subcutânea/metabolismo
11.
J Microbiol Methods ; 152: 69-72, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30071255

RESUMO

We combined spectrophotometry and an original statistical approach to infer bacteria virulence, using the lepidoptera Galleria mellonella as a host model. With this method, it is possible to use a microplate reader to automatize data collection on host survival on batches of 96 samples. The method also allows measurement of pathogen multiplication if GFP labelled bacterial strains are used.


Assuntos
Bactérias/patogenicidade , Modelos Animais de Doenças , Insetos/microbiologia , Lepidópteros/microbiologia , Espectrofotometria/métodos , Animais , Larva/microbiologia , Mariposas/microbiologia , Virulência , Fatores de Virulência
12.
Mol Cell Biol ; 24(14): 6379-92, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15226438

RESUMO

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.


Assuntos
Divisão Celular/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Sobrevivência Celular , Cromossomos Fúngicos , Humanos , Substâncias Macromoleculares , Mutação , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Proteína ran de Ligação ao GTP/genética
13.
Front Syst Neurosci ; 11: 47, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28740466

RESUMO

Dopamine (DA) plays a fundamental role in insect behavior as it acts both as a general modulator of behavior and as a value system in associative learning where it mediates the reinforcing properties of unconditioned stimuli (US). Here we aimed at characterizing the dopaminergic neurons in the central nervous system of the honey bee, an insect that serves as an established model for the study of learning and memory. We used tyrosine hydroxylase (TH) immunoreactivity (ir) to ensure that the neurons detected synthesize DA endogenously. We found three main dopaminergic clusters, C1-C3, which had been previously described; the C1 cluster is located in a small region adjacent to the esophagus (ES) and the antennal lobe (AL); the C2 cluster is situated above the C1 cluster, between the AL and the vertical lobe (VL) of the mushroom body (MB); the C3 cluster is located below the calyces (CA) of the MB. In addition, we found a novel dopaminergic cluster, C4, located above the dorsomedial border of the lobula, which innervates the visual neuropils of the bee brain. Additional smaller processes and clusters were found and are described. The profuse dopaminergic innervation of the entire bee brain and the specific connectivity of DA neurons, with visual, olfactory and gustatory circuits, provide a foundation for a deeper understanding of how these sensory modules are modulated by DA, and the DA-dependent value-based associations that occur during associative learning.

14.
Obesity (Silver Spring) ; 24(5): 1081-9, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26999447

RESUMO

OBJECTIVE: White and brown adipose tissues play a major role in the regulation of metabolic functions. With the explosion of obesity and metabolic disorders, the interest in adipocyte biology is growing constantly. While several studies have demonstrated functional differences between adipose fat pads, especially in their involvement in metabolic diseases, there are no data available on possible heterogeneity within an adipose depot. METHODS: This study investigated the three-dimensional (3-D) organization of the inguinal fat pad in adult mice by combining adipose tissue clearing and autofluorescence signal acquisition by confocal microscopy. In addition, the study analyzed the expression of genes involved in adipocyte biology and browning at the mARN and protein levels in distinct areas of the inguinal adipose tissue, in control conditions and after cold exposure. RESULTS: Semiautomated 3-D image analysis revealed an organization of the fat depot showing two regions: the core was structured into segmented lobules, whereas the periphery appeared unsegmented. Perilipin immunostaining showed that most of the adipocytes located in the core region had smaller lipid droplets, suggesting a brown-like phenotype. qPCR analysis showed a higher expression of the browning markers Ucp1, Prdm16, Ppargc1a, and Cidea in the core region than at the periphery. Finally, cold exposure induced upregulation of thermogenic gene expression associated with an increase of UCP1 protein, specifically in the core region of the inguinal fat depot. CONCLUSIONS: Altogether, these data demonstrate a structural and functional heterogeneity of the inguinal fat pad, with an anatomically restricted browning process in the core area.


Assuntos
Tecido Adiposo Marrom/anatomia & histologia , Gordura Subcutânea/anatomia & histologia , Adipócitos/citologia , Tecido Adiposo Marrom/fisiologia , Adiposidade , Animais , Biomarcadores/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Obesidade , Imagem Óptica , Gordura Subcutânea/química , Gordura Subcutânea/fisiologia , Termogênese , Proteína Desacopladora 1/análise
15.
Int Rev Cell Mol Biol ; 282: 1-90, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20630466

RESUMO

In this review we describe major contributions of light and electron microscopic approaches to the present understanding of functional nuclear architecture. The large gap of knowledge, which must still be bridged from the molecular level to the level of higher order structure, is emphasized by differences of currently discussed models of nuclear architecture. Molecular biological tools represent new means for the multicolor visualization of various nuclear components in living cells. New achievements offer the possibility to surpass the resolution limit of conventional light microscopy down to the nanometer scale and require improved bioinformatics tools able to handle the analysis of large amounts of data. In combination with the much higher resolution of electron microscopic methods, including ultrastructural cytochemistry, correlative microscopy of the same cells in their living and fixed state is the approach of choice to combine the advantages of different techniques. This will make possible future analyses of cell type- and species-specific differences of nuclear architecture in more detail and to put different models to critical tests.


Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Microscopia/tendências , Animais , Núcleo Celular/química , Humanos , Modelos Moleculares
16.
Methods Mol Biol ; 464: 403-13, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18951197

RESUMO

Visualisation of RNA at an ultrastructural level represents a major approach to study organisation and function of the cell nucleus. In addition to methods allowing one to visualise a general distribution of RNA-containing structural constituents, in situ hybridisation (ISH) is a powerful tool for revealing specific RNA sequences or species. In this chapter we describe a method for detecting RNA by electron microscopic in situ hybridisation (EMISH) using anti-sense RNAs as probes. We first present the protocol for preparation of anti-sense RNA probes labeled with different markers, and then describe how such probes are applied to ultrathin sections by a method of ultrastructural ISH. The great advantage of this method is that it does not require denaturing either the specimen or the probe, thus allowing nuclear fine structure to be well preserved. The presence of the marker in the probe can be detected by immunoelectron microscopy using colloidal gold-conjugated antibodies, offering the possibility to evaluate the signal quantitatively. The method can also be combined with cytochemical techniques such as EDTA staining for preferential visualisation of ribonucleoprotein-containing nuclear structural components.


Assuntos
Hibridização In Situ , Microscopia Eletrônica/métodos , RNA/química , RNA/ultraestrutura , Animais , Ácido Edético/química , Humanos
17.
Blood ; 109(3): 1275-83, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17053056

RESUMO

The gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBA-related genes may be involved in the synthesis of the ribosomal subunits.


Assuntos
Anemia de Diamond-Blackfan/genética , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Anemia de Diamond-Blackfan/metabolismo , Células Cultivadas , Fibroblastos/patologia , Células HeLa , Humanos , Mutação , RNA Ribossômico 18S/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia
18.
EMBO J ; 24(16): 2862-72, 2005 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-16037817

RESUMO

It is generally assumed that, in mammalian cells, preribosomal RNAs are entirely processed before nuclear exit. Here, we show that pre-40S particles exported to the cytoplasm in HeLa cells contain 18S rRNA extended at the 3' end with 20-30 nucleotides of the internal transcribed spacer 1. Maturation of this pre-18S rRNA (which we named 18S-E) involves a cytoplasmic protein, the human homolog of the yeast kinase Rio2p, and appears to be required for the translation competence of the 40S subunit. By tracking the nuclear exit of this precursor, we have identified the ribosomal protein Rps15 as a determinant of preribosomal nuclear export in human cells. Interestingly, inhibition of exportin Crm1/Xpo1 with leptomycin B strongly alters processing of the 5'-external transcribed spacer, upstream of nuclear export, and reveals a new cleavage site in this transcribed spacer. Completion of the maturation of the 18S rRNA in the cytoplasm, a feature thought to be unique to yeast, may prevent pre-40S particles from initiating translation with pre-mRNAs in eukaryotic cells. It also allows new strategies for the study of preribosomal transport in mammalian cells.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Transporte de RNA/fisiologia , RNA Ribossômico 18S/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Northern Blotting , Fracionamento Celular , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Primers do DNA , Ácidos Graxos Insaturados/farmacologia , Proteínas de Fluorescência Verde , Humanos , Hibridização in Situ Fluorescente , Carioferinas/antagonistas & inibidores , Camundongos , Interferência de RNA , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Proteínas Ribossômicas , Proteína Exportina 1
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