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1.
J Vis Exp ; (168)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33645569

RESUMO

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.


Assuntos
Dissecação , Cultura Primária de Células/métodos , Epitélio Pigmentado da Retina/citologia , Animais , Bioensaio , Diferenciação Celular , Polaridade Celular , Separação Celular , Impedância Elétrica , Células Epiteliais/citologia , Humanos , Camundongos Endogâmicos C57BL , Fagocitose , Fatores de Tempo
2.
Methods Mol Biol ; 2259: 3-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687705

RESUMO

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.


Assuntos
Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Células-Tronco Mesenquimais/citologia , Proteínas/análise , Células Cultivadas , Cromatografia Líquida/métodos , Meios de Cultura/química , Humanos , Células-Tronco Mesenquimais/química , Microscopia Eletrônica de Transmissão/métodos , Cultura Primária de Células/métodos , Proteínas/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cordão Umbilical/citologia
3.
Methods Mol Biol ; 2244: 51-81, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555582

RESUMO

The extensive tropism of human cytomegalovirus (HCMV) results in the productive infection of multiple cell types within the human host. However, infection of other cell types, such as undifferentiated cells of the myeloid lineage, give rise to nonpermissive infections. This aspect has been used experimentally to model latent infection, which is known to be established in the pluripotent CD34+ hematopoietic progenitor cell population resident in the bone marrow in vivo. The absence of a tractable animal model for studies of HCMV has resulted in a number of laboratories employing experimental infection of cells in vitro to simulate both HCMV lytic and latent infection. Herein, we will focus on the techniques used in our laboratory for the isolation and use of primary cells to study aspects of HCMV latency, reactivation, and lytic infection.


Assuntos
Citomegalovirus/metabolismo , Cultura Primária de Células/métodos , Antígenos CD34/metabolismo , Diferenciação Celular , Infecções por Citomegalovirus/virologia , Células-Tronco Hematopoéticas/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Tropismo Viral/genética , Tropismo Viral/fisiologia , Ativação Viral , Latência Viral
4.
Methods Mol Biol ; 2244: 83-101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555583

RESUMO

Of the many research challenges posed by the study of human cytomegalovirus (HCMV) latency, one of the most notable is the requirement for the use of primary hematopoietic cell culture. Culturing hematopoietic progenitor subpopulations requires that consideration be given to maintaining their physiological relevance. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPC) system as an in vitro model to study HCMV latent infection. Key aspects of the model include infection of primary human CD34+ HPCs prior to ex vivo expansion, a long-term culture with a stromal cell support designed to maintain the ability of stem cells to support hematopoietic reconstitution, and an assay to quantify infectious centers produced prior to and following a reactivation stimulus. Importantly, this system has been used to identify a number of viral determinants of latency or reactivation and findings have been recapitulated in vivo using a humanized mouse model for HCMV latency. Therefore, this system offers a powerful approach to defining virus-host interactions and mechanisms important for HCMV latency and reactivation.


Assuntos
Citomegalovirus/metabolismo , Cultura Primária de Células/métodos , Latência Viral/fisiologia , Antígenos CD34/metabolismo , Diferenciação Celular , Infecções por Citomegalovirus/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Transdução de Sinais , Proteínas Virais , Tropismo Viral/genética , Tropismo Viral/fisiologia , Ativação Viral/genética , Ativação Viral/fisiologia
5.
Methods Mol Biol ; 2244: 103-113, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555584

RESUMO

Human cytomegalovirus (HCMV) can cause severe disease in the immunocompromised. One of the hallmarks of HCMV infection of a human host is the targeted infection of peripheral blood monocytes (but not other leukocyte populations) that in turn serve as the key cell type for hematogenous dissemination and the establishment of persistence following primary infection. Monocytes are also a key cell type associated with viral reactivation and spread following viral reactivation. Because of their importance in the HCMV-host infection cycle and lifelong infection, it is critical to be able to study their infection in controlled in vitro systems in the laboratory. In this chapter, we discuss a viable protocol for harvesting fresh ex vivo blood monocytes from human donors that are pure and unactivated cells and that can be used in a research setting.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , Citomegalovirus/metabolismo , Cultura Primária de Células/métodos , Diferenciação Celular , Infecções por Citomegalovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Monócitos/citologia , Monócitos/metabolismo , Transdução de Sinais , Proteínas Virais , Ativação Viral/genética , Ativação Viral/fisiologia , Internalização do Vírus , Latência Viral/fisiologia
6.
Methods Mol Biol ; 2244: 115-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555585

RESUMO

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.


Assuntos
Citomegalovirus/metabolismo , Técnicas de Silenciamento de Genes/métodos , Cultura Primária de Células/métodos , Linhagem Celular , Infecções por Citomegalovirus/virologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Humanos , RNA Interferente Pequeno/genética , Transfecção/métodos , Proteínas Virais , Replicação Viral/fisiologia
7.
Methods Mol Biol ; 2244: 199-211, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555588

RESUMO

Human cytomegalovirus (HCMV) entry into host cells is a complex process involving interactions between an array of viral glycoproteins with multiple host cell surface receptors. A significant amount of research has been devoted toward identifying these glycoprotein and cellular receptor interactions as the broad cellular tropism of HCMV suggests a highly regulated yet adaptable process that controls viral binding and penetration. However, deciphering the initial binding and cellular receptor activation events by viral glycoproteins remains challenging. The relatively low abundance of receptors and/or interactions with glycoproteins during viral entry, the hydrophobicity of membrane receptors, and the rapid degradation and recycling of activated receptors have complicated the analysis of HCMV entry and the cellular signaling pathways initiated by HCMV engagement to the host membrane. Here, we describe the different methodologies used in our laboratory and others to analyze the interactions between HCMV glycoproteins and host cellular receptors during the entry stage of the viral life cycle.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Linhagem Celular/virologia , Citomegalovirus/genética , Fibroblastos/metabolismo , Humanos , Cultura Primária de Células/métodos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus
8.
Methods Mol Biol ; 2244: 159-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555587

RESUMO

All of the cytomegaloviruses discovered to date encode two or more genes with significant homology to G protein-coupled receptors (GPCRs). The functions of these cytomegalovirus GPCRs continue to be actively studied and it is clear that they exhibit numerous interesting functions in vitro and in vivo. In this chapter, we review the various methodologies that can be used to examine biochemical aspects of viral GPCR signaling in vitro, as well as examine the biological activity of these viral GPCRs in vitro and in vivo in virus infected cells using recombinant cytomegaloviruses.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/genética , Receptores Acoplados a Proteínas-G/genética , Animais , Linhagem Celular/virologia , Citomegalovirus/metabolismo , Humanos , Cultura Primária de Células/métodos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
9.
Methods Mol Biol ; 2235: 169-180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576977

RESUMO

Renal pericytes have a critical importance for angiogenesis and vascular remodeling, medullary blood flow regulation, and development of fibrosis. An emerging role for kidney pericytes is their ability to induce renin expression and synthesis. Here, we present methods for purification of human renal pericytes, their primary culture, and differentiation into renin-producing cells. Possible applications of these protocols include investigations into (1) renin cell recruitment mechanisms, (2) modulation of renin expression/secretion by small molecules, and (3) renin expression/secretion in nonrenal pericytes. A potential therapeutic application of this work is the identification of new players regulating the renin-angiotensin system.


Assuntos
Pericitos/metabolismo , Cultura Primária de Células/métodos , Sistema Renina-Angiotensina/fisiologia , Angiotensinas/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Humanos , Rim/metabolismo , Renina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos
10.
Methods Mol Biol ; 2273: 103-110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604847

RESUMO

Efficient isolation, characterization, and culture of endometrial epithelial cells and stromal fibroblasts from calf uteri collected at the slaughterhouse is key to develop useful 3D culture tissue models to investigate uterine physiology and pathology without the need of performing invasive procedures to recover tissue samples.Here we provide a detail methodology that gives consistently pure and viable populations of distinct primary bovine endometrial cells.


Assuntos
Técnicas de Cultura de Células/métodos , Cultura Primária de Células/métodos , Animais , Bovinos , Células Cultivadas , Endométrio/citologia , Endométrio/crescimento & desenvolvimento , Células Epiteliais/citologia , Feminino , Fibroblastos , Modelos Biológicos , Células Estromais/citologia , Células Estromais/metabolismo
11.
J Vis Exp ; (168)2021 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-33616118

RESUMO

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.


Assuntos
Diferenciação Celular , Colo/citologia , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Cultura Primária de Células/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL
12.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443156

RESUMO

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP2 via the canonical pathway guanylyl cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.


Assuntos
Sinalização do Cálcio/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Feminino , Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiologia , Cultura Primária de Células/métodos
13.
Methods Mol Biol ; 2240: 13-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33423223

RESUMO

Contact allergy is of considerable importance to the toxicologist, and regulatory authorities worldwide require testing for skin sensitization potential and appropriate hazard labeling to enable management of the risk to human health. Although traditionally the identification of skin-sensitizing chemicals has been carried out using animal models, in Europe legislative changes have promoted, and now require, the use of non-animal methods (i.e., Cosmetic Directive, REACH). Several in vitro alternatives for hazard identification have now been validated, but do not provide information on the potency of a skin sensitizer. Here, we describe an animal model, the local lymph node assay (LLNA), and an in vitro model, the RhE IL-18 potency assay, in the context of the identification and potency classification of skin sensitizers. These two assays have been chosen among the different available tests as representative of an alternative in vivo model (the LLNA) and a promising in vitro method with the potential of both hazard identification and potency classification.


Assuntos
Dermatite Alérgica de Contato/etiologia , Interleucina-18/imunologia , Ensaio Local de Linfonodo , Testes de Irritação da Pele/métodos , Alérgenos/imunologia , Alérgenos/toxicidade , Animais , Células Cultivadas , Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/imunologia , Humanos , Irritantes/imunologia , Irritantes/toxicidade , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Camundongos , Cultura Primária de Células/métodos
14.
Methods Mol Biol ; 2240: 31-41, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33423224

RESUMO

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma.


Assuntos
Epiderme/efeitos dos fármacos , Cultura Primária de Células/métodos , Testes de Irritação da Pele/métodos , Células Cultivadas , Humanos
15.
Methods Mol Biol ; 2230: 415-423, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197029

RESUMO

Primary chondrocyte isolation and culture is a useful tool to characterize how cellular perturbations impact chondrocyte behavior and mineralization in vitro. This protocol conveys methods for isolating and culturing primary chondrocytes from costal and growth plate cartilage. Following gross dissection of the neonatal murine anterior rib cage or long bone growth plate cartilage, chondrocytes are isolated via enzymatic digestion and plated at high density. Genetic perturbation of plated primary murine chondrocytes using viral infection of Cre recombinase to excise floxed alleles and/or overexpress genes of interest are also described.


Assuntos
Separação Celular/métodos , Condrócitos/citologia , Cultura Primária de Células/métodos , Animais , Cartilagem/crescimento & desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos
16.
Methods Mol Biol ; 2230: 449-456, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197032

RESUMO

Radiolabeled amino acid uptake assays are a highly sensitive method used to characterize the uptake of amino acids by cells or tissues in culture. This method is an excellent tool to quantify changes in amino acid consumption that are associated with states of cellular differentiation and/or disease. The methods presented here can be adapted to measure the transport of all amino acids and can be applied to cultured cells and bone explants.


Assuntos
Aminoácidos/metabolismo , Marcação por Isótopo/métodos , Cultura Primária de Células/métodos , Trítio/farmacologia , Animais , Transporte Biológico/genética , Linhagem Celular , Transporte de Íons/genética , Leucina/metabolismo , Camundongos
17.
Methods Mol Biol ; 2179: 257-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32939726

RESUMO

The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to posttranslational modifications, to protein turnover. It involves remodeling of polarity and adhesion and enhances migratory capabilities. During physiological events such as embryogenesis or wound healing EMT is used to initiate cell migration, but EMT can also occur in pathological settings. In particular, EMT has been linked to fibrosis and cancer. Neural crest (NC) cells, an embryonic stem cell population whose behavior recapitulates the main steps of carcinoma progression, are a great model to study EMT. In this chapter, we provide a fully detailed protocol to extract NC cells from Xenopus embryos and culture them to study the dynamics of cell-cell adhesion, cell motility, and dispersion.


Assuntos
Rastreamento de Células/métodos , Transição Epitelial-Mesenquimal , Crista Neural/citologia , Cultura Primária de Células/métodos , Animais , Adesão Celular , Movimento Celular , Rastreamento de Células/instrumentação , Xenopus
18.
Methods Mol Biol ; 2179: 315-326, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32939730

RESUMO

Metastasis and chemoresistance, the most lethal features of cancer progression, are strongly associated with a form of cellular plasticity known as the epithelial-to-mesenchymal transition (EMT). Carcinoma cells undergoing EMT lose their epithelial morphology and become more mobile, allowing them to invade and migrate more efficiently. This shift is also associated with a change in vulnerability to chemotherapeutic agents. Importantly, EMT does not involve a single mechanism, but rather encompasses a spectrum of phenotypes with differing degrees of epithelial and mesenchymal characteristics. These hybrid/partial epithelial-mesenchymal states are associated with other important aspects of tumor biology, such as distinct modes of cellular invasion and drug resistance, illustrating the need to further characterize this phenomenon in tumor cells. Although simple in theory, the identification of tumor cells that have undergone EMT in vivo has proven difficult due to their high similarity to other mesenchymal cells that populate tumor stroma, such as cancer-associated fibroblasts. This protocol describes two methods for isolating epithelial and EMT cancer cell populations from primary murine tumors and cultured cancer cells to identify different EMT subtypes. These populations can then be used for several applications, including, but not limited to, functional studies of motility or invasion, gene expression analysis (RNA sequencing and RT-qPCR), DNA sequencing, epigenetic analysis, tumor subtyping, western blotting, immunohistochemistry, etc. Finally, we describe a flow cytometry-based approach to identify and study tumors cells that are undergoing partial EMT.


Assuntos
Transição Epitelial-Mesenquimal , Citometria de Fluxo/métodos , Cultura Primária de Células/métodos , Animais , Camundongos , RNA-Seq/métodos , Células Tumorais Cultivadas
19.
Methods Mol Biol ; 2233: 101-111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222130

RESUMO

The efficient recycling of synaptic vesicles (SVs) during neuronal activity is central for sustaining brain function. During intense neuronal activity, the dominant mechanism of SV retrieval is activity-dependent bulk endocytosis (ADBE). Here, we describe a method to monitor ADBE in isolation from other SV endocytosis modes, via the uptake of large fluorescent fluid-phase markers in primary neuronal culture. Furthermore, we outline how to monitor ADBE using this approach across a field of neurons or in individual neurons.


Assuntos
Endocitose/genética , Neurônios/ultraestrutura , Cultura Primária de Células/métodos , Vesículas Sinápticas/ultraestrutura , Animais , Dextranos/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Corantes Fluorescentes/farmacologia , Humanos , Camundongos , Neurônios/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/ultraestrutura , Vesículas Sinápticas/efeitos dos fármacos
20.
Methods Mol Biol ; 2233: 193-202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222136

RESUMO

Neutrophils are short-lived cells after isolation. The analysis of neutrophil vesicular trafficking requires rapid and gentle handling. Recently developed super-resolution microscopy technologies have generated unparalleled opportunities to help understand the molecular mechanisms regulating neutrophil vesicular trafficking, exocytosis, and associated functions at the molecular level. Here, we describe super-resolution and total internal reflection fluorescence (TIRF) microscopy approaches for the analysis of vesicular trafficking and associated functions of primary neutrophils.


Assuntos
Exocitose/genética , Microscopia de Fluorescência/métodos , Neutrófilos/ultraestrutura , Cultura Primária de Células/métodos , Movimento Celular/genética , Humanos , Neutrófilos/metabolismo , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/genética
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