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1.
Science ; 376(6594): eabl4896, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35549404

RESUMO

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.


Assuntos
Atlas como Assunto , Células , Especificidade de Órgãos , Splicing de RNA , Análise de Célula Única , Transcriptoma , Linfócitos B/metabolismo , Células/metabolismo , Humanos , Especificidade de Órgãos/genética , Linfócitos T/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(22): e2201644119, 2022 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-35605126

RESUMO

Spatial resolution in MRI is ultimately limited by the signal detection sensitivity of NMR, since resolution equal to ρiso in all three dimensions requires the detection of NMR signals from a volume ρiso3. With inductively detected NMR at room temperature, it has therefore proven difficult to achieve isotropic resolution better than ρiso = 3.0 µm, even with radio-frequency microcoils, optimized samples, high magnetic fields, optimized pulse sequence methods, and data acquisition times around 60 h. Here we show that spatial resolution can be improved and data acquisition times can be reduced substantially by performing MRI measurements at 5 K and using dynamic nuclear polarization (DNP) to enhance sensitivity. We describe the experimental apparatus and methods, and we report images of test samples with ρiso = 2.6 µm and ρiso = 1.7 µm, with signal-to-noise ratios greater than 15, acquired in 31.5 and 81.6 h, respectively. Image resolutions are verified by quantitative comparisons with simulations. These results establish a promising direction for high-resolution MRI of small samples. With further improvements in the experimental apparatus and in paramagnetic dopants for DNP, DNP-enhanced low-temperature MRI with ρiso < 1.0 µm is likely to become feasible, potentially enabling informative studies of structures within typical eukaryotic cells, cell clusters, and tissue samples.


Assuntos
Temperatura Baixa , Imageamento por Ressonância Magnética , Células , Eucariotos , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Razão Sinal-Ruído
3.
Curr Opin Chem Biol ; 68: 102151, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35483127

RESUMO

Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research.


Assuntos
Engenharia Celular , Células , Estimulação Elétrica , Genética , Mamíferos , Biologia Sintética , Animais , Engenharia Celular/métodos , Fenômenos Fisiológicos Celulares/genética , Células/metabolismo , Estimulação Elétrica/métodos , Terapia por Estimulação Elétrica , Eletrônica , Regulação da Expressão Gênica , Mamíferos/genética , Biossíntese de Proteínas , Biologia Sintética/métodos , Telemetria
4.
Nanoscale ; 14(11): 4334-4347, 2022 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-35253828

RESUMO

The brush model was introduced to interpret AFM indentation data collected on biological cells in a more consistent way compared just to the traditional Hertz model. It takes into account the presence of non-Hertzian deformation of the pericellular brush-like layer surrounding cells (a mix of glycocalyx molecules and microvilli/microridges). The model allows finding the effective Young's modulus of the cell body in a less depth-dependent manner. In addition, it allows finding the force due to the pericellular brush layer. Compared to simple mechanical models used to interpret the indentation experiments, the brush model has additional complexity. It raises the concern about the possible unambiguity of separation of mechanical properties of the cell body and pericellular layer. Here we present the analysis of the robustness of the brush model and demonstrate a weak dependence of the obtained results on the uncertainties within the model and experimental data. We critically analyzed the use of the brush model on a variety of AFM force curves collected on rather distinct cell types: human cervical epithelial cells, rat neurons, and zebrafish melanocytes. We conclude that the brush model is robust; the errors in the definition of the effective Young's modulus due to possible uncertainties of the model and experimental data are within 4%, which is less than the error, for example, due to a typical uncertainty in the spring constant of the AFM cantilever. We also discuss the errors of parameterization of the force due to the pericellular brush layer.


Assuntos
Células , Microscopia de Força Atômica , Modelos Biológicos , Animais , Módulo de Elasticidade , Glicocálix , Humanos , Ratos , Reprodutibilidade dos Testes , Peixe-Zebra
5.
Sci Rep ; 12(1): 2924, 2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35190567

RESUMO

Classifying and analyzing human cells is a lengthy procedure, often involving a trained professional. In an attempt to expedite this process, an active area of research involves automating cell classification through use of deep learning-based techniques. In practice, a large amount of data is required to accurately train these deep learning models. However, due to the sparse human cell datasets currently available, the performance of these models is typically low. This study investigates the feasibility of using few-shot learning-based techniques to mitigate the data requirements for accurate training. The study is comprised of three parts: First, current state-of-the-art few-shot learning techniques are evaluated on human cell classification. The selected techniques are trained on a non-medical dataset and then tested on two out-of-domain, human cell datasets. The results indicate that, overall, the test accuracy of state-of-the-art techniques decreased by at least 30% when transitioning from a non-medical dataset to a medical dataset. Reptile and EPNet were the top performing techniques tested on the BCCD dataset and HEp-2 dataset respectively. Second, this study evaluates the potential benefits, if any, to varying the backbone architecture and training schemes in current state-of-the-art few-shot learning techniques when used in human cell classification. To this end, the best technique identified in the first part of this study, EPNet, is used for experimentation. In particular, the study used 6 different network backbones, 5 data augmentation methodologies, and 2 model training schemes. Even with these additions, the overall test accuracy of EPNet decreased from 88.66% on non-medical datasets to 44.13% at best on the medical datasets. Third, this study presents future directions for using few-shot learning in human cell classification. In general, few-shot learning in its current state performs poorly on human cell classification. The study proves that attempts to modify existing network architectures are not effective and concludes that future research effort should be focused on improving robustness towards out-of-domain testing using optimization-based or self-supervised few-shot learning techniques.


Assuntos
Células/classificação , Técnicas Citológicas/métodos , Conjuntos de Dados como Assunto , Aprendizado Profundo , Estudos de Viabilidade , Humanos
6.
Clin. transl. oncol. (Print) ; 24(2): 331-341, febrero 2022. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-203438

RESUMO

IntroductionPenile carcinomas are rare tumors throughout Europe. Therefore, little attention is drawn to this disease. That makes it important to study tumor-associated key metrics and relate these to known data on penile neoplasias.Materials and methodsA cohort of 60 well-defined penile invasive carcinomas with known human papillomavirus (HPV) infection status was investigated. Data on tumor type, grading and staging were recorded. Additionally, data on the peri- and intratumoral immune cell infiltrate in a semiquanititave manner applying an HE stain were assessed.ResultsOur study showed a significant correlation of immune cell infiltrate and pT stage with overall survival. Therefore, in a subset of tumors, PD-L1 staining was applied. For tumor proportion score (TPS), 26 of 30 samples (87%) were scored >0%. For the immune cell score (IC), 28 of 30 samples (93%) were defined as >0% and for CPS, 29 of 30 samples (97%) scored >0. PD-L1 expression was not associated with overall survival.ConclusionPD-L1 is expressed in penile carcinomas, providing a rationale for targeted therapy with checkpoint inhibitors. We were able to show that immune reaction appears to be prognostically relevant. These data enhance the need for further studies on the immune cell infiltrate in penile neoplasias and show that PD-L1 expression is existent in our cohort, which may be a potential target for checkpoint inhibitor therapy.


Assuntos
Ciências da Saúde , Carcinoma de Células Escamosas , Microambiente Tumoral , Neoplasias Penianas , Células/imunologia , Sobrevivência , Infecções por Papillomavirus
7.
Acc Chem Res ; 55(3): 381-390, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35040316

RESUMO

Protein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these intermediate species as misfolded protein oligomers, including soluble oligomers and preamyloid oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells.In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multistep protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfolds and aggregates when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugate would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated fluorescent protein (FP) chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that activate different fluorescence emission toward these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor-acceptor linkages are used to control the rotational barriers of fluorophores in the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method.In summary, our work allows the direct monitoring of the misfolded protein oligomers and differentiation of insoluble aggregates from other conformations in live cells, thus enabling studies of many currently unanswered questions in protein aggregation. Future directions are to develop methods that enable quantitative analyses of the protein aggregation process. Further, new methods are needed to detect and to quantify the formation and maturation of protein or RNA condensates that form membraneless organelles.


Assuntos
Amiloide , Técnicas Biossensoriais , Agregados Proteicos , Dobramento de Proteína , Amiloide/química , Células/química , Corantes Fluorescentes/química , Humanos , Hidrolases , Espectrometria de Fluorescência
8.
Elife ; 112022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35001870

RESUMO

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.


Assuntos
Células/citologia , Fluorescência , Espaço Intracelular , Microscopia/métodos , Tomografia Óptica/métodos , Núcleo Celular , Células/ultraestrutura , Células HeLa , Humanos , Refratometria
9.
Clin. transl. oncol. (Print) ; 24(1): 1-12, enero 2022. ilus
Artigo em Inglês | IBECS | ID: ibc-203409

RESUMO

Compared with the traditional forms of cell death—apoptosis, necrosis and autophagy, ferroptosis is a novel form of iron-dependent programmed cell death forms which is different from the above traditional forms of cell death. Brent R Stockwell, a Professor of Columbia University, firstly proposed that this from of cell death was named ferroptosis in 2012. The main characteristics of ferroptosis is increasing iron loading and driving a lot of lipid peroxide generated and ultimately lead to cell death. In this paper, the mechanism of ferroptosis, relationship between ferroptosis and common diseases and immune state of body are reviewed, and the inhibitors and inducers related to ferroptosis that have been found are summarized to provide medicine exploration targeted of ferroptosis and reference for the research in the future.


Assuntos
Ciências da Saúde , Morte Celular , Células , Metabolismo/imunologia , Células/imunologia
10.
Clin. transl. oncol. (Print) ; 24(1): 13-23, enero 2022.
Artigo em Inglês | IBECS | ID: ibc-203410

RESUMO

Rethinking IDH-wildtype glioblastoma through its unique features can help researchers find innovative and effective treatments. It is currently emerging that, after decades of therapeutic impasse, some traditional concepts regarding IDH-wildtype glioblastoma need to be supplemented and updated to overcome therapeutic resistance. Indeed, multiple clinical aspects and recent indirect and direct experimental data are providing evidence that the supratentorial brain parenchyma becomes entirely and quiescently micro-infiltrated long before primary tumor bulk growth. Furthermore, they are indicating that the known micro-infiltration that occurs during the IDH-wildtype glioblastoma growth and evolution is not at the origin of distant relapses. It follows that the ubiquitous supratentorial brain parenchyma micro-infiltration as a source for the development of widespread distant recurrences is actually due to the silent stage that precedes tumor growth rather than to the latter. All this implies that, in addition to the heterogeneity of the primary bulk, there is a second crucial cause of therapeutic resistance that has never hitherto been identified and challenged. In this regard, the ancestral founder cancer stem cell (CSC) appears as the key cell that can link the two causes of resistance.


Assuntos
Ciências da Saúde , Glioblastoma/prevenção & controle , Fatores R , Células , Tecido Parenquimatoso
11.
Clin. transl. oncol. (Print) ; 24(1): 48-56, enero 2022.
Artigo em Inglês | IBECS | ID: ibc-203413

RESUMO

BackgroundPrimary liver cancer cells (PLCs) could more directly simulate the human tumor microenvironment. Compared with liver cancer cell lines, PLCs could reflect the human situation. As in previous studies, tumor stem cells were a small number of cancer cells in the microenvironment and considered to be one of the origins of liver cancer. This study aimed to screen stem cells in PLCs, analyze their biological characteristics, propose the possibility that liver cancer originated from stem cells.MethodsLiver cancer tissues of 17 patients were taken from the Affiliated Hospital of Guangdong Medical College, and PLCs were isolated by tissue slice method. The proliferation, tumor formation in nude mice, stem protein expression of PLCs were observed. C-kit+ liver cancer cells were screened and their biological characteristics were analyzed.ResultsPLCs could be stably passaged. Transmission electron microscopy indicated that the nucleus was irregular, there were many mitochondria, and the endoplasmic reticulum was irregularly distributed. PLCs could express E-Cadherin, Oct-4, β-Catenin, Sox2, CD326, C-kit, GPC3, Nanog. The proliferation curve of PLCs and Hep3B cells were similar, and they all could form tumors in nude mice. Flow-sorted C-kit+ PLCs, as well as C-kit+ Hep3B cells could highly express Bmi1, Sox2, Oct4, Notch1, Nanog, C-kit, β-Catenin, Smo, Nestin, ABCG2, ABCB1. And they also could clone and form tumors in vivo. But C-kit+ PLCs were more sensitive to chemotherapy drugs than C-kit+ liver cancer cell lines.ConclusionC-kit+ PLCs had the characteristics of tumor stem cells and were more sensitive to chemotherapy drugs.


Assuntos
Humanos , Ciências da Saúde , Neoplasias Hepáticas , Células-Tronco , Cultura Primária de Células , Microscopia Eletrônica , Células/imunologia
12.
Nat Commun ; 13(1): 385, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35046414

RESUMO

Mapping cell types across a tissue is a central concern of spatial biology, but cell type abundance is difficult to extract from spatial gene expression data. We introduce SpatialDecon, an algorithm for quantifying cell populations defined by single cell sequencing within the regions of spatial gene expression studies. SpatialDecon incorporates several advancements in gene expression deconvolution. We propose an algorithm harnessing log-normal regression and modelling background, outperforming classical least-squares methods. We compile cell profile matrices for 75 tissue types. We identify genes whose minimal expression by cancer cells makes them suitable for immune deconvolution in tumors. Using lung tumors, we create a dataset for benchmarking deconvolution methods against marker proteins. SpatialDecon is a simple and flexible tool for mapping cell types in spatial gene expression studies. It obtains cell abundance estimates that are spatially resolved, granular, and paired with highly multiplexed gene expression data.


Assuntos
Algoritmos , Células/metabolismo , Transcriptoma/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , Análise dos Mínimos Quadrados , Neoplasias/genética , Neoplasias/imunologia , Análise de Regressão , Microambiente Tumoral/genética
13.
Cell ; 185(2): 345-360.e28, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35063075

RESUMO

We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior.


Assuntos
Células/citologia , Simulação por Computador , Trifosfato de Adenosina/metabolismo , Ciclo Celular/genética , Proliferação de Células/genética , Células/metabolismo , Replicação do DNA/genética , Regulação da Expressão Gênica , Imageamento Tridimensional , Cinética , Lipídeos/química , Redes e Vias Metabólicas , Metaboloma , Anotação de Sequência Molecular , Nucleotídeos/metabolismo , Termodinâmica , Fatores de Tempo
14.
Osteoarthritis Cartilage ; 30(2): 280-290, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34826571

RESUMO

OBJECTIVE: Although cartilage degeneration and invasion of the subchondral bone plate in entheseal lesion has been considered to consequently lead bony ankylosis in ankylosing spondylitis (AS), no evident mechanisms are known. DESIGN: To identify histopathological and physiological changes in enthesitis-related ankylosis in AS, we performed molecular characterization of transcription factors and surface markers, and transcriptome analysis with human tissues. Entheseal tissue containing subchondral bone was obtained from the facet joints of 9 patients with AS and 10 disease controls, and assessed by using differential staining techniques. Enthesis cells were isolated, characterized, stimulated with TNF and/or IL-17A, and analysed by cell-based experimental tools. RESULTS: We found diffusely distributed granular tissue and cartilage in the subchondral bone in AS. Co-expression of SOX9, a specific transcription factor in cartilage, and matrix metalloproteinase 13 (MMP13) was found in the granular tissues within the subchondral bone from AS patients. Intriguingly, SOX9 expression was significantly higher in AS enthesis cells than controls and correlated with TNFR1 and IL-17RA expressions, which is important for high reactivity to TNF and IL-17A cytokines. Co-stimulation by TNF and IL-17A resulted in accelerated mineralization/calcification features, and increased OCN expression in AS enthesis cells. Furthermore, SOX9 overexpression in enthesis leads to promoting mineralization feature by TNF and IL-17A stimuli. Finally, OCN expression is elevated in the destructive enthesis of advanced AS. CONCLUSION: These findings provide insight into the links between inflammation and the mineralization of entheseal tissue as the initiation of spinal ankylosis, emphasizing the importance of SOX9+ enthesis cells.


Assuntos
Anquilose/patologia , Fatores de Transcrição SOX9 , Doenças da Coluna Vertebral/patologia , Espondilite Anquilosante/patologia , Adulto , Células/metabolismo , Feminino , Humanos , Ligamentos Articulares/citologia , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição SOX9/biossíntese , Tendões/citologia
15.
Braz. J. Pharm. Sci. (Online) ; 58: e18984, 2022. graf
Artigo em Inglês | LILACS | ID: biblio-1364429

RESUMO

Interferon-ß-1a (INF-ß-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-ß-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-ß- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-ß-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-ß-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- ß-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO


Assuntos
Pichia/classificação , Interferon beta/agonistas , Carcinoma Hepatocelular/patologia , Otimização de Processos , Códon , Células , Carcinoma Hepatocelular , Concentração de Íons de Hidrogênio
16.
São Paulo; s.n; s.n; 2022. 77 p. graf, tab.
Tese em Português | LILACS | ID: biblio-1379350

RESUMO

A bactéria Gram-negativa Pseudomonas aeruginosa é um patógeno oportunista frequentemente associado a vítimas de queimaduras graves ou indivíduos com fibrose cística, sendo os isolados resistentes a carbepenêmicos dessa espécie considerados pela OMS como uma das maiores ameaças ao controle de infecções. O estabelecimento da infecção por esse patógeno é dependente de uma série de fatores de virulência, entre eles o pilus tipo IV (T4P), que possui papel importante na adesão a superfícies e motilidade do tipo twitching, essenciais para a colonização do hospedeiro. Uma das moléculas importantes na diferenciação entre as formas séssil e planctônica de P. aeruginosa é o segundo mensageiro bis-(3,5)-di-guanosina monofosfato cíclico (c-di-GMP), cuja síntese é feita enzimaticamente por diguanilato ciclases (DGCs). DgcP é uma DGC localizada nos polos da célula, que tem sua atividade de síntese de c-di-GMP aumentada na presença da proteína FimV, essencial para a montagem do T4P em P. aeruginosa. Neste trabalho, ensaios de microscopia de fluorescência, organização e expressão gênica foram realizados com o objetivo de aumentar a compreensão sobre o papel de DgcP em relação a sua expressão e aos fatores que regulam o T4P de P. aeruginosa. A proteína DgcP em fusão com mNeonGreen no C-terminal, expressa a partir do locus cromossômico, se localiza de maneira predominantemente bipolar tanto na linhagem selvagem quanto nos mutantes ΔpilA, ΔpilR e ΔchpA, evidenciando que seu padrão de localização não depende dos sistemas de regulação Pil-Chp e PilS-PilR. Ensaios de RT-PCRmostraram que dgcP se encontra em operon com PA14_72430 e dsbA1, indicando um papel celular conjunto entre esses genes, até o momento, desconhecido. Por fim, ensaios de qRT-PCR revelaram que os níveis de mRNA de dgcP são invariáveis nas linhagens WT, ΔpilA, ΔpilR, ΔchpA e ΔfimV, cultivadas em meio líquido ou meio sólido. Os resultados aqui mostrados, combinados com trabalhos prévios do nosso e de outros grupos, sugerem que DgcP é uma diguanilato ciclase responsável por geração constante de c-di-GMP nos polos da célula, possivelmente, atuando na sinalização local dependente do dinucleotídeo cíclico, cuja localização e atividade não são dependentes dos sistemas de regulação que atuam sobre o T4P


The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe burn victims or individuals with cystic fibrosis, which carbapenem-resistant isolates were classified by th World Health Organization classified one of the greatest threats to infection control. The establishment of infection by this pathogen is dependent on a series of virulence factors, including the type IV pilus (T4P), which plays an important role in adhesion to surfaces and twitching motility, essential features for host colonization. Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger that involved in processes of biofilm formation, motility, and virulence. The diguanylate cyclase DgcP synthetizes cdi-GMP and it is located at the cell poles, and its activity depends on the scaffold protein FimV, essential for T4P assembly in P. aeruginosa. By increasing c-di-GMP levels, DgcP decreases flagellum-dependent motility and increases biofilm formation. In this work, fluorescence microscopy, gene organization and expression assays were performed to understand the whether DgcP localization and expression are under the control of T4P regulatory proteins. Fluorescence microscopy analysis showed that DgcP localizes predominantly at both cell poles in ΔpilA, ΔpilR, and ΔchpA mutants, showing that its localization pattern does not depend on the Pil-Chp and PilS-PilR systems. Furthermore, RT-PCR assays showed that dgcP is found in an operon with PA14_72430 and dsbA1, indicating an unknown putative related cellular role for these genes. Finally, qRT-PCR assays indicated that DgcP expression is invariant in ΔpilA, ΔpilR, ΔchpA, and ΔfimV mutants, either in liquid or solid medium. The results shownhere, combined with previous work by ours and other groups, suggest that DgcP is a diguanylate cyclase responsible for constant generation of c-di-GMP at the cell poles, possibly acting in local signaling dependent on the cyclic dinucleotide, but that is not under the control of the known T4P regulatory systems


Assuntos
Óperon , Pseudomonas aeruginosa/classificação , Controle de Infecções/instrumentação , Organização Mundial da Saúde , Queimaduras , Expressão Gênica/genética , Células , Fatores de Virulência/efeitos adversos , Infecções/complicações , Microscopia de Fluorescência/métodos
17.
São Paulo; s.n; s.n; 2022. 205 p. tab, graf.
Tese em Português | LILACS | ID: biblio-1379336

RESUMO

Dentre os subtipos de câncer de mama, o triplo negativo (TNBC) é o que apresenta as maiores taxas de mortalidade, sendo, portanto, considerado um enorme desafio para a clínica. O uso de moléculas como marcadores tumorais vem auxiliando o clínico no diagnóstico, no prognóstico e, até mesmo, no tratamento do TNBC, sendo essenciais na redução de suas altas taxa de mortalidade. No entanto, um pequeno grupo de marcadores tumorais são validados na prática clínica, estimulando à busca por novos alvos, e sua caracterização funcional, como forma de se entender a Biologia desta doença. Assim, o objetivo deste trabalho é caracterizar funcionalmente o gene codificador de proteína CD14 e o gene não codificador de proteína LINC01133 em linhagens celulares humanas de TNBC, no intuito de descobrir o papel destas moléculas na progressão tumoral. Na primeira parte deste trabalho, analisou-se a expressão do CD14 frente à um painel de linhagens celulares que representam os diferentes subtipos dos tumores mamários. O CD14 exibiu elevados níveis de expressão nas linhagens nãotumorigênicas MCF10A e MCF12A e baixos níveis na linhagem triplo negativa Hs578T. A partir destes resultados, o CD14 foi superexpresso na linhagem Hs578T. Ensaios de caracterização funcional mostraram que a superexpressão do CD14 reduziu a capacidade migratória e invasiva das células, efeito que foi hipoteticamente relacionado ao aumento da expressão da E-caderina. No entanto, observou-se aumento no potencial tumorigênico, levando-nos a sugerir seu envolvimento num possível mecanismo utilizado pelas células para compensar a significativa redução do potencial migratório e invasivo. Os resultados obtidos indicam que o nível basal de expressão do CD14 observado na linhagem Hs578T é importante, podendo contribuir para a desenvolvimento primário do tumor, atuando como um oncogene. Na segunda parte deste trabalho, analisou-se a expressão de 10 RNAs longos não codificadores (lncRNAs), frente ao mesmo painel de linhagens descritoanteriormente. Dentre estes, o lncRNA LINC01133 exibiu baixos níveis de expressão nas linhagens não-tumorigênicas MCF10A e MCF12A e elevados níveis na linhagem triplo negativa Hs578T, sendo, então, escolhido como alvo de estudo. A partir destes resultados, decidimos superexpressar, de forma indutível, o LINC01133 na linhagem MCF10A e nocautear este gene, via sistema CRISPR/Cas9, na linhagem Hs578T. Ensaios de caracterização funcional mostraram que a superexpressão do LINC01133 na linhagem MCF10A reduziu a proliferação celular e inibiu o crescimento de colônias dependente de ancoragem, mas, em contrapartida, aumentou o crescimento de colônias independente de ancoragem e a capacidade migratória e invasiva destas células. No entanto, sugerimos que isto não seja suficiente para tornar estas células tumorigênicas e metastáticas. Por outro lado, o nocauteamento do LINC01133 na linhagem triplo negativa Hs578T aumentou de forma considerável todos os parâmetros de malignidade analisados. Baseado nos dados obtidos, sugerimos que o elevado nível de expressão do LINC01133 na linhagem Hs578T é importante na regulação negativa de processos relacionados com a progressão tumoral, atuando com um supressor tumoral. Os dados obtidos em nosso estudo contribuem para o enriquecimento de informações relacionadas à Biologia do TNBC, auxiliando, desta forma, no desenvolvimento de potenciais protocolos clínicos e terapêuticos utilizandos estes biomarcadores


Among the breast cancer subtypes, the triple negative (TNBC) displays the highest mortality rates, being, therefore, considered a major challenge for the clinic. The use of molecules as tumor markers has helped clinicians in the diagnosis, prognosis and even in treatment of TNBC, being essential in reducing its high mortality rate. However, a small group of tumor markers is validated in clinical practice, stimulating the search for new targets, and their functional characterization, as a way to understand the biology of this disease. Thus, the aim of this work is to functionally characterize the CD14 protein-coding gene and the non-protein-coding LINC01133 gene in human TNBC cell lines, in order to probe into the role of these molecules in tumor progression. In the first part of this work, the expression of CD14 was analyzed in a panel of cell lines that represent the different subtypes of breast tumors. High expression levels of CD14 were observed in the non-tumorigenic MCF10A and MCF12A lineages and low levels in the triple negative Hs578T lineage. Based on these results, CD14 was overexpressed in the Hs578T lineage. Functional characterization assays showed that CD14 overexpression reduced the migratory and invasive capacity of cells, an effect that was hypothetically related to increased E-cadherin expression. However, increased in the tumorigenic potential was observed, leading us to suggest its involvement in a possible mechanism used by cells to compensate for the significant reduction in the migratory and invasive potential. The results obtained indicate that CD14 expression basal level observed in the Hs578T lineage may be important to contribute to the primary development of tumor, thus acting as an oncogene. In the second part of this work, the expression of 10 long non-coding RNAs (lncRNAs) was analyzed against the same lineage panel described above. Among these, the LINC01133 lncRNA exhibited low expression levels in the non-tumorigenic MCF10A and MCF12A lineages and high levels in the triple negative Hs578T lineage, being, then, chosen as a target for this study. Based on these results, we decided toinducibly overexpress LINC01133 in the MCF10A lineage and knockout this gene, via the CRISPR/Cas9 system, in the Hs578T lineage. Functional characterization assays showed that overexpression of LINC01133 in the MCF10A lineage reduced cell proliferation and inhibited anchorage-dependent colony growth, but, on the other hand, increased anchorage-independent colony growth and the migratory and invasive capacity of these cells. However, we suggest that this is not sufficient to render these cells tumorigenic and metastatic. On the other hand, the knockout of LINC01133 in the triple negative Hs578T lineage considerably increased all the analyzed malignancy parameters. Based on the results obtained, we suggest that the high expression level of LINC01133 in the Hs578T lineage is important for down-regulation of processes related to tumor progression, acting as a tumor suppressor. The data obtained in our study contribute to the enrichment of information related to TNBC Biology, thus assisting in the development of potential clinical and therapeutic protocols using these biomarkers


Assuntos
Biomarcadores/análise , Biomarcadores Tumorais/análise , Células/química , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular , Crescimento e Desenvolvimento
18.
São Paulo; s.n; s.n; 2022. 86 p. tab, graf.
Tese em Português | LILACS | ID: biblio-1378701

RESUMO

Responsável por milhões de óbitos anuais e um grande custo para a saúde pública, o câncer é a segunda maior causa de mortes no mundo. Dentre seus diversos tipos, o câncer de pulmão, além da alta incidência, é um dos mais letais. A exposição a substâncias tóxicas provenientes da combustão de matéria orgânica, assim como o consumo de cigarro, são os principais responsáveis pela alta incidência de câncer de pulmão. Dentre estas substâncias, está o benzo[α]pireno (B[α]P), um carcinógeno completo, ou seja, capaz de iniciar e promover o processo de carcinogênese. Resultados anteriores obtidos pelo grupo demonstraram que células BEAS-2B expostas a 1 µM de B[α]P apresentaram alterações das concentrações de metabólitos intracelulares, indução de estresse redox e hipermetilação do DNA. A exposição a 1 µM de nicotinamida ribosídeo (NR), um dos precursores de NAD+, foi capaz de proteger as células BEAS-2B contra a transformação induzida por B[α]P, além de impedir totalmente que células não expostas a B[α]P formassem colônias em soft-agar. A utilização da proteômica neste trabalho permitiu verificar a abundância das proteínas nos quatro diferentes grupos de exposição: Controle, B[α]P, B[α]P + NR e NR. Após 120 h de exposição as células foram coletadas, as proteínas extraídas e preparadas para análise. Foram descobertas 3024 proteínas posteriormente analisadas com o objetivo de elucidar vias possivelmente envolvidas na proteção contra o processo de transfomação maligna. Os grupos NR e Controle demonstram ser mais parecidos em relação ao seu conteúdo, enquanto os grupos B[α]P e B[α]P + NR foram mais semelhantes entre si. A análise de proteínas exclusivas revelou menos processos relacionados ao reparo de DNA no grupo tratado apenas com B[α]P quando comparado com B[α]P + NR. A análise estatística do total de proteínas utilizando o teste ANOVA (p < 0,05, N = 5) revelou 564 proteínas diferencialmente expressas entre os grupos. A clusterização nos permitiu observar a diferença na abundância de proteínas entre os quatro tratamentos. As proteínas estão envolvidas em funções como a regulação do metabolismo, resposta a estresse, transdução de sinal, regulação de expressão gênica e morte celular. Um dos clusters (cluster 1), contendo 59 proteínas, revelou poucos processos na análise de enriquecimento, mas as proteínas contidas nele apresentam funções como controle da divisão celular, apoptose e proteção ao estresse redox. Nele podemos observar que, no geral, o tratamento com B[α]P aumentou a abundância de algumas proteínas, o que foi revertido no grupo B[α]P + NR. O tratamento apenas com NR diminuiu a abundância das proteínas contidas nesse cluster. Outro cluster (cluster 4) apresentou 51 proteínas de abundância diminuída durante a exposição ao B[α]P, o que se reverteu no grupo B[α]P + NR. As proteínas desse cluster estão envolvidas em etapas importantes da via glicolítica, de crescimento, adesão, migração e invasão celular. Apesar de ser descrito que a exposição a NR pode aumentar a eficiência do reparo de DNA, os resultados apresentados nesse trabalho indicam que o efeito protetor pode estar relacionado com a modulação do ciclo celular ou alterações na adesão celular


Responsible for millions of annual deaths and a great health expense, cancer is the second leading cause of death in the world. Among its many types, lung cancer, besides its high incidence, is also one of the most lethal. Exposure to toxic substances resulting from the combustion of organic matter, as well as cigarette consumption, are the mainly responsible for the high incidence of lung cancer. One of these substances is benzo[α]pyrene (B[α]P), a complete carcinogen, able to initiate and promote the carcinogenesis process. Results obtained previously demonstrated that BEAS-2B cells exposed to 1 µM BaP presented alterations in the levels of intracellular metabolites, induction of oxidative stress, and hypermethylation of DNA. The exposure to 1 µM nicotinamide riboside (NR), one of the precursors of NAD+, was able to protect BEAS-2B cells against the transformation induced by B[α]P, moreover, it also totally prevented the colonies formation on soft agar in cells not exposed to B[α]P. The use of proteomics allowed us to verify the abundance of proteins in the four different exposure groups: Control, B[α]P, B[α]P + NR e NR. After 120h of exposure, the cells were collected followed by the extraction of the proteins. A total of 3024 proteins were identified and analyzed aiming to elucidate possible pathways involved in the protective effect against the malignant transformation induced by B[α]P. The NR and Control groups showed to be more similar, while B[α]P and B[α]P + NR were more similar. The analysis of exclusive proteins revealed fewer processes related to DNA repair in B[α]P when compared with B[α]P + NR. The statistical analysis of the total proteins using the ANOVA test (p <0.5, N = 5) revealed 564 proteins differentially expressed between the groups. The heatmap showed the difference in protein abundance between the four treatments. Proteins are involved in functionssuch asthe regulation of metabolism, stress response, signal transduction, regulation of gene expression, and cell death. One of the clusters (cluster 1), containing 59 proteins, revealed a few processes in the enrichment analysis, but the proteins contained in it have functions such as control of cell division, apoptosis, and protection from redox stress. It is possible to observe, in general, treatment with B[α]P increased the abundance of some proteins, which was partially reversed in group B[α]P + NR. On the other hand, the NR treatment decreased the abundance of proteins contained in this cluster. Another cluster (cluster 4) showed 51 proteins of decreased abundance during exposure to B [α] P, which was partially reversed in group B[α]P + NR. The proteins in this cluster are involved in important stages of the glycolytic pathway, also in growth, adhesion, migration, and cell invasion. Although it has been described that exposure to NR can increase the efficiency of DNA repair, the results presented in this work indicate that the protective effect may be related to the modulation of the cell cycle or cell adehsion modifications


Assuntos
Proteômica/classificação , Produtos do Tabaco/classificação , Carcinogênese , Neoplasias , Células/classificação , Análise de Variância , Interpretação Estatística de Dados , Morte Celular , Niacinamida/agonistas , Estresse Oxidativo , Neoplasias Pulmonares/patologia
19.
São Paulo; s.n; s.n; 2022. 74 p. tab, graf, ilus.
Tese em Português | LILACS | ID: biblio-1378473

RESUMO

O neuroblastoma é um tumor sólido muito comum em crianças. O estágio mais avançado da doença é altamente agressivo e invasivo, além de pouco responsivo à terapia, que é limitada por mecanismos de resistência e reincidência relacionados à metástase. Muitos estudos tem sido feitos para identificar mecanismos de invasão e quimioresistência de células tumorais, afim de aumentar a sobrevida dos pacientes com câncer. Nesse trabalho, nós estudamos o efeito dos macrófagos, as células imunes mais abundantes no microambiente tumoral, os TAMs (do inglês tumor-associated macrophage) e do receptor P2X7, um purinoreceptor acionado por ATP, nesses processos. Os TAMs respondem e atuam de acordo com a miríade de fatores que encontram, podendo gerar populações heterogêneas e com funções distintas, tanto antitumorais, como pró-tumorais. Altos níveis de ATP extracelular são encontrados no microambiente tumoral, podendo então ativar o receptor P2X7. Este receptor tem sido relacionado tanto a funções inflamatórias como funções na resolução da inflamação de macrófagos. Além disso, o receptor P2X7 está envolvido em uma variedade de eventos celulares, incluindo a secreção de mediadores pró-inflamatórios, a proliferação celular e a apoptose de células tumorais. Primeiramente, foi avaliado o papel do receptor P2X7 na polarização de macrófagos da derivados medula óssea de camundongos wild-type e nocaute para o P2X7 na presença e ausência de fatores secretados por células de neuroblastoma, e então foi estudada a influência desses diferentes macrófagos polarizados em eventos celulares de grande relevância clínica para o neuroblastoma: a invasividade e quimiorresistência. Os resultados demonstraram que, apesar do reconhecido envolvimento do receptor P2X7 na inflamação, a ausência deste receptor não atenua a expressão de marcadores característicos do fenótipo inflamatório, M1. O aumento da expressão do receptor P2Y2, também envolvido na inflamação, nessas células, sugere um mecanismo genético de compensação para não atenuação da inflamação em macrófagos que não expressam o receptor P2X7. Contudo, a ausência do receptor P2X7 levou a alterações no fenótipo alternativo, M2, de modo que a expressão de Tnf, marcador de M2, não foi reprimido. TAMs noucates para P2X7 tiveram a expressão de arg1, marcador de M2, suprimida, reforçando a importância do receptor P2X7 no estabelecimento de fenótipos ativados alternativamente. Nossos dados também sugerem que ausência do receptor P2X7 em TAMs permite a aquisição de um fenótipo capaz de tornar as células de neuroblastoma que expressam P2X7 mais invasivas e mais quimioresistentes à vincristina. Por outro lado, TAMs, independentemente da presença ou ausência do receptor P2X7, induziram a proliferação e quimioresistência das células de neuroblastoma silenciadas para o receptor P2X7, o que nos leva a concluir que o receptor P2X7 em TAMs é desfavorável à progressão de tumores expressando P2X7


Neuroblastoma is a highly common childhood solid tumor. The most advanced stage of the disease is highly aggressive and invasive, besides from being poorly responsive to therapies, which are limited by resistance and recurrence mechanisms related to metastasis. Several studies attempt to identify invasion and resistance mechanisms of the tumor cells in order to increase overall survival of the patients. On the present work, we investigated the effect of macrophages, the most abundant immune cells on the tumor microenvironment, called TAMs (tumor-associated macrophages), and of the P2X7 receptor, an ATP-gated purinoceptor, on these processes. TAMs and cancer cells crosstalk, and behave accordingly to a miriad of factors present at the TME, generating heterogeneous populations with distinct functionalities, either pro- or antitumor. High extracellular levels of ATP are found in the TME, being able to activate the P2X7 receptor. This receptor mediates both pro- and anti-inflammatory functions in macrophages. In addition, it is involved in several cellular events, including the secretion of pro-inflammatory mediators, cell proliferation and tumor cell apoptosis. At first, we evaluated the role of the P2X7 receptor on the polarization of bone marrow-derived macrophages (BMDM), either wild-type or knockout for the P2X7 receptor, in presence or absence or factors secreted by neuroblastoma cells. Next, we investigated the influence of the polarized macrophages in highly relevant cellular events for neuroblastoma, such as invasiveness and chemoresistance. Our results showed that, despite the known involvement of P2X7 receptor on inflammation, its absence did not decrease the expression if inflammatory markers of M1 macrophage populations. An increase in the expression of the P2Y2 receptor, also involved in inflammation, on these cells suggest a genetic compensation mechanism for preventing attenuation of inflammation when P2X7 is lacking. However, P2X7 receptor absence did compromise the M2 phenotype, driving the expression of Tnf. TAMs knockout for the P2X7 receptor were not able to express arg1, also an M2 marker, reinforcing a role of the P2X7 receptor on establishing alternative macrophage phenotypes. Our data also suggest that TAMs lacking the P2X7 receptor acquire a phenotype capable of turning P2X7R-expressing neuroblastoma cells more invasive and chemoresistant to vincristine. On the other hand, TAMs, independently on the presence of the P2X7 receptor, induced proliferation and resistance of neuroblastoma cells silenced for P2X7 receptor expression, leading us to the conclusion that the P2X7 receptor in TAMs is unfavorable for the progression of P2X7R-expressing tumors


Assuntos
Animais , Masculino , Feminino , Camundongos , Receptores Purinérgicos P2X7/análise , Receptores Purinérgicos P2Y2/análise , Macrófagos Associados a Tumor/patologia , Macrófagos/efeitos dos fármacos , Neuroblastoma/patologia , Apoio ao Desenvolvimento de Recursos Humanos/classificação , Medula Óssea , Células/química , Inflamação
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