RESUMO

Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell number per leaf. Proteomic analysis showed that the biogenesis of the PETC proceeded stepwise in wild-type leaves, with accumulation of light-harvesting proteins preceding that of electron transport components, which might explain the increased energy and electron transfer to oxygen and reactive oxygen species build-up at this stage. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide formation through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation was initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.

Assuntos

RESUMO

The development of fleshy fruits involves changes in size and mass, followed by cell differentiation, which is associated with anatomical and histological changes. Parallel to these changes, metabolic alterations lead to the production of osmolytes and energy that modify cell turgor pressure, thereby promoting cell expansion and fruit growth. Detailed information is known about these processes in climacteric fruits (e.g. tomato); however, the regulation of metabolism and its association with anatomical changes in non-climacteric fruit development are poorly understood. In this study, we used detailed anatomical and histological analyses to define three developmental phases of chili pepper (Capsicum chinense cv. Habanero): cell division, cell expansion, and ripening. We showed that each was marked by distinct metabolic profiles, underpinning the switches in energy metabolism to support cellular processes. Interestingly, mitochondrial activity was high in the early stages of development and declined over time, with a modest increase in O2 consumption by pericarp tissues at the beginning of the ripening stage. This respiratory-like burst was associated with the degradation of starch and malate, which are the sources of energy and carbon required for other processes associated with fruit maturation.

Assuntos

RESUMO

BACKGROUND AND AIMS: Gigantism is a key component of the domestication syndrome, a suite of traits that differentiates crops from their wild relatives. Allometric gigantism is strongly marked in horticultural crops, causing disproportionate increases in the size of edible parts such as stems, leaves or fruits. Tomato (Solanum lycopersicum) has attracted attention as a model for fruit gigantism, and many genes have been described controlling this trait. However, the genetic basis of a corresponding increase in size of vegetative organs contributing to isometric gigantism has remained relatively unexplored. METHODS: Here, we identified a 0.4-Mb region on chromosome 7 in introgression lines (ILs) from the wild species Solanum pennellii in two different tomato genetic backgrounds (cv. 'M82' and cv. 'Micro-Tom') that controls vegetative and reproductive organ size in tomato. The locus, named ORGAN SIZE (ORG), was fine-mapped using genotype-by-sequencing. A survey of the literature revealed that ORG overlaps with previously mapped quantitative trait loci controlling tomato fruit weight during domestication. KEY RESULTS: Alleles from the wild species led to lower cell number in different organs, which was partially compensated by greater cell expansion in leaves, but not in fruits. The result was a proportional reduction in leaf, flower and fruit size in the ILs harbouring the alleles from the wild species. CONCLUSIONS: Our findings suggest that selection for large fruit during domestication also tends to select for increases in leaf size by influencing cell division. Since leaf size is relevant for both source-sink balance and crop adaptation to different environments, the discovery of ORG could allow fine-tuning of these parameters.

Assuntos

RESUMO

In an attempt to find new anti-echinococcal drugs, resveratrol (Rsv) effectiveness against the larval stages of Echinococcus granulosus and E. multilocularis was evaluated. The in vitro effect of Rsv on parasites was assessed via optical and electron microscopy, RT-qPCR and immunohistochemistry. In vivo efficacy was evaluated in murine models of cystic (CE) and alveolar echinococcosis (AE). The impact of infection and drug treatment on the mouse bone marrow hematopoietic stem cell (HSC) population and its differentiation into dendritic cells (BMDCs) was investigated via flow cytometry and RT-qPCR. In vitro treatment with Rsv reduced E. granulosus metacestode and protoscolex viability in a concentration-dependent manner, caused ultrastructural damage, increased autophagy gene transcription, and raised Eg-Atg8 expression while suppressing Eg-TOR. However, the intraperitoneal administration of Rsv was not only ineffective, but also promoted parasite development in mice with CE and AE. In the early infection model of AE treated with Rsv, an expansion of HSCs was observed followed by their differentiation towards BMCDs. The latter showed an anti-inflammatory phenotype and reduced LPS-stimulated activation compared to control BMDCs. We suggest that Rsv ineffectiveness could have been caused by the low intracystic concentration achieved in vivo and the drug's hormetic effect, with opposite anti-parasitic and immunomodulatory responses in different doses.

RESUMO

The processes that contribute to plant organ morphogenesis are spatial-temporally organized. Within the meristem, mitosis produces new cells that subsequently engage in cell expansion and differentiation programs. The latter is frequently accompanied by endoreplication, being an alternative cell cycle that replicates the DNA without nuclear division, causing a stepwise increase in somatic ploidy. Here, we show that the Arabidopsis SCL28 transcription factor promotes organ growth by modulating cell expansion dynamics in both root and leaf cells. Gene expression studies indicated that SCL28 regulates members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) family, encoding cyclin-dependent kinase inhibitors with a role in promoting mitotic cell cycle (MCC) exit and endoreplication, both in response to developmental and environmental cues. Consistent with this role, mutants in SCL28 displayed reduced endoreplication, both in roots and leaves. We also found evidence indicating that SCL28 co-expresses with and regulates genes related to the biogenesis, assembly, and remodeling of the cytoskeleton and cell wall. Our results suggest that SCL28 controls, not only cell proliferation as reported previously but also cell expansion and differentiation by promoting MCC exit and endoreplication and by modulating aspects of the biogenesis, assembly, and remodeling of the cytoskeleton and cell wall.

Assuntos

RESUMO

Gibberellin has been proposed to increase leaf elongation in radish (Raphanus sativus L.) plants, which is associated with decreased tuber growth. Since light intensity can control growth through interaction with gibberellin, investigation of the effect of gibberellin levels on the growth of radish plants would be a step forward towards unraveling factors that underlie biomass accumulation and allocation in response to irradiance levels. Here, we report that the gibberellin biosynthesis inhibitor paclobutrazol (PAC) decreased petiole elongation, but not lamina growth of radish plants grown under full sunlight. However, shading promoted an increase in shoot elongation, while in plants treated with PAC the petiole and leaf lamina fail to elongate. Plants treated with PAC allocated proportionally more biomass to their tubers and less to shoot compared to control under shade. Moreover, PAC decreased the abundance of transcripts encoding cell wall expansion proteins in leaf lamina and petiole of plants grown under shade, which was positively correlated with sugar consumption by the tuber, thereby increasing the mass fraction and concentrations of minerals for tuber. Thus, allocation of biomass during the growth of radish plants and nutritional quality of tubers depend on gibberellin and light intensity.

Assuntos

RESUMO

OBJECTIVE: In order to use fluconazole as an antifungal in cell cultures, we evaluated its possible cytotoxic effects and its influence on the proliferation and viability of canine dental pulp-derived stem cells (cDPSCs). METHODS: Samples from permanent canine teeth were placed in a sterile tube with IMDM, penicillin-streptomycin, sodium heparin, and different concentrations of fluconazole. Dental pulp was digested (collagenase type II) and expanded in vitro. After 12 days of culture, enzymatic dissociation of the cDPSCs was performed to quantify, differentiate, and characterize the cells. Cytotoxicity was evaluated based on cell viability in response to fluconazole treatment using the 7-AAD dye. RESULTS: Characterization of the cDPSCs revealed that fluconazole had no influence on the immunophenotypic characteristics and differentiation of these cells. Cell proliferation assay revealed that fluconazole did not significantly interfere with the replication capacity of the cDPSCs. Cytotoxicity analysis revealed a loss of cell viability as the fluconazole concentration increased. Although there was an increase in cell mortality, the number of dead cells remained low. Though the higher concentration of fluconazole (240 µg/mL) resulted in a higher number of non-viable cells, it remained safe for use. CONCLUSION: To prevent fungal contamination that causes a loss of samples during expansion of cDPSCs and to maintain minimal cell toxicity, we suggest adding 120 µg/mL of fluconazole to the teeth collection medium and cDPSCs culture.

RESUMO

The intriguing questions concerning gall development refer to the processes of the remodelling of the host plant organ. Such processes involve the restructuring of cell walls and can be influenced by phenolics, indole-3-acetic acid (IAA) and reactive oxygen species (ROS). Alterations in cell walls demand the interference in the coupling of cellulose fibrils and hemicelluloses (xyloglucans) at specific stages of gall development. In addition to cell wall remodelling, hemicelluloses, such as the, xyloglucans and heteromannans can act as reserve carbohydrates, while xylans provide rigidity to the secondary cell walls. Developmental traits of the lenticular, fusiform and globoid galls on Inga ingoides (Fabaceae) were analysed using anatomical, cytometric, histochemical and immunocytochemical tools. Phenolics, IAA and ROS accumulated in similar gall tissue compartments, and may have influenced the restructuring of hemicelluloses and pectins. Contrary to expectations, cell wall flexibility regarding the dynamics of xyloglucans and cellulose fibrils does not relate to a temporal scale. The detection of xyloglucans in nutritive cell walls relate to carbohydrate nutritional resources to the galling insect, while xylans were associated to the lignified cell walls. Heteromanans were not detected, either in non-galled or galled tissues. The patterns of cell expansion during gall development relied on the relationship among phenolics, ROS and IAA with the hemicelluloses (xyloglucans and xylans) and cellulose fibrils. Although cell wall dynamics is specific to each gall morphotype in I. ingoides, the xyloglucans function as carbohydrate reserve to the gall inducers, which constitutes a functional trait common to the three morphotypes.

Assuntos

RESUMO

ABSTRACT Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.

Assuntos

RESUMO

Precise coordination of cell expansion and cell proliferation underlies growth in multicellular organisms. In addition to endogenous developmental programs, external environmental signals are integrated to modulate organ growth in plants. Nitrate is a nitrogen nutrient that can act as a potent signal to modulate shoot growth, yet the molecular mechanisms involved are largely unexplored in Arabidopsis thaliana or other plant species. Herein, we show that nitrate regulates vegetative growth by modulating cell size and endoreplication. We identified the LGO gene, a CDK inhibitor, as a key cell cycle regulatory factor influencing ploidy and cell-size depending on external nitrate. Nitrate induces LGO gene expression as early as 3 days after germination in epidermal and mesophyll cell layers, which undergo endoreplication to increment DNA content and cell size. Our results support a dual role for LGO on endoreplication and cell expansion. Surprisingly, although endoreplication and cell size are greatly reduced in lgo-2 mutant plants and increased in LGO-OX plants, cotyledon size remains unchanged relative to wild type and is set by the amount of nitrate. In lgo-2 mutant plants where cells are unable to endoreplicate fully, cotyledon organ size is achieved through cell division. We conclude nitrate generally controls cotyledon and leaf size by increasing ploidy levels and cell expansion but that cell division can substitute for endoreplication without affecting final organ size or growth in plants.

Assuntos

RESUMO

NK cells have been seen as potential agents in adoptive immunotherapy for cancer. The main challenge for the success of this approach is to obtain a great quantity of activated NK cells for adoptive transfer. The present study had aimed to evaluate the effect of a feeder layer of irradiated MSCs in the in vitro expansion of NK cells. MSCs were obtained from the bone marrow (BM) cells remaining in the bag and filter used in the transplantation of hematopoietic stem cells. NK cells were obtained from peripheral blood (PB) of healthy volunteers. NK expansion and activation were stimulated by culture with artificial antigen-presenting cells (aAPCs) and IL-2, in the presence or absence of BM-MSCs. NK cell proliferation, phenotypic expression and cytotoxic activity were evaluated. Both culture conditions showed high NK purity with predominance of NK CD56brightCD16+ subset post expansion. However, cultures without the presence of MSCs showed higher NK proliferation, expression of activation markers (CD16 and NKG2D) and related cytotoxic activity. In this experimental study, the presence of a feeder layer of irradiated BM-MSCs interfered negatively in the expansion of PB-NKs, limiting their growth and activation. Further investigation is needed to understand the mechanisms of NK-MSC interaction and its implications.

Assuntos

RESUMO

INTRODUCTION: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. OBJECTIVES: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. METHODS: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. CONCLUSIONS: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.

RESUMO

The exogenous application of GA3 to atemoya tree flowers induces parthenocarpy, and in association with artificial pollination, it increases the fruit size. Morphological, anatomical, ultrastructural, and chemical aspects were evaluated during development of (1) fruit produced by artificial pollination (AP), (2) fruit from AP followed by the application of 250 ppm GA3, and (3) parthenocarpic fruit induced by the application of 1000 ppm GA3. Fruit growth showed a sigmoidal pattern, with development occurring in three phases: (I) cell division, (II) cell differentiation, and (III) maturation. Phase I presented cells with large nuclear volumes and a large population of organelles, phase II presented cells with a reduction in cytoplasm and an increase in vacuole volume, and phase III presented cells with an increase in plastids with reserve compounds. The application of GA3, in association with pollination, precedes cytological events and delays when applied exclusively. GA3 promotes the growth of pollinated fruits by stimulating cell division and expansion, which occur in association with reduced seed production, and the GA3 induces parthenocarpy by maintaining division and stimulating cell expansion. The absence of seeds accounts for the smaller size of the parthenocarpic fruits, and the lower accumulation of calcium accounts for less firm fruit.

Assuntos

RESUMO

MAIN CONCLUSION: Selenium modulates the formation of primary and lateral roots through alterations in auxin and ethylene, leading to new patterns of root architecture in rice seedlings. Selenium (Se) at low concentrations can control root growth through interaction with hormone biosynthesis. Auxin and ethylene have been shown to control the root architecture, with most of the information obtained from the eudicots such Arabidopsis and Nicotiana tabacum. Here, we presented the effects of Se on auxin and ethylene pathways and examined their impact on primary metabolism and root system architecture in rice (Oryza sativa L.) seedlings. Se treatment increased elongation of primary root, but decreased the number and length of lateral roots. Se led to decreased expression of genes associated with the biosynthesis of auxin and ethylene, concomitantly with reduced production of these hormones by the roots. Moreover, Se decreased the abundance of transcripts encoding auxin transport proteins. Indole-3-acetic acid (IAA) treatment overrode the repressive effect of Se on lateral root growth. The ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine (AVG) increased elongation of primary root, whereas the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) resulted in the opposite effect. Soluble sugars accumulate in roots of rice seedlings under Se treatment. Thus, Se modulates the formation of primary and lateral roots through alterations in auxin and ethylene, leading to new patterns of root architecture in rice seedlings.

Assuntos

RESUMO

BACKGROUND: So far, using human blood-derived components appears to be the most efficient and safest approach available for mesenchymal stromal cell (MSC) expansion. In this paper, we report on the characterization of human AB serum (AB HS) produced by using different plasma sources, and its use as an alternative supplement to MSC expansion. METHODS: Two plasma sources were used for AB HS production: plasma removed from whole blood after 24 h of collection (PC > 24 h) and plasma, cryoprecipitate reduced (PCryoR). The biochemical profile and quality of the produced AB HS batches were analyzed and their ability to support MSC cell growth after different storage times (0, 3, 6, 9 and 12 months) was evaluated. RESULTS: The two plasma sources used showed similar characteristics regarding biochemical constituents and quality parameters and were effective in promoting MSC growth. MSCs cultured in medium supplemented with 10% AB HS presented similar doubling times and cumulative population doublings when compared to the 10% fetal bovine serum(FBS)-supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile. CONCLUSION: Overall, the results indicate that AB HS is an efficient FBS substitute and can be used for at least 12 months after production without impairing cell proliferation and quality.

RESUMO

AtGRP3 is a glycine-rich protein from Arabidopsis thaliana shown to interact with the extracellular domain of the receptor-like kinase (RLK) AtWAK1. Based on previous functional data for AtWAK1, a model was proposed that AtGRP3 when bound to this RLK would negatively regulate its kinase activity, inhibiting cell expansion. Here, we review recent functional studies on AtGRP3 that corroborate this model and suggest that AtGRP3/AtWAK1 complex regulates also defense signaling pathways. On the other hand, we show new data on AtGRP3-overexpressing plants indicating that its role in aluminum signaling pathways, as previously observed for elicitor signaling, seems to be more complex than a simple negative regulator.

Assuntos

RESUMO

Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 µM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

Assuntos

RESUMO

Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins (HRGPs), which include several groups of O-glycoproteins, including extensins (EXTs). Proline hydroxylation, an early post-translational modification (PTM) of HRGPs catalyzed by prolyl 4-hydroxylases (P4Hs), defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architecture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

Assuntos

RESUMO

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost-effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier-based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM-MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM-MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ~4.82 ± 1.18 × 10(5) cell mL(-1) at day 7, representing a 3.9-fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP-compliant large-scale production system for hMSCs for cellular therapy.

Assuntos

RESUMO

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

Assuntos