Seentega lõngavärvimine ja katsed kaneelpruuniku kasvatamiseks

https://www.ester.ee/record=b5143645*es

Avaliação morfofisiológica de Canavalia ensiformis (L.) DC. (Fabaceae) em substrato contaminado com óleo diesel

Orientadora : Profª. Drª. Cleusa BonaCo-orientadores : Profª. Drª. Bárbara Baêsso Moura, Prof. Dr. André Andrian PadialDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Botânica. Defesa: Curitiba, 28/04/2015Inclui referênciasResumo: A contaminação ambiental por óleo diesel gera grande impacto, pois o óleo é tóxico para animais e plantas, além de ser altamente persistente no ambiente e ter degradação lenta. Por conta disso, existem pesquisas para encontrar novas técnicas alternativas de descontaminação. Dentre essas técnicas, existe a fitorremediação, que utiliza plantas e microorganismos associados na descontaminação ambiental, já que algumas espécies vegetais não tem o desenvolvimento comprometido em ambientes poluídos e auxiliam na descontaminação. Antes de serem utilizadas no processo de fitorremediação, são necessários estudos prévios para analisar a tolerância e a sobrevivência das espécies em solo contaminado. O presente estudo teve como objetivo avaliar a tolerância de Canavalia ensiformis (L.) DC. em substrato contaminado com óleo diesel de maneiras distintas. No primeiro experimento, foram analisadas mudas da espécie em substrato contaminado com 95 ml óleo diesel kg-1 por meio de análises de crescimento, teor de clorofila e carotenoides e estrutura anatômica de raízes e folhas. No segundo experimento, os mesmos parâmetros foram testados, além de análise de germinação, em substrato contaminado com diferentes concentrações de óleo diesel (11,8 ml diesel kg-1; 23,7 ml diesel kg-1 e 47,5 ml diesel kg-1). Os resultados encontrados no primeiro experimento mostraram que as plantas em substrato contaminado apresentaram sistema radicular afetado e baixos valores de crescimento e teores de clorofila e carotenoides. Porém, as plantas que sobreviveram neste substrato emitiram novas raízes modificadas anatomicamente, com formação de aerênquima e meristema apical com alta taxa de mitose, além de ausência de danos celulares nos órgãos (raiz e folha) formados após o plantio. Para o segundo experimento, as médias referentes ao número de sementes germinadas, índice de velocidade de germinação (IVG) e valores de crescimento não diferiram estatisticamente entre os tratamentos em substrato contaminado com diferentes níveis de contaminação. Já o comprimento da raiz principal, área do metafilo e comprimento do caule aos 60 e 90 dias após a semeadura apresentaram menores médias quando comparados às plantas do tratamento em substrato não contaminado. Não houve alterações celulares nos órgãos e a planta respondeu ao estresse com o início de formação de aerênquima na raiz. Aos 210 dias após a semeadura, as plantas de todos os tratamentos apresentaram nódulos radiculares e completaram o ciclo com formação de flores e frutos e, até esses níveis, C. ensiformis apresentou potencial para ser utilizada como fitorremediadora. Palavras chave: Anatomia vegetal, Leguminosa, Feijão-de-Porco, Hidrocarboneto do petróleo.Abstract: Environmental contamination by diesel oil generates impact because it is toxic to animals and plants. The diesel oil is highly persistent in the environment and have slow degradation. Because of this, there is research to find new alternative techniques for decontamination. Phytoremediation is a technique that uses plants in environmental decontamination, because there are plant species able to reduce them the environment without having compromised development. Before the plants to be useful to this technique, it needs to be tested for tolerance and survival analysis in contaminated soil. The aim of the study was to evaluate the tolerance of Canavalia ensiformis in contaminated substrate with diesel oil in different ways. The first experimente analyzed seedlings in contaminated soil with 95 ml kg-1 of diesel oil through of growth parameters, chlorophyll and carotenoids levels and morphological structure of roots and leaves. The second section tested the same parameters, and germination analysis too, in contaminated substrate with different concentrations of diesel (11,8 ml diesel kg-1; 23,7 ml diesel kg-1 e 47,5 ml diesel kg-1). In the first section, in contaminated substrate,the roots of the plants were affected and the plants had low growth values and chlorophyll and carotenoids levels. Despite this, the plants were tolerant to contamination by diesel oil because of the growth of anatomically adapted new roots (aerenchyma and high mitotic rate) and no cellular damage in the root and leaf formed after planting. For the second section, the mean values of number of germinated seeds, IVG and growth values did not differ between treatments in contaminated substrate. The main root length, metaphyll área and stem length at 60 and 90 days after sowing had lower mean values. There were no cellular damage in the organ formed and the plants respond to stress through aerenchyma formation. At 210 days after sowing, the plants of all treatments showed root nodules and had flowers and fruits. Canavalia ensiformis (L.) DC. did not drastically affected in substrate contaminated with diesel oil in concentrations of up to 47.5 ml diesel kg-1. Up to this level of contamination, C. ensiformis has the potential to be used as phytoremediation. Key words: Plant anatomy, jack bean, petroleum hydrocarbon

Spontaansuse ja formaalsuse kui dimensionaalse tekstimudeli dimensioonide automaatne hindamine veebitekstides

https://www.ester.ee/record=b5465709*es

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture

Development of a novel 3D culture system for screening features of a complex implantable device for CNS repair

Tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (SCI). We aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three-dimensional (3D) scaffold and optimize the bioprocesses for tissue culture. Because of the complex 3D configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. Thus, we aim to develop scaled down mini-chambers as simplified in vitro simulation systems, to bridge the gap between two-dimensional (2D) cell cultures on structured substrates and three-dimensional (3D) tissue culture. The mini-chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in CNS repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. Since we use CNS myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. It was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. This research demonstrates that the mini-chambers represent a viable, novel, scale down approach for the evaluation of complex 3D scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of SCI

Exploring hypotheses of the actions of TGF-beta 1 in epidermal wound healing using a 3D computational multiscale model of the human epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta 1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta 1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units ( keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta 1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged ( by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta 1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta 1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing

Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation

Background: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine serum

Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension

Blockade of Mast Cell Activation Reduces Cutaneous Scar Formation

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1a, IL-1b, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase b1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound