Involvement of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Skeletal Muscle Stem Cell Differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with NI-guanyl- 1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

Crystalline structure of mangiferin, a C-glycosyl-substituted 9H-xanthen-9-one isolated from the stem bark of Mangifera indica

The crystalline structure of mangiferin (= 2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one; 1), a biologically active xanthenone C-glycoside, isolated from the stem bark of Mangifera indica (Anacardiaceae), was unambiguously determined by single-crystal X-ray diffraction (XRD). The crystal structure is summarized as follows: triclinic, P1, a = 7.6575(5), b = 11.2094(8), c = 11.8749(8) angstrom, alpha = 79.967(5), beta = 87.988(4), gamma = 72.164(4)degrees, V = 955.3(1) angstrom(3), and Z = 2. The structure also shows two molecules in the asymmetric unit cell and five crystallization H2O molecules. The packing is stabilized by several intermolecular H-bonds involving either the two symmetry-independent mangiferin molecules 1a and 1b, or the H2O ones.

Essential Oil from Leaves and Stem Bark of Southeastern Drimys brasiliensis Miers (Winteraceae)

The chemical composition of essential oils obtained from fresh leaves and stem bark of Southeastern Brazilian native Drimys brasiliensis Miers were analyzed by GC and GC/MS and 37 compounds were identified. The oils from fresh leaves showed the presence of monoterpenes (53.9%) and sesquiterpenes (38.4%), with sabinene (9.5%), myrcene (10.5%), limonene (10.6%) and cyclocolorenone (16.0%) being the most abundant. The stern bark oil was characterized by predominance of sesquiterpenoids (87.6%) and the absence of monoterpenes, the main components being cyclocolorenone (28.3%) and spathuleneol (22.9%). A small amount of phenylpropanes (6.8-6.9%) was also detected in both oil samples.

Nytt fordon för transport av bränsleflis : Slutrapport (STEM-projekt 22127-1)

Högskolan Dalarna har i samarbete med Naturbränsle i Mellansverige AB genomfört studier på ett nytt fordon för transport av skogsflis. Fordonet är försett med egen lastningsutrustning (kran och skopa), vilket innebär att flisskördaren kan tippa flisen direkt på marken eller på en i förväg utlagd duk (minskar risken för föroreningar i samband med lastning). Studier har också genomförts på transport av flis med lastväxlarfordon och container (traditionell metod) för att jämförelser skall kunna göras mellan de olika fordonstyperna. Studierna har finansie¬rats via anslag från Statens Energimyndighet och via ”naturabidrag” från deltagande företag.Studierna visar att det nya transportfordonet är ett bättre alternativ än de traditionella last¬växlarfordonen på större objekt med långa transportavstånd. Dessutom pekar studien på att det är sannolikt är bättre även på små objekt under förutsättning att flisningskostnaden kan hållas på en rimlig nivå. Studierna visar också att det bör finnas en viss utvecklingspotential på det nya fordonet (teknik- och metodutveckling), varför det bedöms kunna konkurrera med lastväxlarfordonen även på andra typer av objekt.Jämfört med de traditionella lastväxlarfordonen har det nya fordonet bl.a. följande fördelar:•Transportarbetet blir lättare att planera i och med att beroendeförhållandet mellan in¬blandade maskiner och fordon för flisproduktion och transport upphör.••Risken för störningar i transportflödet minskar.•Miljövinsterna blir större jämfört med lastväxlarfordon som måste ställa ut tomma con¬tainrar innan flisningen kan påbörjas.•Det finns inget behov av lastmaskiner på terminalerna.•Flis kan mellanlagras i skogen.Till nackdelarna med det nya fordonet hör bl.a. följande:•Framkomligheten är något sämre än för lastväxlarfordon på mycket smala och kurviga skogsbilvägar.•Det finns en viss risk för att föroreningar följer med vid lastning av fordonet. Studies were carried out on a new vehicle for transport of fuel chips from the forest. The vehicle was equipped with a crane and a bucket meaning that the chipper may tip the fuel chips right on the ground or on a mat (vira from wood processing industry) to prevent from dirt such as sand and stones when loading. Studies were also carried out on traditional main hauling with transport bins. Transport speed was the same for all vehicles except for the new self-loader on forest roads with lower quality.The studies show that the new system probably is a better alternative on large sites with long transport distances and on sites with only little parking place for transport bins. It is also likely that the new vehicle may be used on very small sites if they are close to each other and if moving cost for the chipper is low.The studies show that the new vehicle has the following advantages:•Transport and other work may be planned in a better way leading to that stress de¬creases.•Dependence between chipper operators and truck drivers decreases.•The risk for disturbances in transport flow decreases.•Environment benefits compared to traditional system with higher traffic intensity (less exhaust gases and lower stress on roads and bridges).•No need for loading machines on terminals.•Easier to store fuel chips on landing.

Långa toppar - metod för uttag av skogsbränsle i slutavverkningar : Slutrapport (STEM-projekt 21937-1)

SammanfattningHögskolan Dalarna har i samarbete med Skogsägarna Mellanskog, Naturbränsle i Mellan¬sverige AB och GDE-Net genomfört studier på en ny metod för uttag av skogsbränsle från slutavverkningar. Metoden går ut på att timmer tas ut som enda rundvirkessortiment. Resten av trädet, samt klenare träd som inte håller timmerdimension, tas ut som ett bränslesortiment. Metoden har jämförts med en konventionell slutavverkning med uttag av timmer, massaved och GROT-flis.Enligt genomförda försök skulle en avverkning enligt den nya metoden (långa toppar) ge ett högre drivningsnetto och drygt dubbelt så mycket bränsleflis som en konventionell avverk¬ning. En anledning till det högre drivningsnettot är att kostnaden för flisning blir lägre än vid flisning av GROT och att flisen betalas bättre än GROT-flis. Resultaten är beroende av de faktiska beståndsförutsättningarna och gällande prisrelationer mellan massaved och bränsle¬flis.Faktorer som har en positiv inverkan på drivningsnettot vid uttag av ”långa toppar” är t.ex. stora uttagsvolymer och korta terrängtransportavstånd samt bestånd med en hög andel virke av låg kvalitet eller udda sortiment som betalas dåligt på rundvirkesmarknaden.SummaryIn Sweden forest energy from final felling is traditionally harvested as logging residues after harvesting of timber (saw logs) and pulpwood, but depending on the market situation other methods with higher yield of forest energy might be of interest. Dalarna University has study a new method called “Undelimbed long tops” where only saw timber was taken out as an industrial assortment. The rest of the trees and smaller trees that don’t hold timber dimensions was left intact on the clear-felled area and been chipped later on. The study was done in different stands with some different conditions. The results have been compared with the traditional method for final felling. The surplus (forest owners net income) was higher in almost all stands when the method with “undelimbed long tops” was used, compared to the traditional method for taking out forest energy, and the volume of chips was more than doubled. A reason for the higher income from long tops is that the costs for chipping is lower and the prize of chips is higher compared to chips from logging residues. Other reason is that forest owners will not be paid for wasted pulpwood, but will be fully paid for the chips from such pulpwood. Factors that will have a positive influence on the ULT-method are for example large logging volumes and short distance between the logging area and the landing, different kinds of price reductions on pulpwood and large volumes of rotten wood or low paid industrial assortments.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Anatomy of relationship significance: a critical realist exploration

Markets-as-networks (MAN) theorists contend, at least tacitly, the significance of business relationships to the firm – that is, business relationships contribute somewhat to corporate survival or growth. One does not deny the existence of significant business relationships but sustain, in contrast to the consensus within the MAN theory, that relationship significance should not be a self-evident assumption. For significance cannot be a taken-for-granted property of each and every one of the firm’s business relationships. The authors adopt explicitly a critical realist metatheoretical position in this conceptual paper and claim that relationship significance is an event of the business world, whose causes remain yet largely unidentified. Where the powers and liabilities of business relationships (i.e., relationship functions and dysfunctions) are put to work, inevitably under certain contingencies (namely the surrounding networks and markets), relationship effects ensue for the firm (often benefits in excess of sacrifices, i.e., relationship value) and as a consequence relationship significance is likely to be brought about. In addition, relationship significance can result from the dual impact that business relationships may have on the structure and powers and liabilities of the firm, that is, on corporate nature and scope, respectively.

Leaf anatomy of rubber-tree clones

A seringueira é uma planta de fácil reconhecimento por ser lenhosa, de porte mediano a grande, que apresenta um padrão característico de desfolha e reenfolhamento e, sobretudo, pela produção de látex. O objetivo do trabalho foi efetuar um estudo anatômico e morfológico foliar, comparando os clones RRIM 600 e GT 1 de seringueira &91;Hevea brasiliensis (Wild. ex Adr. de Juss.) Muell.- Arg&93;, desenvolvidos sob as mesmas condições edáficas e climáticas, para obtenção de informações que possam fornecer subsídios para correlações com dados fisiológicos e também diferenciar os clones em relação ao conteúdo de fibras, espessamento de tecidos do parênquima paliçádico e do parênquima lacunoso, caracterização anatômica do pecíolo, número e tamanho de estômatos e fornecer dados referentes a morfologia foliolar. Foram realizadas secções transversais na região do mesófilo, nervura central e pecíolo, seguindo-se os métodos usuais de preparação de lâminas permanentes. Foram realizadas análises biométricas de extensões de tecidos dos parênquimas paliçádico e lacunoso e contagem do número de células do parênquima lacunoso. Paralelamente foram realizadas análises biométricas para aferições de estômatos. Não houve diferenças para a altura das células epidérmicas, altura e número de camadas do parênquima lacunoso e para o comprimento e para a maior largura do limbo foliolar. Porém houve variação para a espessura das células do parênquima paliçádico, sendo que GT 1 apresentou maior espessura em relação a RRIM 600. GT1 apresentou maior número de estômatos em relação a RRIM 600, porém com menor tamanho. GT1 apresentou maior diâmetro da nervura central da folha e do pecíolo e maior quantidade de fibras de esclerênquima que RRIM 600.

Sclerocorneal limbal stem cell autograft transplantation in dogs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cardiac function in dogs with chronic Chagas cardiomyopathy undergoing autologous stem cell transplantation into the coronary arteries

This study assessed the effects of a single intracoronary injection of autologous stem cells on the cardiac function of dogs with Chagas cardiomyopathy. Bone-marrow-derived stem cells were delivered into the right and left coronary arteries of 5 mature dogs with mildly compromised cardiac function due to chronic Chagas cardiomyopathy. Blood pressure and electrocardiographic and echocardiographic parameters were recorded at monthly intervals for 6 mo in the 3 dogs that survived. Although no changes were observed in the electrocardiogram and blood pressure, there was a significant increase in peak velocity of aortic flow 3 mo after stem cell transplantation. Pre-ejection period, isovolumic relaxation time, and the Tei index of myocardial performance were reduced significantly 4 mo after the procedure. All significant changes persisted to the end of the study. The results suggest that the transplantation of autologous bone-marrow-derived stem cells into the coronary arteries of dogs with Chagas cardiomyopathy may have a beneficial effect but the small number of dogs studied was a limitation.

Seed anatomy and germination of Phoenix roebelenii O'Brien (Arecaceae)

O objetivo desse trabalho foi estudar a morfologia, anatomia e o processo germinativo de sementes de Phoenix roebelenii. Para o levantamento dos dados biométricos foram utilizadas 100 sementes de frutos recém-colhidos, deixados secar ao ar por um dia. Para a germinação, quatro repetições de 50 sementes tratadas com Vitavax-Thiran foram semeadas em bandejas de plástico, contendo Sphagnum sp. como substrato e mantidas sob condições ambientais de laboratório. Detalhes da morfologia da semente foram documentados com o uso de microscopia eletrônica de varredura e esquematizados com auxílio de câmara clara, acoplada ao estereomicroscópio. Foram confeccionadas lâminas permanentes com cortes do embrião, para o estudo de sua anatomia. As dimensões médias das sementes foram: 10,32mm comprimento, 5,21mm largura e 3,91mm espessura. O peso de 1000 sementes foi de 151,1g e 1kg continha 6600 unidades. O início da germinação variou entre 27 e 58 dias. As sementes são albuminosas, com endosperma duro e o embrião é pouco diferenciado, lateral e periférico. A germinação inicia-se pela abertura de um opérculo, através do qual é emitido o pecíolo cotiledonar com o eixo embrionário na extremidade. O pecíolo funciona, internamente, como um haustório, digerindo gradativamente o endosperma. Na sua parte posterior, desenvolve-se a plúmula, que emerge através de uma fenda. Nota-se o aparecimento de raízes secundárias na porção anterior da raiz primária.

Características anatômicas da lâmina foliar e do colmo de gramíneas forrageiras tropicais, em função do nível de inserção no perfilho, da idade e da estação de crescimento

Foram avaliados os efeitos da idade, do nível de inserção da folha no perfilho e da estação de crescimento (verão ou outono), sobre a proporção de tecidos e a espessura da parede celular em lâminas foliares e segmentos de colmo de capim-braquiária (Brachiaria decumbens), capim-gordura (Melinis minutiflora) e capim-tifton 85 (Cynodon sp). Lâminas foliares das posições inferior e superior do perfilho foram colhidas no dia da exposição da lígula e 20 dias após. Foram determinadas as proporções relativas de epiderme, xilema, floema, bainha parenquimática dos feixes, esclerênquima, parênquima no colmo e mesofilo na lâmina foliar. Foram medidas as espessuras das paredes dos vasos de metaxilema e do esclerênquima da lâmina e do colmo. A proporção de tecidos em lâminas foliares não foi alterada pela idade nem pela estação de crescimento, sendo a espessura da parede de células do esclerênquima da lâmina a única característica modificada pela idade. Lâminas do nível de inserção superior apresentaram mais elevadas proporções de esclerênquima, bainha parenquimática dos feixes e xilema e células do esclerênquima e do metaxilema com paredes mais espessas, enquanto as lâminas do nível de inserção inferior se destacaram por apresentar mais elevada proporção de mesofilo e paredes celulares mais delgadas. Enquanto a proporção de parênquima decresceu, a área relativa de esclerênquima e as espessuras das paredes celulares variaram diretamente com a idade do colmo e foram maiores, em geral, no verão.