Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

Misturas betuminosas temperadas, do tipo SMA, com incorporação de material betuminoso reciclado e fibras celulósicas : contribuição para a sua caracterização

A crescente necessidade de reabilitação de pavimentos rodoviários tem conduzido ao aumento dos materiais resultantes das intervenções e ao consumo de quantidades significativas de energia e de novos materiais. Umas das formas de reduzir o consumo de energia decorrente da execução de trabalhos de reabilitação, e consequentemente a emissão de gases poluentes associada, é a produção de Misturas Betuminosas Temperadas. Estas são produzidas a temperaturas inferiores a 140ºC, o que permite poupar energia em relação às fabricadas a quente a uma temperatura superior a 140ºC. Por outro lado, a adição de Misturas Betuminosas Recuperadas (MBR) na produção de novas misturas, em substituição de parte dos agregados naturais e do betume novo, contribui para a diminuição do consumo de novos materiais, o que constitui uma prática ambientalmente sustentável e economicamente vantajosa. Reunindo os aspetos referidos no fabrico de misturas Stone Mastic Asphalt (SMA), caracterizadas pela sua elevada durabilidade e baixo custo de manutenção ao longo do seu ciclo de vida, pode obter-se uma mistura com inúmeras vantagens do ponto de vista económico e ambiental, destacando-se a redução do consumo e do custo de matérias-primas naturais, a redução do volume de resíduos a transportar a vazadouro, a redução do consumo de energia e a redução dos custos na manutenção e reabilitação dos pavimentos. Ao longo deste trabalho, descrevem-se as diversas tecnologias usadas para produzir misturas betuminosas temperadas, caracterizam-se as misturas do tipo Stone Mastic Asphalt, e mencionam-se os aspetos mais relevantes na incorporação de MBR na produção de novas misturas. Por último, foram realizados estudos experimentais com o intuito de investigar as propriedades volumétricas e o comportamento mecânico de misturas temperadas do tipo SMA com incorporação de MBR, contribuindo para o desenvolvimento deste tipo de misturas não tradicionais, com o objetivo de promover a sua utilização futura em trabalhos de pavimentação rodoviária.

Caratterizzazione dinamica di miscele bituminose SMA contenenti polverino di gomma da Pneumatici Fuori Uso

Lo Stone Matrix Asphalt (SMA) è un tipo di miscela chiusa costituita da uno scheletro litico di aggregato grosso, assortito in modo tale da ottenere una distribuzione granulometrica indicata con il termine gap-graded, e da un mastice, con funzione riempitiva, ottenuto dalla miscelazione di bitume, filler ed additivi stabilizzanti. In ambito di progettazione delle miscele per conglomerati bituminosi sta assumendo sempre più importanza l’utilizzo di materiali derivanti dalla frantumazione degli Pneumatici Fuori Uso, quali granulato e polverino di gomma. Quest’ultimo può essere impiegato come valida alternativa alla modifica polimerica del bitume garantendo maggiori prestazioni in termini di resistenza all’ormaiamento, a fatica e durabilità, con un conseguente contenimento dei costi di manutenzione della sovrastruttura nel medio e lungo periodo. Il presente studio è stato condotto con lo scopo di valutare le prestazioni meccaniche che una miscela di conglomerato bituminoso può esplicare a seguito della sua mescolazione con il polverino di gomma. In particolare, è stata impiegata una miscela bituminosa di tipo SMA che, data la sua composizione interna, conferisce allo strato di usura della pavimentazione ottime qualità soprattutto in termini di resistenza alle sollecitazioni, durabilità, fonoassorbenza e macrotessitura superficiale. Al fine di rendere più esaustiva la fase sperimentale, sono state messe a confronto due miscele di tipo SMA differenti tra loro per l’aggiunta del polverino di gomma. I dati ottenuti e le considerazioni effettuate al termine della fase sperimentale hanno permesso di affermare che la miscela indagata possiede proprietà meccaniche idonee per essere impiegata nella realizzazione di nuove infrastrutture o nella manutenzione delle pavimentazioni esistenti.

Septenario doloroso de María Sma. ; con un Sermon (al fin) de gracias por el hallazgo de la imagen de N. Sra. de la Assumpcion /

Mode of access: Internet.

Vida de la Sma. Virgen /

Mode of access: Internet.

Vida ejemplar de la excelentísma senõra D.A Dorotea de Chopitea, viuda de Serra /

Mode of access: Internet.

Translation of the Céna Upanishad : one of the chapters of the Sáma Véda according to the gloss of the celebrated Shancaráchárya:establishing the unity and the sole omnipotence of the Supreme Being and that He alone is the object of worship /

Mode of access: Internet.

Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene to maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA) which vary in size and shape were used. Our results show that several polymers can be used to extract membrane proteins comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular weight (7.5-10 kDa) is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification SMA 2000 was found to be the best choice for yield, purity and function. However the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

Australian consumers' attitudes about sports sponsorship during an economic downturn

This paper provides an overview of the prevailing attitudes held by Australian residents as they relate to sports sponsorship during a global financial downturn. A survey of 1,158 Australians assessed changes in attitudes from 2008 to 2009; then it addressed issues specific to the economic conditions of 2009. In general, Australians view sports sponsorship favourably.

The pursuit of thinness : an outcome study of anorexia nervosa

INTRODUCTION: Anorexia nervosa (AN) is a growing problem among young female Singaporeans. We studied the demographics and follow-up data of AN patients referred to dietitians for nutritional intervention. METHODS: A retrospective nutritional notes review was done on 94 patients seen from 1992 to 2004. All patients were given nutritional intervention, which included individualised counselling for weight gain, personalised diet plan, correction of poor dietary intake and correction of perception towards healthy eating. We collected data on body mass index (BMI), patient demographics and outcome. RESULTS: 96 percent of the patients were female and 86.2 percent were Chinese. The median BMI at initial consultation was 14.7 kilogramme per square metre (range, 8.6-18.8 kilogramme per square metre). 76 percent were between 13 and 20 years old. 83 percent of the patients came back for follow-up appointments with the dietitians in addition to consultation with the psychiatrist. Overall, there was significant improvement in weight and BMI from an average 37 kg to 41 kg and 14.7 kilogramme per square metre to 16.4 kilogramme per square metre, respectively, between the fi rst and fi nal consultations (p-value is less than 0.001). The average duration of followup was about eight months. Among the patients on follow-up, 68 percent showed improvement with an average weight gain of 6 kg. Patients that improved had more outpatient follow-up sessions with the dietitians (4.2 consultations versus 1.6 consultations; p-value is less than 0.05), lower BMI at presentation (14.2 kilogramme per square metre versus 15.7 kilogramme per square metre; p-value is less than 0.01) and shorter duration of disease at presentation (one year versus three years; p-value is less than 0.05) compared with those who did not improve. Seven patients with the disease for more than two years did not show improvement with follow-up. CONCLUSION: We gained valuable understanding of the AN patients referred to our tertiary hospital for treatment, two-thirds of whom improved with adequate follow-up treatment. Patients that had suffered AN longer before seeking help appeared more resistant to improvement.

Pre-clinical evaluation of amphiphilic silicone oligomers for scar remediation

The formation of hypertrophic scars is a frequent outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS; Perkins et al., 1983). Although widely used, knowledge regarding SGS and their mechanism of action on hypertrophic scars is limited. Furthermore, SGS require consistent application for at least twelve hours a day for up to twelve consecutive months, beginning as soon as wound reepithelialisation has occurred. Preliminary research at QUT has shown that some species of silicone present in SGS have the ability to permeate into collagen gel skin mimetics upon exposure. An analogue of these species, GP226, was found to decrease both collagen synthesis and the total amount of collagen present following exposure to cultures of cells derived from hypertrophic scars. This silicone of interest was a crude mixture of silicone species, which resolved into five fractions of different molecular weight. These five fractions were found to have differing effects on collagen synthesis and cell viability following exposure to fibroblasts derived from hypertrophic scars (HSF), keloid scars (KF) and normal skin (nHSF and nKF). The research performed herein continues to further assess the potential of GP226 and its fractions for scar remediation by determining in more detail its effects on HSF, KF, nHSF, nKF and human keratinocytes (HK) in terms of cell viability and proliferation at various time points. Through these studies it was revealed that Fraction IV was the most active fraction as it induced a reduction in cell viability and proliferation most similar to that observed with GP226. Cells undergoing apoptosis were also detected in HSF cultures exposed to GP226 and Fraction IV using the Tunel assay (Roche). These investigations were difficult to pursue further as the fractionation process used for GP226 was labour-intensive and time inefficient. Therefore a number of silicones with similar structure to Fraction IV were synthesised and screened for their effect following application to HSF and nHSF. PDMS7-g-PEG7, a silicone-PEG copolymer of low molecular weight and low hydrophilic-lipophilic balance factor, was found to be the most effective at reducing cell proliferation and inducing apoptosis in cultures of HSF, nHSF and HK. Further studies investigated gene expression through microarray and superarray techniques and demonstrated that many genes are differentially expressed in HSF following treatment with GP226, Fraction IV and PDMS7-g-PEG7. In brief, it was demonstrated that genes for TGFβ1 and TNF are not differentially regulated while genes for AIFM2, IL8, NSMAF, SMAD7, TRAF3 and IGF2R show increased expression (>1.8 fold change) following treatment with PDMS7-g-PEG7. In addition, genes for αSMA, TRAF2, COL1A1 and COL3A1 have decreased expression (>-1.8 fold change) following treatment with GP226, Fraction IV and PDMS7-g-PEG7. The data obtained suggest that many different pathways related to apoptosis and collagen synthesis are affected in HSF following exposure to PDMS7-g-PEG7. The significance is that silicone-PEG copolymers, such as GP226, Fraction IV and PDMS7-g-PEG7, could potentially be a non-invasive substitute to apoptosis-inducing chemical agents that are currently used as scar treatments. It is anticipated that these findings will ultimately contribute to the development of a novel scar therapy with faster action and improved outcomes for patients suffering from hypertrophic scars.

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background: Mesenchymal stromal cells (MSC) with similar properties to bone marrow derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We presently contribute to this novel area of research by evaluating methods for culturing human limbal MSC (L-MSC). Methods: Four basic strategies are compared: serum-supplemented medium (10% foetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor, and fibroblast growth factor 2, or one of two commercial serum-free media including Defined Keratinocyte Serum Free Medium (Invitrogen), and MesenCult-XF (Stem Cell Technologies). The phenotype of resulting cultures was examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141, CD271), immunocytochemistry (α-sma), differentiation assays (osteogenesis, adipogenesis, chrondrogenesis), and co-culture experiments with human limbal epithelial (HLE) cells. Results: While all techniques supported to varying degrees establishment of cultures, sustained growth and serial propagation was only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70-80% CD34-/CD45-/CD90+/CD73+/CD105+, approximately 25% α-sma+, and displayed multi-potency. Cultures established in MesenCult-XF were >95% CD34-/CD45-/CD90+/CD73+/CD105+, 40% CD141+, rarely expressed α-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3. Conclusions: We conclude that MesenCult-XF® is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.