Search results for: small cell carcinoma of rectum

Authors: Purva Nemavarkar, Badri Narain Pandey, Jitendra Kumar

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Introduction: The objective was to understand the effect of various radioprotectors on DNA damage repair enzyme and survival in gamma-irradiated wild and cdc mutants of S. pombe (fission yeast) cultured under permissive and restrictive conditions. DNA repair process, as influenced by radioprotectors, was measured by activity of DNA polymerase in the cells. The use of single cell gel electrophoresis assay (SCGE) or Comet Assay to follow gamma-irradiation induced DNA damage and effect of radioprotectors was employed. In addition, studying the effect of caffeine at different concentrations on S-phase of cell cycle was also delineated. Materials and Methods: S. pombe cells grown at permissive temperature (250C) and/or restrictive temperature (360C) were followed by gamma-radiation. Percentage survival and activity of DNA Polymerase (yPol II) were determined after post-irradiation incubation (5 h) with radioprotectors such as Caffeine, Curcumin, Disulphiram, and Ellagic acid (the dose depending on individual D 37 values). The gamma-irradiated yeast cells (with and without the radioprotectors) were spheroplasted by enzyme glusulase and subjected to electrophoresis. Radio-resistant cells were obtained by arresting cells in S-phase using transient treatment of hydroxyurea (HU) and studying the effect of caffeine at different concentrations on S-phase of cell cycle. Results: The mutants of S. pombe showed insignificant difference in survival when grown under permissive conditions. However, growth of these cells under restrictive temperature leads to arrest in specific phases of cell cycle in different cdc mutants (cdc10: G1 arrest, cdc22: early S arrest, cdc17: late S arrest, cdc25: G2 arrest). All the cdc mutants showed decrease in survival after gamma radiation when grown at permissive and restrictive temperatures. Inclusion of the radioprotectors at respective concentrations during post irradiation incubation showed increase in survival of cells. Activity of DNA polymerase enzyme (yPol II) was increased significantly in cdc mutant cells exposed to gamma-radiation. Following SCGE, a linear relationship was observed between doses of irradiation and the tail moments of comets. The radioprotection of the fission yeast by radioprotectors can be seen by the reduced tail moments of the yeast comets. Caffeine also exhibited its radio-protective ability in radio-resistant S-phase cells obtained after HU treatment. Conclusions: The radioprotectors offered notable radioprotection in cdc mutants when added during irradiation. The present study showed activation of DNA damage repair enzyme (yPol II) and an increase in survival after treatment of radioprotectors in gamma irradiated wild type and cdc mutants of S. pombe cells. Results presented here showed feasibility of applying SCGE in fission yeast to follow DNA damage and radioprotection at high doses, which are not feasible with other eukaryotes. Inclusion of caffeine at 1mM concentration to S phase cells offered protection and did not decrease the cell viability. It can be proved that at minimal concentration, caffeine offered marked radioprotection.

Keywords: radiation protection, cell cycle, fission yeast, comet assay, s-phase, DNA repair, radioprotectors, caffeine, curcumin, SCGE

Procedia PDF Downloads 113

Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

Procedia PDF Downloads 79

Authors: Hyuk Sang Yoo, Young Ju Son, Wei Mao, Myung Gu Kang, Sol Lee

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Biomedical applications of electrospun nanofibrous meshes have been received tremendous attentions because of their unique structures and versatilities as biomaterials. Incorporation of growth factors in fibrous meshes can be performed by surface-modification and encapsulation. Those growth factors stimulate differentiation and proliferation of specific types of cells and thus lead tissue regenerations of specific cell types. Topographical cues of electrospun nanofibrous meshes also increase differentiation of specific cell types according to alignments of fibrous structures. Wound healing treatments of diabetic ulcers were performed using nanofibrous meshes encapsulating multiple growth factors. Aligned nanofibrous meshes and those with random configuration were compared for differentiating mesenchymal stem cells into neuronal cells. Thus, nanofibrous meshes can be applied to drug delivery carriers and matrix for promoting cellular proliferation.

Keywords: nanofiber, tissue, mesh, drug

Procedia PDF Downloads 339

Authors: Fitri Ayu, Nadia, Tanti, Putri, Fatkhan, Pasid Harlisa, Suparmi

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Acne is a skin abnormal conditions experienced by many teens, this is caused by various factors such as the climate is hot, humid and excessive sun exposure can aggravate acne because it will lead to excess oil production. Flavonoids form complex compounds against extracellular proteins that disrupt the integrity of bacterial cell membrane in a way denature bacterial cell proteins and bacterial cell membrane damage. This study aimed to test the antibacterial activity of corn silk extract with a concentration of 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % and 100 % in vitro by measuring the inhibition of the growth of bacteria Propionibacterium acne, Staphylococcus aureus and Staphylococcus epidermis then compared with the standard antibiotic clindamycin. Extracts tested by Disk Diffusion Method, in which the blank disc soaked with their respective corn silk extract concentration for 15-30 minutes and then the medium of bacteria that have been planted with Propionibacterium acne, Staphylococcus aureus and Staphylococcus epidermis in the given disk that already contains extracts with various concentration. Incubated for 24 hours and then measured the growth inhibition zone Propionibacterium acne, Staphylococcus aureus and Staphylococcus epidermidis. Corn silk contains flavonoids, is shown by the test of flavonoids in corn silk extract by using a tube heating and without heating. Flavonoid in corn silk potentially as anti acne by inhibiting the growth of bacteria that cause acne. Corn silk extract concentration which has the highest antibacterial activity is then performed in a cream formulation and evaluation test of physical and chemical properties of the resulting cream preparation.

Keywords: antibacterial, flavonoid, corn silk, acne

Procedia PDF Downloads 509

Authors: Mariana Panayotova-Pencheva, Anetka Trifonova, Vassilena Dakova

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790 lungs from brown hares (Lepus europeus L.) from different regions of Bulgaria were investigated during the period 2009-2017. The parasitological status and pathomorphological features in the lungs were recorded. The following parasite species were established: one nematode - Protostrongylus tauricus (7.59% prevalence), one tapeworm – larva of Taenia pisiformis – Cysticercus pisiformis (3.04% prevalence) and one arthropod – larva of Linguatula serrata – Pentastomum dentatum (0.89% prevalence). Macroscopic lesions in the lungs were different depending on the causative agents. The infections with C. pisiformis and P. dentatum were attended with small, mainly superficial changes in the lungs. Protostrongylid infections were connected with different in appearance and burden macroscopic changes. In 77.7%, they were nodular, and in the rest of cases, they diffuse. The consistency of the lesions was compact. In most of the cases, alterations were grey in colour, rarely were dark-red or marble-like. In 91.7% of these cases, they were spread on the apical parts of large lung lobes. In 36.7% middle parts of the large lung lobes, and, in 26.7% small lung lobes, were also affected. The small lung lobes were never independently infected.

Keywords: Cysticercus pisiformis, Lepus europeus, lung lesions, Pentastomum dentatum, Protostrongylus tauricus

Procedia PDF Downloads 212

Authors: Hani Belhadj, Daoud Harzallah, Seddik Khennouf, Saliha Dahamna, Mouloud Ghadbane

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In this study, Lactobacillus strains isolated from pollen were identified by means of phenotypic and genotypic methods, At pH 2, most strains proved to be acid resistants, with losses in cell viability ranging from 0.77 to 4.04 Log orders. In addition, at pH 3 all strains could grew and resist the acidic conditions, with losses in cell viability ranging from 0.40 to 3.61 Log orders. It seems that, 0.3% and 0.5% of bile salts does not affect greatly the survival of most strains, excluding Lactobacillus sp. BH1398. Survival ranged from 81.0±3.5 to 93.5±3.9%. In contrast, in the presence of 1.0% bile salts, survival of five strains was decreased by more than 50%. Lactobacillus fermentum BH1509 was considered the most tolerant strain (77.5% for 1% bile) followed by Lactobacillus plantarum BH1541 (59.9% for 1% bile). Furthermore, all strains were resistant to colistine, clindamycine, chloramphenicol, and ciprofloxacine, but most of the strains were susceptible to Peniciline, Oxacillin, Oxytetracyclin, and Amoxicillin. Functionally interesting Lactobacillus isolates may be used in the future as probiotic cultures for manufacturing fermented foods and as bioactive delivery systems.

Keywords: probiotics, lactobacillus, pollen, bile, acid tolerance

Procedia PDF Downloads 420

Authors: Vaclav Mentl, P. Zlabek, J. Volak

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New methods of mechanical testing were developed recently that are based on making use of miniature test specimens (e.g. Small Punch Test). The most important advantage of these method is the nearly non-destructive withdrawal of test material and small size of test specimen what is interesting in cases of remaining lifetime assessment when a sufficient volume of the representative material cannot be withdrawn of the component in question. In opposite, the most important disadvantage of such methods stems from the necessity to correlate test results with the results of standardised test procedures and to build up a database of material data in service. The correlations among the miniature test specimen data and the results of standardised tests are necessary. The paper describes the results of fatigue tests performed on miniature tests specimens in comparison with traditional fatigue tests for several steels applied in power producing industry. Special miniature test specimens fixtures were designed and manufactured for the purposes of fatigue testing at the Zwick/Roell 10HPF5100 testing machine. The miniature test specimens were produced of the traditional test specimens. Seven different steels were fatigue loaded (R = 0.1) at room temperature.

Keywords: mechanical properties, miniature test specimens, correlations, small punch test, micro-tensile test, mini-charpy impact test

Procedia PDF Downloads 538

Authors: Adegboyega Adeolapo Ola, Gerelene Jagganath

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One of the greatest challenges of human right in the world, especially African states is the use of child soldiers in armed conflict, constituting a major source of destruction of lives and properties. Mostly, they are in developing countries with the situation in Sub-Saharan Africa, the abduction and employment of children as soldiers is a form of exploitative labour that is tantamount to slavery. Since the end of cold war, Child soldier has increased in Africa countries like Angola, Liberia, Sierra Leone and Uganda. This study examines the main cause of the recruitment and use of child soldiers and its challenges to human right. It further assesses the role of international regional bodies and various governments in curbing child soldiers with a view to proffer suggestions on how to address some of the resultant threat of human right. The study posits that the control of small arms and light weapons is essential in curtailing the spread of child soldier and abuse of human right. This hopefully should result in the sustainability of human/child right in African continent. It is a recommendation of this study that, in order to sustain human right in the region, all Africa leaders, government and regional bodies; such as African Union, Economic Community of West African States, South African Development Community among others, should cooperate and work together to address the issue of illicit small arms, which could eventually lead to child soldier.

Keywords: arms control, child soldier, human right, small arms

Procedia PDF Downloads 207

Authors: Cagla Gul Guldiken, Levent Akyalcin, Hasan Ferdi Gercel

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Fuel cells are electrochemical devices which convert the chemical energy of hydrogen into the electricity. Among the types of fuel cells, polymer electrolyte membrane fuel cells (PEMFCs) are attracting considerable attention as non-polluting power generators with high energy conversion efficiencies in mobile applications. Polymer electrolyte membrane (PEM) is one of the essential components of PEMFCs. Perfluorosulfonic acid based membranes known as Nafion® is widely used as PEMs. Nafion® membranes water dependent proton conductivity which limits the operating temperature below 100ᵒC. At higher temperatures, proton conductivity and mechanical stability of these membranes decrease because of dehydration. Polybenzimidazole (PBI), which has good anhydrous proton conductivity after doped with acids, as well as excellent thermal stability, shows great potential in the application of high temperature PEMFCs. In the present study, PBI polymers were synthesized by solution polycondensation at 190 and 210ᵒC. The synthesized polymers were characterized by FTIR, 1H NMR, and TGA. Phosphoric acid doped PBI membranes were prepared and tested in a PEMFC. The influences of reaction temperature on structural properties of synthesized polymers were investigated. Mechanical properties, acid-doping level, proton conductivity, and fuel cell performances of prepared phosphoric acid doped PBI membranes were evaluated. The maximum power density was found as 32.5 mW/cm² at 120ᵒC.

Keywords: fuel cell, high temperature polymer electrolyte membrane, polybenzimidazole, proton exchange membrane fuel cell

Procedia PDF Downloads 185

Authors: Vivian Uchechi Okpala

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Background: Millions of workers die every year as a result of occupational hazards, accidents and injuries, which are as a result of non- compliance to the laws or legislations guiding the health, safety and welfare of workers in the industries. This and many more lead to the assessment of implementation of the health and safety contents of the Nigerian Factories Act (NFA) by small and medium scale industries in Anambra State. Objectives: The study is aimed at achieving the following specific objectives; to assess the extent of implementation of Part-II Health and Part -III Safety (General Provisions), implementation of Part II Health and Part -III Safety (General Provisions Nigerian Factories Acts based on the age of the industries, locations of the industries and level of education of the workers of the small and medium scale industries Methods: the research design that was used for this study was descriptive survey research design, Area of this study was Anambra state, The population for this study comprised 180 chairmen/presidents of union workers of manufacturing industries in Anambra State, The instrument used for this study was structured questionnaire titled ‘assessment of implementation of NFA health and safety contents by small and medium scale industries, results: From the analysis, the following findings were made: Results: The medium scale industries implemented the Part-II Health and Part III Safety (General provisions) better than the small scale industries in Anambra state, the age of the industries, location of the industries and the level of education of the workers in the industries significantly influenced the implementation of the Part III Safety (General Provisions) of NFA, the location of the industries significantly influenced the implementation of the Part II-Health (General Provisions) of NFA. Conclusion: there was generally a certain level of implementation of the factories Act, there is need for more improvement, strict inspection by the regulatory agencies. Implications of the study were highlighted and several suggestions for further studies were made. Based on the findings, several recommendations were made including that the Ministry of Labour and Productivity and the Ministry of Health should strengthen planned information, strict policies to sanction the offenders. Keywords: Occupational Health and Safety, Nigerian Factories Act

Keywords: occupational health and safety, Nigerian factories act, workers, welfare

Procedia PDF Downloads 141

Authors: Hilal Turkoglu Sasmazel, Ozan Ozkan, Seda Surucu

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Tissue engineering is the field of treating defects caused by injuries, trauma or acute/chronic diseases by using artificial scaffolds that mimic the extracellular matrix (ECM), the natural biological support for the tissues and cells within the body. The main aspects of a successful artificial scaffold are (i) large surface area in order to provide multiple anchorage points for cells to attach, (ii) suitable porosity in order to achieve 3 dimensional growth of the cells within the scaffold as well as proper transport of nutrition, biosignals and waste and (iii) physical, chemical and biological compatibility of the material in order to obtain viability throughout the healing process. By hybrid scaffolds where two or more different materials were combined with advanced fabrication techniques into complex structures, it is possible to combine the advantages of individual materials into one single structure while eliminating the disadvantages of each. Adding this to the complex structure provided by advanced fabrication techniques enables obtaining the desired aspects of a successful artificial tissue scaffold. In this study, fibroblast compatibility of poly(ε-caprolactone) (PCL)/chitosan core-shell electrospun hybrid scaffolds with proper mechanical, chemical and physical properties successfully developed in our previous study was investigated. Standard 7-day cell culture was carried out with L929 fibroblast cell line. The viability of the cells cultured with the scaffolds was monitored with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay for every 48 h starting with 24 h after the initial seeding. In this assay, blank commercial tissue culture polystyrene (TCPS) Petri dishes, single electrospun PCL and single electrospun chitosan mats were used as control in order to compare and contrast the performance of the hybrid scaffolds. The adhesion, proliferation, spread and growth of the cells on/within the scaffolds were observed visually on the 3rd and the 7th days of the culture period with confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). The viability assay showed that the hybrid scaffolds caused no toxicity for fibroblast cells and provided a steady increase in cell viability, effectively doubling the cell density for every 48 h for the course of 7 days, as compared to TCPS, single electrospun PCL or chitosan mats. The cell viability on the hybrid scaffold was ~2 fold better compared to TCPS because of its 3D ECM-like structure compared to 2D flat surface of commercially cell compatible TCPS, and the performance was ~2 fold and ~10 fold better compared to single PCL and single chitosan mats, respectively, even though both fabricated similarly with electrospinning as non-woven fibrous structures, because single PCL and chitosan mats were either too hydrophobic or too hydrophilic to maintain cell attachment points. The viability results were verified with visual images obtained with CSLM and SEM, in which cells found to achieve characteristic spindle-like fibroblast shape and spread on the surface as well within the pores successfully at high densities.

Keywords: chitosan, core-shell, fibroblast, electrospinning, PCL

Procedia PDF Downloads 176

Authors: Rohaya Abdul Wahab, Raja Mohd Fuad Tengku Aziz, Nazaliza Othman, Sharifah Saleh, Nabihah Razali, Rozaimah Baharim, Md Hanif Md Nasir

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This paper will discuss flip chip methodology, in which I/O pads, standard cells, macros and bump cells array are placed in the floorplan, then routed using Astro place and route tool. Final DRC and LVS checking is done using Calibre verification tool. The design vehicle to run this methodology is an OpenRISC design targeted to Silterra 0.18 micrometer technology with 6 metal layers for routing. Astro has extensive support for flip chip placement and routing. Astro tool commands for flip chip are straightforward approach like the conventional standard wire bond packaging. However since we do not have flip chip commands in our Astro tool, no LEF file for bump cell and no LEF file for flip chip I/O pad, we create our own methodology to prepare for future flip chip tapeout. 

Keywords: methodology, flip chip, bump cell, LEF, astro, calibre, SCHEME, TCL

Procedia PDF Downloads 488

Authors: Neha Singh, Anuj Ranjan, Tanu Jindal

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Over the last decade, the exponential growth of mobile communication has been accompanied by a parallel increase in density of electromagnetic fields (EMF). The continued expansion of mobile phone usage raises important questions as EMF, especially radio frequency (RF), have long been suspected of having biological effects. In the present experiments, we studied the effects of RF-EMF on cell death (apoptosis) and DNA damage of a well- tested biological model, Drosophila melanogaster exposed to 2400 MHz frequency for different time duration i.e. 2 hrs, 4 hrs, 6 hrs,8 hrs, 10 hrs, and 12 hrs each day for five continuous days in ambient temperature and humidity conditions inside an exposure chamber. The flies were grouped into control, sham-exposed, and exposed with 100 flies in each group. In this study, well-known techniques like Comet Assay and TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) Assay were used to detect DNA damage and for apoptosis studies, respectively. Experiments results showed DNA damage in the brain cells of Drosophila which increases as the duration of exposure increases when observed under the observed when we compared results of control, sham-exposed, and exposed group which indicates that EMF radiation-induced stress in the organism that leads to DNA damage and cell death. The process of apoptosis and mutation follows similar pathway for all eukaryotic cells; therefore, studying apoptosis and genotoxicity in Drosophila makes similar relevance for human beings as well.

Keywords: cell death, apoptosis, Comet Assay, DNA damage, Drosophila, electromagnetic fields, EMF, radio frequency, RF, TUNEL assay

Procedia PDF Downloads 169

Authors: Julius Denafas, Irina Kliopova, Gintaras Denafas

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Three opportunities for implementation of industrial ecology principles in the real industrial production of c-Si solar cells and modules are presented in this study. It includes: material flow dematerialisation, product modification and industrial symbiosis. Firstly, it is shown how the collaboration between R&D institutes and industry helps to achieve significant reduction of material consumption by a) refuse from phosphor silicate glass cleaning process and b) shortening of SiNx coating production step. This work was performed in the frame of Eco-Solar project, where Soli Tek R&D is collaborating together with the partners from ISC-Konstanz institute. Secondly, it was shown how the modification of solar module design can reduce the CO2 footprint for this product and enhance waste prevention. It was achieved by implementing a frameless glass/glass solar module design instead of glass/backsheet with aluminium frame. Such a design change is possible without purchasing new equipment and without loss of main product properties like efficiency, rigidity and longevity. Thirdly, industrial symbiosis in the solar cell production is possible in such case when manufacturing waste (silicon wafer and solar cell breakage) are collected, sorted and supplied as raw-materials to other companies involved in the production chain of c-Si solar cells. The obtained results showed that solar cells produced from recycled silicon can have a comparable electrical parameters like produced from standard, commercial silicon wafers. The above mentioned work was performed at solar cell producer Soli Tek R&D in the frame of H2020 projects CABRISS and Eco-Solar.

Keywords: solar cells and solar modules, manufacturing, waste prevention, recycling

Procedia PDF Downloads 213

Authors: Davinder Singh, Jaimal Singh Khamba, Tarun Nanda

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Growth of the Indian economy has accelerated to 8% and efforts are on to further propel it to 10%. Undoubtedly, all the segments of the economy, viz. agriculture, industry and services have to improve their contribution to the economy. Growth of Micro-small and medium enterprises (MSMEs) is a sine qua non for the growth of industry, exports and other segments of the economy. Furthermore, promotion of entrepreneurship is also vital for sustenance and upward movement of the current growth trajectory of the economy. The MSME sector acts as a catalyst in upholding and encouraging the creation of the innovative spirit and entrepreneurship in the economy, thereby helping in laying the foundation for rapid industrial development. In this competitive world, they need to be able to confront the increasing competition from developed and emerging economies and to plug into the new market opportunities.

Keywords: characteristics, management, MSMEs, technology infrastructure

Procedia PDF Downloads 642

Authors: I. Bochev, A. Shterev, S. Kyurkchiev

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One of the factors associated with poor success rates in human in vitro fertilization (IVF) is the suboptimal culture conditions in which fertilization and early embryonic growth occur. Co-culture systems with helper cell lines appear to enhance the in vitro conditions and allow embryos to demonstrate improved in vitro development. The co-culture of human embryos with monolayers of autologous endometrial stromal cell (EnSCs) results in increased blastocyst development with a larger number of blastomeres, lower incidence of fragmentation and higher pregnancy rates in patients with recurrent implantation failure (RIF). The aim of the study was to examine the influence of autologous endometrial stromal cell (EnSC) co-culture on day 3 embryo quality by comparing the morphological status of the embryos from the same patients undergoing consecutive IVF/Intracytoplasmic sperm injection (ICSI) cycles without and with EnSC co-culture. This retrospective randomized study (2015-2017) includes 20 couples and a total of 46 IVF/ICSI cycles. Each patient couple included had at least two IVF/ICSI procedures – one with and one without autologous EnSC co-culture. Embryo quality was assessed at 68±1 hours in culture, according to Istanbul consensus criteria (2010). Day 3 embryos were classified into three groups: good – grade 1; fair – grade 2; poor – grade 3. Embryos from all cycles were divided into two groups (A – co-cultivated; B – not co-cultivated) and analyzed. Second, for each patient couple, embryos from matched IVF/ICSI cycles (with and without co-culture) were analyzed separately. When an analysis of co-cultivated day 3 embryos from all cycles was performed (n=137; group A), 43.1% of the embryos were graded as “good”, which was not significantly different from the respective embryo quality rate of 42.2% (p = NS) in group B (n=147) with non-co-cultivated embryos. The proportions of fair and poor quality embryos in group A and group B were similar as well – 11.7% vs 10.2% and 45.2% vs 47.6% (p=NS), respectively. Nevertheless, the separate embryo analysis by matched cycles for each couple revealed that in 65% of the cases the proportion of morphologically better embryos was increased in cycles with co-culture in comparison with those without co-culture. A decrease in this proportion after endometrial stromal cell co-cultivation was found in 30% of the cases, whereas no difference was observed in only one couple. The results demonstrated that there is no marked difference in the overall morphological quality between co-cultured and non-co-cultured embryos on day 3. However, in significantly greater percentage of couples the process of autologous EnSC co-culture could increase the proportion of morphologically improved day 3 embryos. By mimicking the in vivo relationship between embryo and maternal environment, co-culture in autologous EnSC system represents a perspective approach to improve the quality of embryos in cases with elevated risk for development of embryos with impaired morphology.

Keywords: autologous endometrial stromal cells, co-culture, day 3 embryo, morphological quality

Procedia PDF Downloads 234

Authors: M. Cardenas-Garcia, P. P. Gonzalez-Perez

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Intercellular communication is a necessary condition for cellular functions and it allows a group of cells to survive as a population. Throughout this interaction, the cells work in a coordinated and collaborative way which facilitates their survival. In the case of cancerous cells, these take advantage of intercellular communication to preserve their malignancy, since through these physical unions they can send signs of malignancy. The Wnt/β-catenin signaling pathway plays an important role in the formation of intercellular communications, being also involved in a large number of cellular processes such as proliferation, differentiation, adhesion, cell survival, and cell death. The modeling and simulation of cellular signaling systems have found valuable support in a wide range of modeling approaches, which cover a wide spectrum ranging from mathematical models; e.g., ordinary differential equations, statistical methods, and numerical methods– to computational models; e.g., process algebra for modeling behavior and variation in molecular systems. Based on these models, different simulation tools have been developed from mathematical ones to computational ones. Regarding cellular and molecular processes in cancer, its study has also found a valuable support in different simulation tools that, covering a spectrum as mentioned above, have allowed the in silico experimentation of this phenomenon at the cellular and molecular level. In this work, we simulate and explore the complex interaction patterns of intercellular communication in cancer cells using the Cellulat bioinformatics tool, a computational simulation tool developed by us and motivated by two key elements: 1) a biochemically inspired model of self-organizing coordination in tuple spaces, and 2) the Gillespie’s algorithm, a stochastic simulation algorithm typically used to mimic systems of chemical/biochemical reactions in an efficient and accurate way. The main idea behind the Cellulat simulation tool is to provide an in silico experimentation environment that complements and guides in vitro experimentation in intra and intercellular signaling networks. Unlike most of the cell signaling simulation tools, such as E-Cell, BetaWB and Cell Illustrator which provides abstractions to model only intracellular behavior, Cellulat is appropriate for modeling both intracellular signaling and intercellular communication, providing the abstractions required to model –and as a result, simulate– the interaction mechanisms that involve two or more cells, that is essential in the scenario discussed in this work. During the development of this work we made evident the application of our computational simulation tool (Cellulat) for the modeling and simulation of intercellular communication between normal and cancerous cells, and in this way, propose key molecules that may prevent the arrival of malignant signals to the cells that surround the tumor cells. In this manner, we could identify the significant role that has the Wnt/β-catenin signaling pathway in cellular communication, and therefore, in the dissemination of cancer cells. We verified, using in silico experiments, how the inhibition of this signaling pathway prevents that the cells that surround a cancerous cell are transformed.

Keywords: cancer cells, in silico approach, intercellular communication, key molecules, modeling and simulation

Procedia PDF Downloads 249

Authors: Aroonsri Priprem, Sucharat Limsitthichaikoon, Suttasinee Thappasarapong

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Anthocyanins are natural pigments with effective UV protection but their topical use could be limited due to their physicochemical characteristics. An attempt to overcome such limitations by complexation of 2 major anthocyanin-rich sources, C. ternatea, and Z. mays, for investigation on potential use as topical anti-inflammatory. Cell studies indicate no cytotoxicity of the anthocyanin complex (AC) up to 1 mg/ml tested in HaCaT and human forehead fibroblasts by MTT. Croton oil-induced ear edema in Wistar rats suggests an effective dose of 5 mg/cm2 of AC as a topical anti-inflammatory in comparison to 0.5 mg/cm2 of fluocinolone acetonide. Niosomal encapsulation of the AC significantly prolonged the anti-inflammatory activity particularly at 8 h after topical application (p = 0.0001). The AC was not cytotoxic and its anti-inflammatory and activity was dose-dependent and prolonged by niosomal encapsulation. It has also shown to promote collagen type 1 production in cell culture. Thus, AC could be a potential candidate for topical anti-inflammatory agent from natural resources.

Keywords: anthocyanin complex, ear edema, inflammation, niosomes, skin

Procedia PDF Downloads 328

Authors: Hilal T. Sasmazel, Seda Surucu

Abstract:

Skin tissue engineering is a promising field for the treatment of skin defects using scaffolds. This approach involves the use of living cells and biomaterials to restore, maintain, or regenerate tissues and organs in the body by providing; (i) larger surface area for cell attachment, (ii) proper porosity for cell colonization and cell to cell interaction, and (iii) 3-dimensionality at macroscopic scale. Recent studies on this area mainly focus on fabrication of scaffolds that can closely mimic the natural extracellular matrix (ECM) for creation of tissue specific niche-like environment at the subcellular scale. Scaffolds designed as ECM-like architectures incorporating into the host with minimal scarring/pain and facilitate angiogenesis. This study is related to combining of synthetic PCL and natural chitosan polymers to form 3D PCL/Chitosan core-shell structures for skin tissue engineering applications. Amongst the polymers used in tissue engineering, natural polymer chitosan and synthetic polymer poly(ε-caprolactone) (PCL) are widely preferred in the literature. Chitosan has been among researchers for a very long time because of its superior biocompatibility and structural resemblance to the glycosaminoglycan of bone tissue. However, the low mechanical flexibility and limited biodegradability properties reveals the necessity of using this polymer in a composite structure. On the other hand, PCL is a versatile polymer due to its low melting point (60°C), ease of processability, degradability with non-enzymatic processes (hydrolysis) and good mechanical properties. Nevertheless, there are also several disadvantages of PCL such as its hydrophobic structure, limited bio-interaction and susceptibility to bacterial biodegradation. Therefore, it became crucial to use both of these polymers together as a hybrid material in order to overcome the disadvantages of both polymers and combine advantages of those. The scaffolds here were fabricated by using electrospinning technique and the characterizations of the samples were done by contact angle (CA) measurements, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-Ray Photoelectron spectroscopy (XPS). Additionally, gas permeability test, mechanical test, thickness measurement and PBS absorption and shrinkage tests were performed for all type of scaffolds (PCL, chitosan and PCL/chitosan core-shell). By using ImageJ launcher software program (USA) from SEM photographs the average inter-fiber diameter values were calculated as 0.717±0.198 µm for PCL, 0.660±0.070 µm for chitosan and 0.412±0.339 µm for PCL/chitosan core-shell structures. Additionally, the average inter-fiber pore size values exhibited decrease of 66.91% and 61.90% for the PCL and chitosan structures respectively, compare to PCL/chitosan core-shell structures. TEM images proved that homogenous and continuous bead free core-shell fibers were obtained. XPS analysis of the PCL/chitosan core-shell structures exhibited the characteristic peaks of PCL and chitosan polymers. Measured average gas permeability value of produced PCL/chitosan core-shell structure was determined 2315±3.4 g.m-2.day-1. In the future, cell-material interactions of those developed PCL/chitosan core-shell structures will be carried out with L929 ATCC CCL-1 mouse fibroblast cell line. Standard MTT assay and microscopic imaging methods will be used for the investigation of the cell attachment, proliferation and growth capacities of the developed materials.

Keywords: chitosan, coaxial electrospinning, core-shell, PCL, tissue scaffold

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Authors: Cristina Busuioc, Georgeta Voicu, Sorin-Ion Jinga

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The human bone presents in its dry form piezoelectric properties, which means that a mechanical stress results in electric polarization and an applied electric field causes strain. The immediate consequence was the revealing of piezoelectricity role in bone remodelling, as well as the integration of ceramic materials with piezoelectric behaviour in the composition of unitary or composite biomaterials. Thus, we prepared hydroxyapatite - collagen hybrid materials with barium titanate addition in order to achieve a better osseointegration. Barium titanate powder synthesized by a combined sol-gel-hydrothermal method, commercial hydroxyapatite and laboratory extracted collagen gel were employed as starting materials. Before the composites, fabrication, the powder with piezoelectric features was characterized in detail from the compositional, structural, morphological and electrical point of view. The next step was to elucidate the influence of barium titanate presence especially on the biological properties of the final materials. The biocompatibility of the hybrid supports without or with piezoelectric addition was investigated on mouse osteoblast cells through LDH cytotoxicity assay, LIVE/DEAD cell viability assay, and MTT cell proliferation assay. All results indicated that the analysed materials do not exert cytotoxic effects and present the ability to sustain cell survival and to promote their proliferation. In conclusion, barium titanate nanoparticles exhibit a good biocompatibility and osteoinductive properties, while the derived composite materials based on hydroxyapatite as oxide phase and collagen as polymeric phase can be successfully used for tissue engineering applications.

Keywords: barium titanate, hybrid composites, piezoelectricity, tissue engineering

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Authors: V. Marzal, J. C. Torres, B. Garcia-Camara, Manuel Cano-Garcia, Xabier Quintana, I. Perez Garcilopez, J. M. Sanchez-Pena

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At the present time, the use of nanostructures in complex media, like liquid crystals, is widely extended to manipulate their properties, either electrical or optical. In addition, these media can also be used to control the optical properties of the nanoparticles, for instance when they are resonant. In this work, the change on electrical properties of a liquid crystal cell by adding TiO₂ nanoparticles on one of the alignment layers has been analyzed. These nanoparticles, with a diameter of 100 nm and spherical shape, were deposited in one of the substrates (ITO + polyimide) by spin-coating in order to produce a homogeneous layer. These substrates were checked using an optical microscope (objective x100) to avoid potential agglomerates. The liquid crystal cell is then fabricated, using one of these substrates and another without nanoparticles, and filled with E7. The study of the electrical response was done through impedance measurements in a long range of frequencies (3 Hz- 6 MHz) and at ambient temperature. Different nanoparticle concentrations were considered, as well as pure E7 and an empty cell for comparison purposes. Results about the effective dielectric permittivity and conductivity are presented along with models of equivalent electric circuits and its physical interpretation. As a summary, it has been observed the clear influence of the presence of the nanoparticles, strongly modifying the electric response of the device. In particular, a variation of both the effective permittivity and the conductivity of the device have been observed. This result requires a deep analysis of the effect of these nanoparticles on the trapping of free ions in the device, allowing a controlled manipulation and frequency tuning of the electrical response of these devices.

Keywords: alignment layer, electrical behavior, liquid crystal, TiO₂ nanoparticles

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Authors: P. S. Dhananjaya Swamy, B. Chinnappa, G. B. Ramesh, Naveen P. Kumar

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Kodagu district is one of the most densely forested districts in the India as around sixty five per cent of geographical areas under tree cover. Nearly 53 per cent of the flora of Kodagu is endemic. The district is also a hotspot of endemic orchids found mainly in the Thadiandamol. Shade grown, eco-friendly coffee farms are perhaps a selected few places on this planet where nature runs wild. The Kodagu accounts for more than 8.8 per cent of floral diversity of Karnataka state. Estimation of unit cost of cultivation plays a vital role in determining the governmental program their market intervention policies. On an average, planters incurred around Rs. 17041 per acre. The extent of production risk was highest among small category of planters (66 %) compared to other two exhibiting production instability. The result shows that, the coffee productivity in medium plantations was 1051.2 kg per acre as against 758.5 and 789.2 kg in the case of small and large plantations. An annual net return per acre was highest in the case of medium planters (Rs. 26109.3) as against Rs. 20566.7 and Rs. 18572.7 in the case of small and large planters. Cost of production was lowest in the case of small planters (Rs. 18.9 per kg of output) followed by medium planters (Rs. 21.2 per kg of output) and large planters (Rs. 22.5 per kg of output). The productivity of coffee is less whenever it is grown under high shade and native tree cover; it is around 6 quintals per acre when compared with low shade conditions, which is around 8.9 quintals per acre, without a significant difference in the amount invested for growing coffee. Net gain was lower by Rs. 15.5 per kg for the planters growing under high shade and native trees cover when compared with low shade and exotic trees cover.

Keywords: coffee, cultivation, economics, Kodagu

Procedia PDF Downloads 196

Authors: P. Slepicka, N. Slepickova Kasalkova, I. Michaljanicova, O. Nedela, Z. Kolska, V. Svorcik

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Polymers exposed to laser or plasma treatment or modified with different wet methods which enable the introduction of nanoparticles or biologically active species, such as amino-acids, may find many applications both as biocompatible or anti-bacterial materials or on the contrary, can be applied for a decrease in the number of cells on the treated surface which opens application in single cell units. For the experiments, two types of materials were chosen, a representative of non-biodegradable polymers, polyethersulphone (PES) and polyhydroxybutyrate (PHB) as biodegradable material. Exposure of solid substrate to laser well below the ablation threshold can lead to formation of various surface structures. The ripples have a period roughly comparable to the wavelength of the incident laser radiation, and their dimensions depend on many factors, such as chemical composition of the polymer substrate, laser wavelength and the angle of incidence. On the contrary, biopolymers may significantly change their surface roughness and thus influence cell compatibility. The focus was on the surface treatment of PES and PHB by pulse excimer KrF laser with wavelength of 248 nm. The changes of physicochemical properties, surface morphology, surface chemistry and ablation of exposed polymers were studied both for PES and PHB. Several analytical methods involving atomic force microscopy, gravimetry, scanning electron microscopy and others were used for the analysis of the treated surface. It was found that the combination of certain input parameters leads not only to the formation of optimal narrow pattern, but to the combination of a ripple and a wrinkle-like structure, which could be an optimal candidate for cell attachment. The interaction of different types of cells and their interactions with the laser exposed surface were studied. It was found that laser treatment contributes as a major factor for wettability/contact angle change. The combination of optimal laser energy and pulse number was used for the construction of a surface with an anti-cellular response. Due to the simple laser treatment, we were able to prepare a biopolymer surface with higher roughness and thus significantly influence the area of growth of different types of cells (U-2 OS cells).

Keywords: cell response, excimer laser, polymer treatment, periodic pattern, surface morphology

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Authors: Susumu Fukita, Hiromi Kawasaki, Satoko Yamasaki, Kotomi Yamashita, Tomoko Iki

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The increase in the number of workers with mental health problems has become an issue. Many workers work in small- and medium-sized enterprises, which often support local employment and economy, especially in underpopulated mountainous areas. It is important for managers to take mental health measures for employees since there is no budget to hire health staff in small- and medium-sized enterprises. It is necessary to understand the manager's attitude toward the mental health of employees and to publicly support the manager in promoting mental health measures for employees. The purpose of this study was to study the awareness of managers of small- and medium-sized enterprises regarding the mental health of employees and to consider support for managers to take measures for the mental health of employees. Semi-structured interviews were conducted with six managers of small- and medium-sized enterprises in underpopulated mountainous areas in November 2019. Managers were asked about their awareness of the mental health of their employees. Qualitative descriptive analysis was used, and subcategories and categories were extracted. Four categories emerged. Regarding the mental health of employees, the managers acknowledged that if the appearance and behavior of the employees do not interfere with their lives, the manager judges that the employees’ mental health is normal. It was also found that the managers acknowledged that there is a comfortable working environment due to the characteristics of the underpopulated mountainous area. On the other hand, the managers acknowledged that employees are dissatisfied with salaries and management systems. In addition, it was found the manager acknowledged that some employees retire due to mental health problems. Although managers recognized that employees may be dissatisfied with salaries, they also recognized that there was a comfortable working environment due to the characteristics of the areas, with good interpersonal relationships. Economic challenges are difficult to solve in underpopulated mountainous areas. It is useful to consider measures that take advantage of the characteristics of the areas where it is easy to work because of good relations with each other, for example, to create a family-like workplace culture where managers and employees can engage in daily conversation. The managers judged that the employees were in good health if there was no interference with their lives. However, it is too late to take measures at the stage when it becomes an obstacle to life. Therefore, it is necessary to provide training for managers to learn observation techniques by which they quickly notice changes in the situation of employees and give appropriate responses; and to set up a contact point for managers to consult. Local governments should actively provide public support such as training for managers and establishing consultation desks to maintain valuable employment and local economics in underpopulated mountainous areas.

Keywords: employer, mental health, small- and medium- sized enterprises, underpopulated areas

Procedia PDF Downloads 145

Authors: Agnieszka Stempniewicz, Piotr Ceranowicz, Wojciech Macyk, Jakub Cieszkowski, Beata Kuśnierz-Cabała, Katarzyna Gałązka, Zygmunt Warzecha

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Objectives: There are numerous strategies for the prevention or treatment of oral mucositis. However, their effectiveness is limited and does not correspond to expectations. Recent studies have shown that obestatin exhibits a protective effect and accelerates the healing of gastrointestinal mucosa. The aim of the present study was to examine the influence of obestatin administration on oral ulcers in rats. Methods: lingual ulcers were induced by the use of acetic acid. Rats were treated twice a day intraperitoneally with saline or obestatin(4, 8, or 16 nmol/kg/dose) for five days. The study determined: lingual mucosa morphology, cell proliferation, mucosal blood flow, and mucosal pro-inflammatory interleukin-1β level(IL-1β). Results: In animals without induction of oral ulcers, treatment with obestatin was without any effect. Obestatin administration in rats with lingual ulcers increased the healing rate of these ulcers. Obestatin given at the dose of 8 or 16 nmol/kg/dose caused the strongest and similar therapeutic effect. This result was associated with a significant increase in blood flow and cell proliferation in gingival mucosa, as well as a significant decrease in IL-1β level. Conclusions: Obestatin accelerates the healing of lingual ulcers in rats. This therapeutic effect is well-correlated with an increase in blood flow and cell proliferation in oral mucosa, as well as a decrease in pro-inflammatory IL-1β levels. Obestatin is a potentially useful candidate for the prevention and treatment of oral mucositis. Acknowledgment: Agnieszka Stempniewicz acknowledges the support of InterDokMed project no. POWR.03.02.00- 00-I013/16.

Keywords: oral mucositis, ulcers, obestatin, lingual mucosa

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Authors: Selma Sbit, Mohamed Bechir Dadi, Belgacem Chibani Rhaimi

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Incorporating Home eNodeB (HeNB) in cellular networks, e.g. Long Term Evolution Advanced (LTE-A), is beneficial for extending coverage and enhancing capacity at low price especially within the non-line-of sight (NLOS) environments such as homes. HeNB or femtocell is a small low powered base station which provides radio coverage to the mobile users in an indoor environment. This deployment results in a heterogeneous network where the available spectrum becomes shared between two layers. Therefore, a problem of Inter Cell Interference (ICI) appears. This issue is the main challenge in LTE-A. To deal with this challenge, various techniques based on frequency, time and power control are proposed. This paper deals with the impact of carrier aggregation and higher order MIMO (Multiple Input Multiple Output) schemes on the LTE-Advanced performance. Simulation results show the advantages of these schemes on the system capacity (4.10⁹ b/s/Hz when bandwidth B=100 MHz and when applying MIMO 8x8 for SINR=30 dB), maximum theoretical peak data rate (more than 4 Gbps for B=100 MHz and when MIMO 8x8 is used) and spectral efficiency (15 b/s/Hz and 30b/s/Hz when MIMO 4x4 and MIMO 8x8 are applying respectively for SINR=30 dB).

Keywords: capacity, carrier aggregation, LTE-Advanced, MIMO (Multiple Input Multiple Output), peak data rate, spectral efficiency

Procedia PDF Downloads 256

Authors: Man Kyu Kim, Hansu Shin

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This study is an analysis on gravel of sand-gravel bar that stretches variously in the Duchon and Naechon stream basins, which are situated on Hong-Cheon River (a well-developed sand-gravel bar in upstream river) basins in Korea. Naechon stream mostly flows through granite zone but Duchon stream mostly flows through gneiss zone. The characteristics of gravel in the sand-gravel bar of these two branches in the upper Hongcheon River were analyzed in this study in order to understand the geomorphic development of streams depending on the differences of bedrock. Through the analysis on the roundness and flatness of gravel, we figured out an irregular trend following the increase in supply of granite gravel and gneiss gravel as we traveled downstream. The result shows that the two basins have uppermost small basin condition reflecting the mountain valley environment although it may be difficult to do an equivalent comparison to other roundness researches in Korea or in Europe. This study conducted an analysis on gravels found in small scale streams unlike the previous studies trend which mostly studies large rivers. The research provides an opportunity to offer basic data for continuous comparison research on various small basins.

Keywords: flatness, geology, roundness, sand-gravel bar

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Authors: Hiba Mezaache

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The current study aimed to know the impact of using information and communication technology on the process of re-engineering small and medium enterprises, as the world witnessed the speed development of the latter in its field of work and the diversity of its objectives and programs, that also made its process important for the growth and development of the institution and also gaining the flexibility to face the changes that may occur in the environment of work, so in order to know the impact of information and communication technology on the success of this process, we prepared an electronic questionnaire that included (70) items, and we also used the SPSS statistical calendar to analyze the data obtained. In the end of our study, our conclusion was that there was a positive correlation between the four dimensions of information and communication technology, i.e., hardware and equipment, software, communication networks, databases, and the re-engineering process, in addition to the fact that the studied institutions attach great importance to formal communication, for its positive advantages that it achieves in reducing time and effort and costs in performing the business. We could also say that communication technology contributes to the process of formulating objectives related to the re-engineering strategy. Finally, we recommend the necessity of empowering workers to use information technology and communication more in enterprises, and to integrate them more into the activity of the enterprise by involving them in the decision-making process, and also to keep pace with the development in the field of software, hardware, and technological equipment.

Keywords: information and communication technology, re-engineering, small and medium enterprises, the impact

Procedia PDF Downloads 177

Authors: Wael M. Rabeh, Cesar Carrasco-Lopez, Juliana C. Ferreira, Pance Naumov

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Bioluminescence, the emission of light from a biological process, is found in various living organisms including bacteria, fireflies, beetles, fungus and different marine organisms. Luciferase is an enzyme that catalyzes a two steps oxidation of luciferin in the presence of Mg2+ and ATP to produce oxyluciferin and releases energy in the form of light. The luciferase assay is used in biological research and clinical applications for in vivo imaging, cell proliferation, and protein folding and secretion analysis. The luciferase enzyme consists of two domains, a large N-terminal domain (1-436 residues) that is connected to a small C-terminal domain (440-544) by a flexible loop that functions as a hinge for opening and closing the active site. The two domains are separated by a large cleft housing the active site that closes after binding the substrates, luciferin and ATP. Even though all insect luciferases catalyze the same chemical reaction and share 50% to 90% sequence homology and high structural similarity, they emit light of different colors from green at 560nm to red at 640 nm. Currently, the majority of the structural and biochemical studies have been conducted on green-emitting firefly luciferases. To address the color emission mechanism, we expressed and purified two luciferase enzymes with blue-shifted green and red emission from indigenous Brazilian species Amydetes fanestratus and Phrixothrix, respectively. The two enzymes naturally emit light of different colors and they are an excellent system to study the color-emission mechanism of luciferases, as the current proposed mechanisms are based on mutagenesis studies. Using a vapor-diffusion method and a high-throughput approach, we crystallized and solved the crystal structure of both enzymes, at 1.7 Å and 3.1 Å resolution respectively, using X-ray crystallography. The free enzyme adopted two open conformations in the crystallographic unit cell that are different from the previously characterized firefly luciferase. The blue-shifted green luciferase crystalized as a monomer similar to other luciferases reported in literature, while the red luciferases crystalized as an octamer and was also purified as an octomer in solution. The octomer conformation is the first of its kind for any insect’s luciferase, which might be relate to the red color emission. Structurally designed mutations confirmed the importance of the transition between the open and close conformations in the fine-tuning of the color and the characterization of other interesting mutants is underway.

Keywords: bioluminescence, enzymology, structural biology, x-ray crystallography

Procedia PDF Downloads 326

Authors: Bhawna Chandravanshi, Ramesh Bhonde

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In the present study, we focused on the improvisation of islet survival in hypoxia. The Islet-like cell aggregates (ICAs) derived from Wharton's jelly mesenchymal stem cells (WJ-MSC) were cultured with and without WJ-MSC for 48h in hypoxia and normoxia and tested for their direct trophic effect on β cell survival. The WJ MSCs themselves secreted insulin upon glucose challenge and expressed the pancreatic markers at both transcription and translational level (C-peptide, Insulin, Glucagon and Glut 2). Direct contact of MSCs with ICAs facilitate the highest viability under hypoxia as evidenced by fluorescein diacetate/propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytokine analysis of the co-cultured ICAs revealed amplification of anti-inflammatory cytokine-like TGFβ and TNFα accompanied by depletion of pro-inflammatory cytokines. The increment in VEGF and PDGFa was also seen showing their ability to vascularize upon transplantation. This was further accompanied by reduction in total reactive oxygen species, nitric oxide, and super oxide ions and down-regulation of Caspase3, Caspase8, p53 and up regulation of Bcl2 confirming prevention of apoptosis in ICAs. There was a significant reduction in the expression of p38 protein in the presence of MSCs making the ICAs responsive to glucose. Taken together our data demonstrate for the first time that the WJ-MSC expressed pancreatic markers and their supplementation protected engineered islets against hypoxia, oxidative stress, and inflammatory cytokines by inhibiting p38 MAPK protein.

Keywords: hypoxia, islet-like cell aggregates, inflammatory cytokines, oxidative stress

Procedia PDF Downloads 261