Search results for: mature embryo

Authors: S. Mamoucha, N. Tsafantakis, Α. Ioannidis, S. Chatzipanagiotou, C. Nikolaou, L. Skaltsounis, N. Fokialakis, N. Christodoulakis

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Introduction: Plant secondary metabolites (SM) can be grouped into three chemically distinct groups: terpenes, phenolics, and nitrogen-containing compounds. For many years the adaptive significance of SM was unknown. They were thought to be functionless end-products. Currently it is accepted that many secondary metabolites (also known as natural products) have important ecological roles in plants. For instance, they serve as attractants (odor, color, taste) for pollinators and seed-dispersing animals. Moreover, they protect plants from herbivores, microbial pathogens and from environmental stress (high and low temperatures, drought, alkalinity, salinity, radiation etc). It is well known that both biotic and abiotic stress often increase the accumulation of SM. The local climatic conditions, seasonal changes, external factors such as light, temperature, humidity affect the biosynthesis and composition of secondary metabolites. A well known dioecious evergreen plant, Pistacia lentiscus L. (mastic tree), was selected in order to study the metabolic variations occur in response to the different climate conditions, due to the seasonal variation and its effect on the biosynthesis of bioactive compounds. Materials-methods: Young and mature leaves were collected in January and July 2014, dried and extracted by accelerated solvent extraction (Dionex ASE™ 350) using solvents of increased polarity (DCM, MeOH, and H2O). GC-MS and UHPLC-HRMS analysis were carried out in order to define the nature and the relative abundance of SM. The antibacterial activity was evaluated by using the Agar Disc Diffusion Assay against ATCC and clinical isolates strains: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Streptococcus mutans and Klebsiella pneumoniae. All tests were carried out in duplicate and the average radii of the inhibition zones were calculated for each extract. Results: According to the phytochemical profile obtained from each extract, the biosynthesis of SM varied both qualitatively and quantitatively under the two different types of seasonal stress. With exception of the biologically inactive nonpolar DCM extract of July, all extracts inhibited the growth of most of the investigated microorganisms. A clear positive correlation has been observed between the relative abundance of SM and the bioactivity of the DCM extracts of January and July. Observed changes during phytochemical analysis were mainly focused on the triterpenoid content. On the other hand, the bioactivity of the polar extracts (MeOH and H2O) of January and July resulted practically invariable against most of the microorganisms, besides the significant variation of the SM content due to the seasonal variation. Conclusion: Our results clearly confirmed the hypothesis of abiotic stress as an important regulating factor that significantly affects the biosynthesis of secondary metabolites and thus the presence of bioactive compounds. Acknowledgment: This work was supported by IKY - State Scholarship Foundation, Athens, Greece.

Keywords: antibacterial screening, phytochemical profile, Pistacia lentiscus, abiotic stress

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Authors: S. Najafi, E. Bertini, M. Pezzotti, G.B. Tornielli, S. Zenoni

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Grapevine is an important agricultural fruit crop plant consumed worldwide and with a key role in the global economy. Grapevine is strongly affected by both biotic and abiotic stresses, which impact grape growth at different stages, such as during plant and berry development and pre- and post-harvest, consequently causing significant economic losses. Recently global warming has propelled the anticipation of the onset of berry ripening, determining the reduction of a grape color and increased volatilization of aroma compounds. Climate change could negatively alter the physiological characteristics of the grape and affect the berry and wine quality. Modern plant breeding can provide tools such as genome editing for improving grape resilience traits while maintaining intact the viticultural and oenological quality characteristics of the genotype. This study aims at developing a platform for genome editing application in grapevine plants with the final goal to improve berry quality, biotic, and abiotic resilience traits. We chose to directly deliver ribonucleoproteins (RNP, preassembled Cas protein and guide RNA) into plant protoplasts, and, from these cell structures, regenerate grapevine plants edited in specific selected genes controlling traits of interest. Edited plants regenerated by somatic embryogenesis from protoplasts will then be sequenced and molecularly characterized. Embryogenic calli of Sultana and Shiraz cultivars were initiated from unopened leaves of in-vitro shoot tip cultures and from stamens, respectively. Leaves were placed on NB2 medium while stamens on callus initiation medium (PIV) medium and incubated in the dark at 28 °C for three months. Viable protoplasts, tested by FDA staining, isolated from embryogenic calli were cultured by disc method at 1*105 protoplasts/ml. Mature well-shaped somatic embryos developed directly in the protoplast culture medium two months later and were transferred in the light into to shooting medium for further growth. Regenerated plants were then transferred to the greenhouse; no phenotypic alterations were observed when compared to non in-vitro cultured plants. The performed experiments allowed to established an efficient protocol of embryogenic calli production, protoplast isolation, and regeneration of the whole plant through somatic embryogenesis in both Sultana and Shiraz. Regenerated plants, through direct somatic embryogenesis deriving from a single cell, avoid the risk of chimerism during the regeneration process, therefore improving the genome editing process. As pre-requisite of genome editing, an efficient method for transfection of protoplast by yellow fluorescent protein (YFP) marker genes was also established and experiments of direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) in protoplasts to achieve efficient DNA-free targeted mutations are in progress.

Keywords: CRISPR-cas9, plant regeneration, protoplast isolation, Vitis vinifera

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Authors: G. Cunningham, E. Cantor, X. Wu, F. Shen, G. Jiang, S. Philips, C. Bales, Y. Xiao, T. R. Cummins, J. C. Fehrenbacher, B. P. Schneider

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Background: Taxane-induced peripheral neuropathy (TIPN) is the most devastating survivorship issue for patients receiving therapy. Dose reductions due to TIPN in the curative setting lead to inferior outcomes for African American patients, as prior research has shown that this group is more susceptible to developing severe neuropathy. The mechanistic underpinnings of TIPN, however, have not been entirely elucidated. While it would be appealing to use primary tissue to study the development of TIPN, procuring nerves from patients is not realistically feasible, as nerve biopsies are painful and may result in permanent damage. Therefore, our laboratory has investigated paclitaxel-induced neuronal morphological and molecular changes using an ex vivo model of human-induced pluripotent stem cell (iPSC)-derived neurons. Methods: iPSCs are undifferentiated and endlessly dividing cells that can be generated from a patient’s somatic cells, such as peripheral blood mononuclear cells (PBMCs). We successfully reprogrammed PBMCs into iPSCs using the Erythroid Progenitor Reprograming Kit (STEMCell Technologiesᵀᴹ); pluripotency was verified by flow cytometry analysis. iPSCs were then induced into neurons using a differentiation protocol that bypasses the neural progenitor stage and uses selected small-molecule modulators of key signaling pathways (SMAD, Notch, FGFR1 inhibition, and Wnt activation). Results: Flow cytometry analysis revealed expression of core pluripotency transcription factors Nanog, Oct3/4 and Sox2 in iPSCs overlaps with commercially purchased pluripotent cell line UCSD064i-20-2. Trilineage differentiation of iPSCs was confirmed with immunofluorescent imaging with germ-layer-specific markers; Sox17 and ExoA2 for ectoderm, Nestin, and Pax6 for mesoderm, and Ncam and Brachyury for endoderm. Sensory neuron markers, β-III tubulin, and Peripherin were applied to stain the cells for the maturity of iPSC-derived neurons. Patch-clamp electrophysiology and calcitonin gene-related peptide (CGRP) release data supported the functionality of the induced neurons and provided insight into the timing for which downstream assays could be performed (week 4 post-induction). We have also performed a cell viability assay and fluorescence-activated cell sorting (FACS) using four cell-surface markers (CD184, CD44, CD15, and CD24) to select a neuronal population. At least 70% of the cells were viable in the isolated neuron population. Conclusion: We have found that these iPSC-derived neurons recapitulate mature neuronal phenotypes and demonstrate functionality. Thus, this represents a patient-derived ex vivo neuronal model to investigate the molecular mechanisms of clinical TIPN.

Keywords: chemotherapy, iPSC-derived neurons, peripheral neuropathy, taxane, paclitaxel

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Authors: Saba Vahedi, Qiuyan Yuan

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A vast majority of small municipalities and First Nations communities in Manitoba operate facultative or aerated lagoons for wastewater treatment, and most of them use Ferric Chloride (FeCl3) or alum (usually in the form of Al2(SO4)3 ·18H2O) as coagulant for phosphorous removal. The insoluble particles that form during the coagulation process result in a massive volume of sludge which is typically left in the lagoons. Therefore, phosphorous, which is a valuable nutrient, is lost in the process. In this project, the complete recovery of phosphorous from the sludge that is produced in the process of phosphorous removal from wastewater lagoons by using a controlled composting process is investigated. Objective The main objective of this project is to compost alum precipitated sludge that is produced in the process of phosphorous removal in wastewater treatment lagoons in Manitoba. The ultimate goal is to have a product that will meet the characteristics of Class A biosolids in Canada. A number of parameters, including the bioavailability of nutrients in the composted sludge and the toxicity of the sludge, will be evaluated Investigating the bioavailability of phosphorous in the final compost product. The compost will be used as a source of P compared to a commercial fertilizer (monoammonium phosphate MAP) Experimental setup Three different batches of composts piles have been run using the Alum sludge and Ferric sludge. The alum phosphate sludge was collected from an innovative phosphorous removal system at the RM of Taché . The collected sludge was sent to ALS laboratory to analyze the C/N ratio, TP, TN, TC, TAl, moisture contents, pH, and metals concentrations. Wood chips as the bulking agent were collected at the RM of Taché landfill The sludge in the three piles were mixed with 3x dry woodchips. The mixture was turned every week manually. The temperature, the moisture content, and pH were monitored twice a week. The temperature of the mixtures was remained above 55 °C for two weeks. Each pile was kept for ten weeks to get mature. The final products have been applied to two different plants to investigate the bioavailability of P in the compost product as well as the toxicity of the product. The two types of plants were selected based on their sensitivity, growth time, and their compatibility with the Manitoba climate, which are Canola, and switchgrass. The pots are weighed and watered every day to replenish moisture lost by evapotranspiration. A control experiment is also conducted by using topsoil soil and chemical fertilizers (MAP). The experiment will be carried out in a growth room maintained at a day/night temperature regime of 25/15°C, a relative humidity of 60%, and a corresponding photoperiod of 16 h. A total of three cropping (seeding to harvest) cycles need be completed, with each cycle at 50 d in duration. Harvested biomass must be weighed and oven-dried for 72 h at 60°C. The first cycle of growth Canola and Switchgrasses in the alum sludge compost, harvested at the day 50, oven dried, chopped into bits and fine ground in a mill grinder (< 0.2mm), and digested using the wet oxidation method in which plant tissue samples were digested with H2SO4 (99.7%) and H2O2 (30%) in an acid block digester. The digested plant samples need to be analyzed to measure the amount of total phosphorus.

Keywords: wastewater treatment, phosphorus removal, composting alum sludge, bioavailibility of pohosphorus

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Authors: Iraklis Argyriou

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The transition to net-zero emission urban bus (ZEB) systems is receiving increased attention in research and policymaking throughout the globe. Most studies in this area tend to address techno-economic aspects and the perspectives of a narrow group of stakeholders, while they largely overlook analysis of current bus system dynamics. This offers limited insight into the types of ZEB governance challenges and opportunities that are encountered in real-world contexts, as well as into some of the immediate actions that need to be taken to set off the transition over the longer term. This research offers a multi-stakeholder perspective into both the technical and non-technical factors that influence ZEB transitions within a particular context, the UK. It does so by drawing from a recent transition visioning stakeholder workshop (June 2023) with key public, private and civic actors of the urban bus transportation system. Using NVivo software to qualitatively analyze the workshop discussions, the research examines the key technological and funding aspects, as well as the short-term actions (over the next five years), that need to be addressed for supporting the ZEB transition in UK cities. It finds that ZEB technology has reached a mature stage (i.e., high efficiency of batteries, motors and inverters), but important improvements can be pursued through greater control and integration of ZEB technological components and systems. In this regard, telemetry, predictive maintenance and adaptive control strategies pertinent to the performance and operation of ZEB vehicles have a key role to play in the techno-economic advancement of the transition. Yet, more pressing gaps were identified in the current ZEB funding regime. Whereas the UK central government supports greater ZEB adoption through a series of grants and subsidies, the scale of the funding and its fragmented nature do not match the needs for a UK-wide transition. Funding devolution arrangements (i.e., stable funding settlement deals between the central government and the devolved administrations/local authorities), as well as locally-driven schemes (i.e., congestion charging/workplace parking levy), could then enhance the financial prospects of the transition. As for short-term action, three areas were identified as critical: (1) the creation of whole value chains around the supply, use and recycling of ZEB components; (2) the ZEB retrofitting of existing fleets; and (3) integrated transportation that prioritizes buses as a first-choice, convenient and reliable mode while it simultaneously reduces car dependency in urban areas. Taken together, the findings point to the need for place-based transition approaches that create a viable techno-economic ecosystem for ZEB development but at the same time adopt a broader governance perspective beyond a ‘net-zero’ and ‘bus sectoral’ focus. As such, multi-actor collaborations and the coordination of wider resources and agency, both vertically across institutional scales and horizontally across transport, energy and urban planning, become fundamental features of comprehensive ZEB responses. The lessons from the UK case can inform a broader body of empirical contextual knowledge of ZEB transition governance within domestic political economies of public transportation.

Keywords: net-zero emission transition, stakeholders, transition governance, UK, urban bus transportation

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Authors: Khin Thi Htun, James Jain, Tim Andrews

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This study examines the under-researched phenomenon of crowdfunding use and effectiveness in developing non-western markets. More precisely, using an institutional theoretical lens, the research explores the attitudes, motivations, and practice surrounding the initiation, development, and receipt of crowdfunding campaignsin a business context symptomatic of widely dissimilar regulatory, normative cognitive institutional ‘pillars’ to those studied – and utilized in practice - to date. As, in essence, a form of alternative finance, crowdfunding is used primarily to fund a wide range of projects through the securement of small amounts of money from a large pool of investors/participants. Being tied almost inextricably to e-commerce channels, the practice of crowdfunding typically sources its means and communicates the purpose of each venture mainly, though not exclusively, online. The wide range of projects supported to date span social entrepreneurship, community benefits initiatives, creative and artistic endeavors, assistance to disadvantaged social cohorts, and small business start-ups. Adopting a longitudinal, comparative approach, the study reported here embodies an investigation centered on six case start-up campaigns within the Thai societal context, covering a range of fundings calls and cause choices. Data was sourced from a variety of respondents using semi-structured interviews, observation (direct and participant), and company information. Results suggest that the motives and effectiveness of crowdfunding campaigns differ significantly in non-western consumer contexts from the norms that have evolved to date in mature Western contexts(particularly the US and UK). Specifically, whereas data on the different regulatory pressures showed relatively insignificant variation, the results regarding cognitive and, especially, normative dissimilarities between the Thai and US/UK institutional profiles surfaced potentially important differences with far-reaching implications. Particular issuesto emerge from our data concerned consumer motivation in terms of support and engagement with different types of campaigns. This was found to stem from social norms symptomatic of ‘collectivist’ and ‘relations based/particularist’ cultural assistance behavior, in turn, linked to deeply-held societal values regarding interpersonal network (‘in group’) reciprocity. This research serves to refine and extend the limited body of knowledge to date on crowdfunding by exploring the phenomenon in a non-western, non-developed country contextswhere social norms and values differ. This was achieved through uncovering and explicating the effects of cultural dissimilarity on motivation, decision-making, construed ethics, and general engagement with crowdfunding ideas. Implications for theory into e-marketing and cross-cultural marketing, as well as for practitioners seeking to develop effective crowdfunding campaigns in a Southeast Asian cultural environment, are discussed to conclude the paper.

Keywords: crowdfunding, national culture, e-marketing, cross-cultural business

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Authors: Zahirah Dhurmeea, Iker Zudaire, Heidi Pethybridge, Emmanuel Chassot, Maria Cedras, Natacha Nikolic, Jerome Bourjea, Wendy West, Chandani Appadoo, Nathalie Bodin

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Scientific advice on the status of fish stocks relies on indicators that are based on strong assumptions on biological parameters such as condition, maturity and fecundity. Currently, information on the biology of albacore tuna, Thunnus alalunga, in the Indian Ocean is scarce. Consequently, many parameters used in stock assessment models for Indian Ocean albacore originate largely from other studied stocks or species of tuna. Inclusion of incorrect biological data in stock assessment models would lead to inappropriate estimates of stock status used by fisheries manager’s to establish future catch allowances. The reproductive biology of albacore tuna in the western Indian Ocean was examined through analysis of the sex ratio, spawning season, length-at-maturity (L50), spawning frequency, fecundity and fish condition. In addition, the total lipid content (TL) and lipid class composition in the gonads, liver and muscle tissues of female albacore during the reproductive cycle was investigated. A total of 923 female and 867 male albacore were sampled from 2013 to 2015. A bias in sex-ratio was found in favour of females with fork length (LF) <100 cm. Using histological analyses and gonadosomatic index, spawning was found to occur between 10°S and 30°S, mainly to the east of Madagascar from October to January. Large females contributed more to reproduction through their longer spawning period compared to small individuals. The L50 (mean ± standard error) of female albacore was estimated at 85.3 ± 0.7 cm LF at the vitellogenic 3 oocyte stage maturity threshold. Albacore spawn on average every 2.2 days within the spawning region and spawning months from November to January. Batch fecundity varied between 0.26 and 2.09 million eggs and the relative batch fecundity (mean  standard deviation) was estimated at 53.4 ± 23.2 oocytes g-1 of somatic-gutted weight. Depending on the maturity stage, TL in ovaries ranged from 7.5 to 577.8 mg g-1 of wet weight (ww) with different proportions of phospholipids (PL), wax esters (WE), triacylglycerol (TAG) and sterol (ST). The highest TL were observed in immature (mostly TAG and PL) and spawning capable ovaries (mostly PL, WE and TAG). Liver TL varied from 21.1 to 294.8 mg g-1 (ww) and acted as an energy (mainly TAG and PL) storage prior to reproduction when the lowest TL was observed. Muscle TL varied from 2.0 to 71.7 g-1 (ww) in mature females without a clear pattern between maturity stages, although higher values of up to 117.3 g-1 (ww) was found in immature females. TL results suggest that albacore could be viewed predominantly as a capital breeder relying mostly on lipids stored before the onset of reproduction and with little additional energy derived from feeding. This study is the first one to provide new information on the reproductive development and classification of albacore in the western Indian Ocean. The reproductive parameters will reduce uncertainty in current stock assessment models which will eventually promote sustainability of the fishery.

Keywords: condition, size-at-maturity, spawning behaviour, temperate tuna, total lipid content

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Authors: Ananya Hazare, Ranjit Kamble

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Introduction: Patients with Cleft lip and palate (CL/P) can present with a maxillary retrution after cleft repair. Anterior Maxillary distraction osteogenesis (AMD) is a technique that provides simultaneous skeletal advancement and expansion of the soft tissues related to an anterior segment of the maxilla. This case presented is a case of AMD. The advantage of this technique is that the occlusion in the posterior segment can be maintained, and only the segment in cross bite is advanced for correction of the midfacial deficiency. The other alternative treatment is anterior movement by a Lefort 1 osteotomy. When a Lefort 1 osteotomy is compared with the Distraction osteogenesis or AMD, the disadvantages of the Le Fort 1 include a higher risk of morbidity, requirement of fixation, relapse tendency and unexpected changes in the nasal form. These complications were eliminated by AMD technique. This was followed by placement of the implant in the bone formed after AMD. Hence complete surgical, orthodontic and prosthodontics rehabilitation of the patient was done by an interdisciplinary approach. Methods: Patient presented with repaired UCL/P of the right side with midfacial retrusion. Intro-oral examination revealed a good occlusion in the posterior arch and anterior Crossbite from canine to canine. Patient's both maxillary lateral incisors were missing. The lower arch was well aligned with all teeth present. The study models when scored according to GOSLON yardstick received a score of 4. After pre-surgical orthodontic phase was completed an intraoral distractor was fabricated by modification of HYRAX expansion screw. After surgery, low subapical osteotomy cuts were placed and the distractor was fixed. The latency period of 5 days was observed after which the distraction was started. Distraction was done at a rate of 1 mm/day with a rhythm of 0.5mm in morning and 0.5mm in the evening. The total distraction of 12 mm was done. After a consolidation period, the distractor was removed, and retention by a removable partial denture was given. Radiographic examination confirmed mature bone formation in the distracted segment. Implants were placed and allowed to osseointegrate for approximately 4 months and were then loaded with abutments. Results: Total distraction done was 12mm and after relapse it was 8mm. After consolidation phase the radiographic examination revealed a B2 quality of bone according to the Misch's classification and sufficient height from the maxillary sinus. These findings were indicative for placement of implants in the distracted bone formed in premolar region. Implants were placed and after radiographic evidence of osseointegration was seen they were loaded with abutments. Thus resulting in a complete rehabilitation of a cleft patient by an interdisciplinary approach. Conclusion: Anterior maxillary distraction can be used as an alternative method instead of complete distraction osteogenesis or Lefort 1 advancement of maxilla in cases where the advancement needed is minimum. Use of HYRAX expansion screw modified as intra-oral distractor can be used in such cases, which significantly reduces the cost of treatment, as expensive distractors are not used. This technique is very useful and efficient in countries like India where the patient cannot afford expensive treatment options.

Keywords: cleft lip and palate, distraction osteogenesis, anterior maxillary distraction, orthodontics and dentofacial orthopaedics, hyrax expansion screw modification

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Authors: Faruk Inaltekin, Imran Gurakan

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This paper will aim to discuss the importance of value-added services provided by Science and Technology Parks for entrepreneurship development in Turkey. Entrepreneurship is vital subject for all countries. It has not only fostered economic development but also promoted innovation at local and international levels. To foster high tech entrepreneurship ecosystem, Technopark (Science and Technology Park/STP) concept was initiated with the establishment of Silicon Valley in the 1950s. The success and rise of Silicon Valley led to the spread of technopark activities. Developed economies have been setting up projects to plan and build STPs since the 1960s and 1970s. To promote the establishment of STPs, necessary legislations were made by Ministry of Science, Industry, and Technology in 2001, Technology Development Zones Law (No. 4691) and it has been revised in 2016 to provide more supports. STPs’ basic aim is to provide customers high-quality office spaces with various 'value added services' such as business development, network connections, cooperation programs, investor/customers meetings and internationalization services. For this aim, STPs should help startups deal with difficulties in the early stages and to support mature companies’ export activities in the foreign market. STPs should support the production, commercialization and more significantly internationalization of technology-intensive business and foster growth of companies. Nowadays within this value-added services, internationalization is very popular subject in the world. Most of STPs design clusters or accelerator programs in order to support their companies in the foreign market penetration. If startups are not ready for international competition, STPs should help them to get ready for foreign market with training and mentoring sessions. These training and mentoring sessions should take a goal based approach to working with companies. Each company has different needs and goals. Therefore the definition of ‘success' varies for each company. For this reason, it is very important to create customized value added services to meet the needs of startups. After local supports, STPs should also be able to support their startups in foreign market. Organizing well defined international accelerator program plays an important role in this mission. Turkey is strategically placed between key markets in Europe, Russia, Central Asia and the Middle East. Its population is young and well educated. So both government agencies and the private sectors endeavor to foster and encourage entrepreneurship ecosystem with many supports. In sum, the task of technoparks with these and similar value added services is very important for developing entrepreneurship ecosystem. The priorities of all value added services are to identify the commercialization and growth obstacles faced by entrepreneurs and get rid of them with the one-to-one customized services. Also, in order to have a healthy startup ecosystem and create sustainable entrepreneurship, stakeholders (technoparks, incubators, accelerators, investors, universities, governmental organizations etc.) should fulfill their roles and/or duties and collaborate with each other. STPs play an important role as bridge for these stakeholders & entrepreneurs. STPs always should benchmark and renew services offered to how to help the start-ups to survive, develop their business and benefit from these stakeholders.

Keywords: accelerator, cluster, entrepreneurship, startup, technopark, value added services

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Authors: Stanislav Babaev, Vladimir Cuk, Sjef Cobben, Jan Desmet

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The procedures for performing harmonic studies for medium-voltage distribution feeders have become relatively mature topics since the early 1980s. The efforts of various electric power engineers and researchers were mainly focused on handling large harmonic non-linear loads connected scarcely at several buses of medium-voltage feeders. In order to assess the impact of these loads on the voltage quality of the distribution system, specific modeling and simulation strategies were proposed. These methodologies could deliver a reasonable estimation accuracy given the requirements of least computational efforts and reduced complexity. To uphold these requirements, certain analysis assumptions have been made, which became de facto standards for establishing guidelines for harmonic analysis. Among others, typical assumptions include balanced conditions of the study and the negligible impact of impedance frequency characteristics of various power system components. In latter, skin and proximity effects are usually omitted, and resistance and reactance values are modeled based on the theoretical equations. Further, the simplifications of the modelling routine have led to the commonly accepted practice of neglecting phase angle diversity effects. This is mainly associated with developed load models, which only in a handful of cases are representing the complete harmonic behavior of a certain device as well as accounting on the harmonic interaction between grid harmonic voltages and harmonic currents. While these modelling practices were proven to be reasonably effective for medium-voltage levels, similar approaches have been adopted for low-voltage distribution systems. Given modern conditions and massive increase in usage of residential electronic devices, recent and ongoing boom of electric vehicles, and large-scale installing of distributed solar power, the harmonics in current low-voltage grids are characterized by high degree of variability and demonstrate sufficient diversity leading to a certain level of cancellation effects. It is obvious, that new modelling algorithms overcoming previously made assumptions have to be accepted. In this work, a simulation approach aimed to deal with some of the typical assumptions is proposed. A practical low-voltage feeder is modeled in PowerFactory. In order to demonstrate the importance of diversity effect and harmonic interaction, previously developed measurement-based models of photovoltaic inverter and battery charger are used as loads. The Python-based script aiming to supply varying voltage background distortion profile and the associated current harmonic response of loads is used as the core of unbalanced simulation. Furthermore, the impact of uncertainty of feeder frequency-impedance characteristics on total harmonic distortion levels is shown along with scenarios involving linear resistive loads, which further alter the impedance of the system. The comparative analysis demonstrates sufficient differences with cases when all the assumptions are in place, and results indicate that new modelling and simulation procedures need to be adopted for low-voltage distribution systems with high penetration of non-linear loads and renewable generation.

Keywords: electric power system, harmonic distortion, power quality, public low-voltage network, harmonic modelling

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Authors: Majed Al Otaibi, Melissa Lessard-Beaudoin, Amel Loudghi, Raphael Chouinard-Watkins, Melanie Plourde, Frederic Calon, C. Alexandre Castellano, Stephen Cunnane, Helene Payette, Pierrette Gaudreau, Denis Gris, Rona K. Graham

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Introduction: Alzheimer disease (AD) is a chronic disorder that affects millions of individuals worldwide. Symptoms include memory dysfunction, and also alterations in attention, planning, language and overall cognitive function. Olfactory dysfunction is a common symptom of several neurological disorders including AD. Studying the mechanisms underlying the olfactory dysfunction may therefore lead to the discovery of potential biomarkers and/or treatments for neurodegenerative diseases. Objectives: To determine if olfactory dysfunction predicts future cognitive impairment in the aging population and to characterize the olfactory system in a murine model expressing a genetic factor of AD. Method: For the human study, quantitative olfactory tests (UPSIT and OMT) have been done on 93 subjects (aged 80 to 94 years) from the Quebec Longitudinal Study on Nutrition and Successful Aging (NuAge) cohort accepting to participate in the ORCA secondary study. The telephone Modified Mini Mental State examination (t-MMSE) was used to assess cognition levels, and an olfactory self-report was also collected. In a separate cohort, olfactory cortical volume was calculated using MRI results from healthy old adults (n=25) and patients with AD (n=18) using the AAL single-subject atlas and performed with the PNEURO tool (PMOD 3.7). For the murine study, we are using Western blotting, RT-PCR and immunohistochemistry. Result: Human Study: Based on the self-report, 81% of the participants claimed to not suffer from any problem with olfaction. However, based on the UPSIT, 94% of those subjects showed a poor olfactory performance and different forms of microsmia. Moreover, the results confirm that olfactory function declines with age. We also detected a significant decrease in olfactory cortical volume in AD individuals compared to controls. Murine study: Preliminary data demonstrate there is a significant decrease in expression levels of the proform of caspase-3 and the caspase substrate STK3, in the olfactory bulb of mice expressing human APOE4 compared with controls. In addition, there is a significant decrease in the expression level of the caspase-9 proform and caspase-8 active fragment. Analysis of the mature neuron marker, NeuN, shows decreased expression levels of both isoforms. The data also suggest that Iba-1 immunostaining is increased in the olfactory bulb of APOE4 mice compared to wild type mice. Conclusions: The activation of caspase-3 may be the cause of the decreased levels of STK3 through caspase cleavage and may play role in the inflammation observed. In the clinical study, our results suggest that seniors are unaware of their olfactory function status and therefore it is not sufficient to measure olfaction using the self-report in the elderly. Studying olfactory function and cognitive performance in the aging population will help to discover biomarkers in the early stage of the AD.

Keywords: Alzheimer's disease, APOE4, cognition, caspase, brain atrophy, neurodegenerative, olfactory dysfunction

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Authors: Dilnee D. Suraweera, Tim Groom, Brian Chung, Brendan Bond, Andrew Schipp, Marc E. Nicolas

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Pyrethrum (Tanacetum cinerariifolium) is a perennial plant belongs to family Asteraceae. This is cultivated commercially for extraction of natural insecticide pyrethrins, which accumulates in their flower head achenes. Approximately 94% of the pyrethrins are produced within secretory ducts and trichomes of achenes of the mature pyrethrum flower. This is the most widely used botanical insecticide in the world and Australia is the current largest pyrethrum producer in the world. Rainfall in pyrethrum growing regions in Australia during pyrethrum flowering period, in late spring and early summer is significantly less. Due to lack of adequate soil moisture and under elevated temperature conditions during post-anthesis period, resulting in yield reductions. Therefore, understanding of yield responses of pyrethrum to irrigation is important for Pyrethrum as a commercial crop. Irrigation management has been identified as a key area of pyrethrum crop management strategies that could be manipulated to increase yield. Pyrethrum is a comparatively drought tolerant plant and it has some ability to survive in dry conditions due to deep rooting. But in dry areas and in dry seasons, the crop cannot reach to its full yield potential without adequate soil moisture. Therefore, irrigation is essential during the flowering period prevent crop water stress and maximise yield. Irrigation during the water deficit period results in an overall increased rate of water uptake and growth by the plant which is essential to achieve the maximum yield benefits from commercial crops. The effects of irrigation treatments applied at post-anthesis period on pyrethrum yield responses were studied in two irrigation methods. This was conducted in a first harvest commercial pyrethrum field in Waubra, Victoria, during 2012/2013 season. Drip irrigation and overhead sprinkler irrigation treatments applied during whole flowering period were compared with ‘rainfed’ treatment in relation to flower yield and pyrethrin yield responses. The results of this experiment showed that the application of 180mm of irrigation throughout the post-anthesis period, from early flowering stages to physiological maturity under drip irrigation treatment increased pyrethrin concentration by 32%, which combined with the 95 % increase in the flower yield to give a total pyrethrin yield increase of 157%, compared to the ‘rainfed’ treatment. In contrast to that overhead sprinkler irrigation treatment increased pyrethrin concentration by 19%, which combined with the 60 % increase in the flower yield to give a total pyrethrin yield increase of 91%, compared to the ‘rainfed’ treatment. Irrigation treatments applied throughout the post-anthesis period significantly increased flower yield as a result of enhancement of number of flowers and flower size. Irrigation provides adequate soil moisture for flower development in pyrethrum which slows the rate of flower development and increases the length of the flowering period, resulting in a delayed crop harvest (11 days) compared to the ‘rainfed’ treatment. Overall, irrigation has a major impact on pyrethrin accumulation which increases the rate and duration of pyrethrin accumulation resulting in higher pyrethrin yield per flower at physiological maturity. The findings of this study will be important for future yield predictions and to develop advanced agronomic strategies to maximise pyrethrin yield in pyrethrum.

Keywords: achene, drip irrigation, overhead irrigation, pyrethrin

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Authors: Pedro Llanos, Diego García

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Spaceflight is considered the last frontier in terms of science, technology, and engineering. But it is also the next frontier in terms of human physiology and performance. After more than 200,000 years humans have evolved under earth’s gravity and atmospheric conditions, spaceflight poses environmental stresses for which human physiology is not adapted. Hypoxia, accelerations, and radiation are among such stressors, our research involves suborbital flights aiming to develop effective countermeasures in order to assure sustainable human space presence. The physiologic baseline of spaceflight participants is subject to great variability driven by age, gender, fitness, and metabolic reserve. The objective of the present study is to characterize different physiologic variables in a population of STEM practitioners during an aerobatic flight. Cardiovascular and pulmonary responses were determined in Science Astronaut Candidates (SACs) during unusual attitude aerobatic flight indoctrination. Physiologic data recordings from 20 subjects participating in high-G flight training were analyzed. These recordings were registered by wearable sensor-vest that monitored electrocardiographic tracings (ECGs), signs of dysrhythmias or other electric disturbances during all the flight. The same cardiovascular parameters were also collected approximately 10 min pre-flight, during each high-G/unusual attitude maneuver and 10 min after the flights. The ratio (pre-flight/in-flight/post-flight) of the cardiovascular responses was calculated for comparison of inter-individual differences. The resulting tracings depicting the cardiovascular responses of the subjects were compared against the G-loads (Gs) during the aerobatic flights to analyze cardiovascular variability aspects and fluid/pressure shifts due to the high Gs. In-flight ECG revealed cardiac variability patterns associated with rapid Gs onset in terms of reduced heart rate (HR) and some scattered dysrhythmic patterns (15% premature ventricular contractions-type) that were considered as triggered physiological responses to high-G/unusual attitude training and some were considered as instrument artifact. Variation events were observed in subjects during the +Gz and –Gz maneuvers and these may be due to preload and afterload, sudden shift. Our data reveal that aerobatic flight influenced the breathing rate of the subject, due in part by the various levels of energy expenditure due to the increased use of muscle work during these aerobatic maneuvers. Noteworthy was the high heterogeneity in the different physiological responses among a relatively small group of SACs exposed to similar aerobatic flights with similar Gs exposures. The cardiovascular responses clearly demonstrated that SACs were subjected to significant flight stress. Routine ECG monitoring during high-G/unusual attitude flight training is recommended to capture pathology underlying dangerous dysrhythmias in suborbital flight safety. More research is currently being conducted to further facilitate the development of robust medical screening, medical risk assessment approaches, and suborbital flight training in the context of the evolving commercial human suborbital spaceflight industry. A more mature and integrative medical assessment method is required to understand the physiology state and response variability among highly diverse populations of prospective suborbital flight participants.

Keywords: g force, aerobatic maneuvers, suborbital flight, hypoxia, commercial astronauts

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Authors: Sanaz Alizadeh, Yves Comeau, Arshath Abdul Rahim, Sunhasis Ghoshal

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The application of silver nanoparticles (AgNPs) in personal care products, various household and industrial products has resulted in an inevitable environmental exposure of such engineered nanoparticles (ENPs). Ag ENPs, released via household and industrial wastes, reach water resource recovery facilities (WRRFs), yet the fate and transport of ENPs in WRRFs and their potential risk in the biological wastewater processes are poorly understood. Accordingly, our main objective was to elucidate the impact of long-term continuous exposure to AgNPs on biological activity of aerobic wastewater biofilm. The fate, transport and toxicity of 10 μg.L-1and 100 μg.L-1 PVP-stabilized AgNPs (50 nm) were evaluated in an attached growth biological treatment process, using lab-scale moving bed bioreactors (MBBRs). Two MBBR systems for organic matter removal were fed with a synthetic influent and operated at a hydraulic retention time (HRT) of 180 min and 60% volumetric filling ratio of Anox-K5 carriers with specific surface area of 800 m2/m3. Both reactors were operated for 85 days after reaching steady state conditions to develop a mature biofilm. The impact of AgNPs on the biological performance of the MBBRs was characterized over a period of 64 days in terms of the filtered biodegradable COD (SCOD) removal efficiency, the biofilm viability and key enzymatic activities (α-glucosidase and protease). The AgNPs were quantitatively characterized using single-particle inductively coupled plasma mass spectroscopy (spICP-MS), determining simultaneously the particle size distribution, particle concentration and dissolved silver content in influent, bioreactor and effluent samples. The generation of reactive oxygen species and the oxidative stress were assessed as the proposed toxicity mechanism of AgNPs. Results indicated that a low concentration of AgNPs (10 μg.L-1) did not significantly affect the SCOD removal efficiency whereas a significant reduction in treatment efficiency (37%) was observed at 100 μg.L-1AgNPs. Neither the viability nor the enzymatic activities of biofilm were affected at 10 μg.L-1AgNPs but a higher concentration of AgNPs induced cell membrane integrity damage resulting in 31% loss of viability and reduced α-glucosidase and protease enzymatic activities by 31% and 29%, respectively, over the 64-day exposure period. The elevated intercellular ROS in biofilm at a higher AgNPs concentration over time was consistent with a reduced biological biofilm performance, confirming the occurrence of a nanoparticle-induced oxidative stress in the heterotrophic biofilm. The spICP-MS analysis demonstrated a decrease in the nanoparticles concentration over the first 25 days, indicating a significant partitioning of AgNPs into the biofilm matrix in both reactors. The concentration of nanoparticles increased in effluent of both reactors after 25 days, however, indicating a decreased retention capacity of AgNPs in biofilm. The observed significant detachment of biofilm also contributed to a higher release of nanoparticles due to cell-wall destabilizing properties of AgNPs as an antimicrobial agent. The removal efficiency of PVP-AgNPs and the biofilm biological responses were a function of nanoparticle concentration and exposure time. This study contributes to a better understanding of the fate and behavior of AgNPs in biological wastewater processes, providing key information that can be used to predict the environmental risks of ENPs in aquatic ecosystems.

Keywords: biofilm, silver nanoparticle, single particle ICP-MS, toxicity, wastewater

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Authors: Viktorya Sargsyan, Arax Hovhannesyan, Karine Abelyan

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Introduction: During the last decade, scientific studies have proven the importance of Early Childhood Development (ECD) interventions. These interventions are shown to create strong foundations for children’s intellectual, emotional and physical well-being, as well as the impact they have on learning and economic outcomes for children as they mature into adulthood. Many children in rural Armenia fail to reach their full development potential due to lack of early brain stimulation (playing, singing, reading, etc.) from their parents, and lack of community tools and services to follow-up children’s neurocognitive development. This is exacerbated by high rates of stunting and anemia among children under 3(CU3). This research study tested the effectiveness of an integrated ECD and Maternal, Newborn and Childhood Health (MNCH) model, called “Go Baby, Go!” (GBG), against the traditional (MNCH) strategy which focuses solely on preventive health and nutrition interventions. The hypothesis of this quasi-experimental study was: Children exposed to GBG will have better neurocognitive and nutrition outcomes compared to those receiving only the MNCH intervention. The secondary objective was to assess the effect of GBG on parental child care and nutrition practices. Methodology: The 14 month long study, targeted all 1,300 children aged 0 to 23 months, living in 43 study communities the in Gavar and Vardenis regions (Gegharkunik province, Armenia). Twenty-three intervention communities, 680 children, received GBG, and 20 control communities, 630 children, received MCHN interventions only. Baseline and evaluation data on child development, nutrition status and parental child care and nutrition practices were collected (caregiver interview, direct child assessment). In the intervention sites, in addition to MNCH (maternity schools, supportive supervision for Health Care Providers (HCP), the trained GBG facilitators conducted six interactive group sessions for mothers (key messages, information, group discussions, role playing, video-watching, toys/books preparation, according to GBG curriculum), and two sessions (condensed GBG) for adult family members (husbands, grandmothers). The trained HCPs received quality supervision for ECD counseling and screening. Findings: The GBG model proved to be effective in improving ECD outcomes. Children in the intervention sites had 83% higher odd of total ECD composite score (cognitive, language, motor) compared to children in the control sites (aOR 1.83; 95 percent CI: 1.08-3.09; p=0.025). Caregivers also demonstrated better child care and nutrition practices (minimum dietary diversity in intervention site is 55 percent higher compared to control (aOR=1.55, 95 percent CI 1.10-2.19, p =0.013); support for learning and disciplining practices (aOR=2.22, 95 percent CI 1.19-4.16, p=0.012)). However, there was no evidence of stunting reduction in either study arm. he effect of the integrated model was more prominent in Vardenis, a community which is characterised by high food insecurity and limited knowledge of positive parenting skills. Conclusion: The GBG model is effective and could be applied in target areas with the greatest economic disadvantages and parenting challenges to improve ECD, care practices and developmental outcomes. Longitudinal studies are needed to view the long-term effects of GBG on learning and school readiness.

Keywords: early childhood development, integrated interventions, parental practices, quasi-experimental study

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Authors: Todd H. Buxton, Yong G. Lai

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Temperature management in regulated rivers can involve significant expenditures of water to meet the cold-water requirements of species in summer. For this purpose, flows released from Lewiston Dam on the Trinity River in Northern California are 12.7 cms with temperatures around 11oC in July through September to provide adult spring Chinook cold water to hold in deep pools and mature until spawning in fall. The releases are more than double the flow and 10oC colder temperatures than the natural conditions before the dam was built. The high, cold releases provide springers the habitat they require but may suppress the stream food base and limit future populations of salmon by reducing the juvenile fish size and survival to adults via the positive relationship between the two. Field and modeling research was undertaken to explore whether lowering summer releases from Lewiston Dam may promote thermal stratification in river pools so that both the cold-water needs of adult salmon and warmer water requirements of other organisms in the stream biome may be met. For this investigation, a three-dimensional (3D) computational fluid dynamics (CFD) model was developed and validated with field measurements in two deep pools on the Trinity River. Modeling and field observations were then used to identify the flows and temperatures that may form and maintain thermal stratification under different meteorologic conditions. Under low flows, a pool was found to be well mixed and thermally homogenous until temperatures began to stratify shortly after sunrise. Stratification then strengthened through the day until shading from trees and mountains cooled the inlet flow and decayed the thermal gradient, which collapsed shortly before sunset and returned the pool to a well-mixed state. This diurnal process of stratification formation and destruction was closely predicted by the 3D CFD model. Both the model and field observations indicate that thermal stratification maintained the coldest temperatures of the day at ≥2m depth in a pool and provided water that was around 8oC warmer in the upper 2m of the pool. Results further indicate that the stratified pool under low flows provided almost the same daily average temperatures as when flows were an order of magnitude higher and stratification was prevented, indicating significant water savings may be realized in regulated streams while also providing a diversity in water temperatures the ecosystem requires. With confidence in the 3D CFD model, the model is now being applied to a dozen pools in the Trinity River to understand how pool bathymetry influences thermal stratification under variable flows and diurnal temperature variations. This knowledge will be used to expand the results to 52 pools in a 64 km reach below Lewiston Dam that meet the depth criteria (≥2 m) for spring Chinook holding. From this, rating curves will be developed to relate discharge to the volume of pool habitat that provides springers the temperature (<15.6oC daily average), velocity (0.15 to 0.4 m/s) and depths that accommodate the escapement target for spring Chinook (6,000 adults) under maximum fish densities measured in other streams (3.1 m3/fish) during the holding time of year (May through August). Flow releases that meet these goals will be evaluated for water savings relative to the current flow regime and their influence on indicator species, including the Foothill Yellow-Legged Frog, and aspects of the stream biome that support salmon populations, including macroinvertebrate production and juvenile Chinook growth rates.

Keywords: 3D CFD modeling, flow regulation, thermal stratification, chinook salmon, foothill yellow-legged frogs, water managment

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Authors: Ananya Barui, Provas Banerjee, Jyotirmoy Chatterjee

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Application of optical technology in medicine and biology has a long track-record. In this endeavor, OCT is able to attract both engineers and biologists to work together in the field of photonics for establishing a striking non-invasive imaging technology. In contrast to other in vivo imaging modalities like Raman imaging, confocal imaging, two-photon microscopy etc. which can perform in vivo imaging upto 100-200 micron depth due to limitation in numerical aperture or scattering, however, OCT can achieve high-resolution imaging upto few millimeters of tissue structures depending on their refractive index in different anatomical location. This tomographic system depends on interference of two light waves in an interferometer to produce a depth profile of specimen. In wound healing, frequent collection of biopsies for follow-up of repair process could be avoided by such imaging technique. Real time skin OCT (the optical biopsy) has efficacy in deeper and faster illumination of cutaneou tissue to acquire high resolution cross sectional images of their internal micro-structure. Swept Source-OCT (SS-OCT), a novel imaging technique, can generate high-speed depth profile (~ 2 mm) of wound at a sweeping rate of laser with micron level resolution and optimum coherent length of 5-6 mm. Normally multi-layered skin tissue depicts different optical properties along with variation in thickness, refractive index and composition (i.e. keratine layer, water, fat etc.) according to their anatomical location. For instance, stratum corneum, the upper-most and relatively dehydrated layer of epidermis reflects more light and produces more lucid and a sharp demarcation line with rest of the hydrated epidermal region. During wound healing or regeneration, optical properties of cutaneous tissue continuously altered with maturation of wound bed. More mature and less hydrated tissue component reflects more light and becomes visible as a brighter area in comparison to immature region which content higher amount water or fat that depicts as a darker area in OCT image. Non-healing wound possess prolonged inflammation and inhibits nascent proliferative stage. Accumulation of necrotic tissues also prevents the repair of non-healing wounds. Due to high resolution and potentiality to reflect the compositional aspects of tissues in terms of their optical properties, this tomographic method may facilitate in differentiating non-healing and acute wounds in addition to clinical observations. Non-invasive OCT offers better insight regarding specific biological status of tissue in health and pathological conditions, OCT images could be associated with histo-pathological ‘gold standard’. This correlated SS-OCT and microscopic evaluation of the wound edges can provide information regarding progressive healing and maturation of the epithelial components. In the context of searching analogy between two different imaging modalities, their relative performances in imaging of healing bed were estimated for probing an alternative approach. Present study validated utility of SS-OCT in revealing micro-anatomic structure in the healing bed with newer information. Exploring precise correspondence of OCT images features with histo-chemical findings related to epithelial integrity of the regenerated tissue could have great implication. It could establish the ‘optical biopsy’ as a potent non-invasive diagnostic tool for cutaneous pathology.

Keywords: histo-pathology, non invasive imaging, OCT, wound healing

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Authors: Savvas Rogotis, Nikolaos Kalatzis, Stergios Dimou-Sakellariou, Nikolaos Marianos

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As precision holds the key for the introduction of distinct benefits in agriculture (e.g., energy savings, reduced labor costs, optimal application of inputs, improved products, and yields), it steadily becomes evident that new initiatives should focus on rendering Precision Agriculture (PA) more accessible to the average farmer. PA leverages on technologies such as the Internet of Things (IoT), earth observation, robotics and positioning systems (e.g., the Global Navigation Satellite System – GNSS - as well as individual positioning systems like GPS, Glonass, Galileo) that allow: from simple data georeferencing to optimal navigation of agricultural machinery to even more complex tasks like Variable Rate Applications. An identified customer pain point is that, from one hand, typical triangulation-based positioning systems are not accurate enough (with errors up to several meters), while on the other hand, high precision positioning systems reaching centimeter-level accuracy, are very costly (up to thousands of euros). Within this paper, a Ground-Based Augmentation System (GBAS) is introduced, that can be adapted to any existing IoT field-sensing station infrastructure. The latter should cover a minimum set of requirements, and in particular, each station should operate as a fixed, obstruction-free towards the sky, energy supplying unit. Station augmentation will allow them to function in pairs with GNSS rovers following the differential GNSS base-rover paradigm. This constitutes a key innovation element for the proposed solution that encompasses differential GNSS capabilities into an IoT field-sensing infrastructure. Integrating this kind of information supports the provision of several additional PA beneficial services such as spatial mapping, route planning, and automatic field navigation of unmanned vehicles (UVs). Right at the heart of the designed system, there is a high-end GNSS toolkit with base-rover variants and Real-Time Kinematic (RTK) capabilities. The GNSS toolkit had to tackle all availability, performance, interfacing, and energy-related challenges that are faced for a real-time, low-power, and reliable in the field operation. Specifically, in terms of performance, preliminary findings exhibit a high rover positioning precision that can even reach less than 10-centimeters. As this precision is propagated to the full dataset collection, it enables tractors, UVs, Android-powered devices, and measuring units to deal with challenging real-world scenarios. The system is validated with the help of Gaiatrons, a mature network of agro-climatic telemetry stations with presence all over Greece and beyond ( > 60.000ha of agricultural land covered) that constitutes part of “gaiasense” (www.gaiasense.gr) smart farming (SF) solution. Gaiatrons constantly monitor atmospheric and soil parameters, thus, providing exact fit to operational requirements asked from modern SF infrastructures. Gaiatrons are ultra-low-cost, compact, and energy-autonomous stations with a modular design that enables the integration of advanced GNSS base station capabilities on top of them. A set of demanding pilot demonstrations has been initiated in Stimagka, Greece, an area with a diverse geomorphological landscape where grape cultivation is particularly popular. Pilot demonstrations are in the course of validating the preliminary system findings in its intended environment, tackle all technical challenges, and effectively highlight the added-value offered by the system in action.

Keywords: GNSS, GBAS, precision agriculture, RTK, smart farming

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Authors: Jolene M. Helena, Annie M. Joubert, Peace Mabeta, Magdalena Coetzee, Roy Lakier, Anne E. Mercier

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Targeting the distant tumour and its microenvironment whilst preserving bone density is important in improving the outcomes of patients with bone metastases. 2-Ethyl-3-O-sulphamoyl-estra1,3,5(10)16-tetraene (ESE-16) is an in-silico-designed 2- methoxyestradiol analogue which aimed at enhancing the parent compound’s cytotoxicity and providing a more favourable pharmacokinetic profile. In this study, the potential radiosensitization effects of ESE-16 were investigated in an in vitro bone metastasis model consisting of murine pre-osteoblastic (MC3T3-E1) and pre-osteoclastic (RAW 264.7) bone cells, metastatic prostate (DU 145) and breast (MDA-MB-231) cancer cells, as well as human umbilical vein endothelial cells (HUVECs). Cytotoxicity studies were conducted on all cell lines via spectrophotometric quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. The experimental set-up consisted of flow cytometric analysis of cell cycle progression and apoptosis detection (Annexin V-fluorescein isothiocyanate) to determine the lowest ESE-16 and radiation doses to induce apoptosis and significantly reduce cell viability. Subsequent experiments entailed a 24-hour low-dose ESE-16-exposure followed by a single dose of radiation. Termination proceeded 2, 24 or 48 hours thereafter. The effect of the combination treatment was investigated on osteoclasts via tartrate-resistant acid phosphatase (TRAP) activity- and actin ring formation assays. Tumour cell experiments included investigation of mitotic indices via haematoxylin and eosin staining; pro-apoptotic signalling via spectrophotometric quantification of caspase 3; deoxyribonucleic acid (DNA) damage via micronuclei analysis and histone H2A.X phosphorylation (γ-H2A.X); and Western blot analyses of bone morphogenetic protein-7 and matrix metalloproteinase-9. HUVEC experiments included flow cytometric quantification of cell cycle progression and free radical production; fluorescent examination of cytoskeletal morphology; invasion and migration studies on an xCELLigence platform; and Western blot analyses of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor receptor 1 and 2. Tumour cells yielded half-maximal growth inhibitory concentration (GI50) values in the nanomolar range. ESE-16 concentrations of 235 nM (DU 145) and 176 nM (MDA-MB-231) and a radiation dose of 4 Gy were found to be significant in cell cycle and apoptosis experiments. Bone and endothelial cells were exposed to the same doses as DU 145 cells. Cytotoxicity studies on bone cells reported that RAW 264.7 cells were more sensitive to the combination treatment than MC3T3-E1 cells. Mature osteoclasts were more sensitive than pre-osteoclasts with respect to TRAP activity. However, actin ring morphology was retained. The mitotic arrest was evident in tumour and endothelial cells in the mitotic index and cell cycle experiments. Increased caspase 3 activity and superoxide production indicated pro-apoptotic signalling in tumour and endothelial cells. Increased micronuclei numbers and γ-H2A.X foci indicated increased DNA damage in tumour cells. Compromised actin and tubulin morphologies and decreased invasion and migration were observed in endothelial cells. Western blot analyses revealed reduced metastatic and angiogenic signalling. ESE-16-induced radiosensitization inhibits metastatic signalling and tumour cell survival whilst preferentially preserving bone cells. This low-dose combination treatment strategy may promote the quality of life of patients with metastatic bone disease. Future studies will include 3-dimensional in-vitro and murine in-vivo models.

Keywords: angiogenesis, apoptosis, bone metastasis, cancer, cell migration, cytoskeleton, DNA damage, ESE-16, radiosensitization.

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Authors: Elhadi Nasr, Ibrahim Ramadan

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Biostratigraphical analyses of well sections from the Maragh Low in the Eastern Sirt Basin has allowed high resolution correlations to be undertaken. Full integration of this data with available palaeoenvironmental, lithological, gravity, seismic, aeromagnetic, igneous, radiometric and wireline log information and a geochemical analysis of source rock quality and distribution has led to a more detailed understanding of the geological and the structural history of this area. Pre Sirt Unconformity two superimposed rifting cycles have been identified. The oldest is represented by the Amal Group of sediments and is of Late Carboniferous, Kasimovian / Gzelian to Middle Triassic, Anisian age. Unconformably overlying is a younger rift cycle which is represented the Sarir Group of sediments and is of Early Cretaceous, late Neocomian to Aptian in age. Overlying the Sirt Unconformity is the marine Late Cretaceous section. An assessment of pyrolysis results and a palynofacies analysis has allowed hydrocarbon source facies and quality to be determined. There are a number of hydrocarbon source rock horizons in the Maragh Low, these are sometimes vertically stacked and they are of fair to excellent quality. The oldest identified source rock is the Triassic Shale, this unit is unconformably overlain by sandstones belonging to the Sarir Group and conformably overlies a Triassic Siltstone unit. Palynological dating of the Triassic Shale unit indicates a Middle Triassic, Anisian age. The Triassic Shale is interpreted to have been deposited in a lacustrine palaeoenvironment. This particularly is evidenced by the dark, fine grained, organic rich nature of the sediment and is supported by palynofacies analysis and by the recovery of fish fossils. Geochemical analysis of the Triassic Shale indicates total organic carbon varying between 1.37 and 3.53. S2 pyrolysate yields vary between 2.15 mg/g and 6.61 mg/g and hydrogen indices vary between 156.91 and 278.91. The source quality of the Triassic Shale varies from being of fair to very good / rich. Linked to thermal maturity it is now a very good source for light oil and gas. It was once a very good to rich oil source. The Early Barremian Shale was also deposited in a lacustrine palaeoenvironment. Recovered palynomorphs indicate an Early Cretaceous, late Neocomian to early Barremian age. The Early Barremian Shale is conformably underlain and overlain by sandstone units belonging to the Sarir Group of sediments which are also of Early Cretaceous age. Geochemical analysis of the Early Barremian Shale indicates that it is a good oil source and was originally very good. Total organic carbon varies between 3.59% and 7%. S2 varies between 6.30 mg/g and 10.39 mg/g and the hydrogen indices vary between 148.4 and 175.5. A Late Barremian Shale unit of this age has also been identified in the central Maragh Low. Geochemical analyses indicate that total organic carbon varies between 1.05 and 2.38%, S2 pyrolysate between 1.6 and 5.34 mg/g and the hydrogen index between 152.4 and 224.4. It is a good oil source rock which is now mature. In addition to the non marine hydrocarbon source rocks pre Sirt Unconformity, three formations in the overlying Late Cretaceous section also provide hydrocarbon quality source rocks. Interbedded shales within the Rachmat Formation of Late Cretaceous, early Campanian age have total organic carbon ranging between, 0.7 and 1.47%, S2 pyrolysate varying between 1.37 and 4.00 mg/g and hydrogen indices varying between 195.7 and 272.1. The indication is that this unit would provide a fair gas source to a good oil source. Geochemical analyses of the overlying Tagrifet Limestone indicate that total organic carbon varies between 0.26% and 1.01%. S2 pyrolysate varies between 1.21 and 2.16 mg/g and hydrogen indices vary between 195.7 and 465.4. For the overlying Sirt Shale Formation of Late Cretaceous, late Campanian age, total organic carbon varies between 1.04% and 1.51%, S2 pyrolysate varies between 4.65 mg/g and 6.99 mg/g and the hydrogen indices vary between 151 and 462.9. The study has proven that both the Sirt Shale Formation and the Tagrifet Limestone are good to very good and rich sources for oil in the Maragh Low. High resolution biostratigraphical interpretations have been integrated and calibrated with thermal maturity determinations (Vitrinite Reflectance (%Ro), Spore Colour Index (SCI) and Tmax (ºC) and the determined present day geothermal gradient of 25ºC / Km for the Maragh Low. Interpretation of generated basin modelling profiles allows a detailed prediction of timing of maturation development of these source horizons and leads to a determination of amounts of missing section at major unconformities. From the results the top of the oil window (0.72% Ro) is picked as high as 10,700’ and the base of the oil window (1.35% Ro) assuming a linear trend and by projection is picked as low as 18,000’ in the Maragh Low. For the Triassic Shale the early phase of oil generation was in the Late Palaeocene / Early to Middle Eocene and the main phase of oil generation was in the Middle to Late Eocene. The Early Barremian Shale reached the main phase of oil generation in the Early Oligocene with late generation being reached in the Middle Miocene. For the Rakb Group section (Rachmat Formation, Tagrifet Limestone and Sirt Shale Formation) the early phase of oil generation started in the Late Eocene with the main phase of generation being between the Early Oligocene and the Early Miocene. From studying maturity profiles and from regional considerations it can be predicted that up to 500’ of sediment may have been deposited and eroded by the Sirt Unconformity in the central Maragh Low while up to 2000’ of sediment may have been deposited and then eroded to the south of the trough.

Keywords: Geochemical analysis of the source rocks from wells in Eastern Sirt Basin.

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