Transfection optimization and gene editing method establishment for fish cells

Aquaculture is one of the fastest-growing industries around the world, and salmonoid aquaculture is a very big industry for Norway. Ensuring fish health and welfare is essential for aquaculture, also to avoid economic loss. To solve these challenges, researchers need to build new knowledge and understanding through research. To avoid the overuse of fish for in-vivo experiments, researchers need good in vitro cell models. Such cell models can be used to study the effects of infectious diseases, environmental impact, and functions of associated genes with the help of gene editing. Effective and optimized methods are essential for successful gene editing using the CRISPR Cas9 method. Electroporation is a very effective method for the transfection of cell lines. This project was aimed to optimize transfection protocols for fish gill cell lines using two electroporation methods and attempting to perform genome sediting based on the CRISPR/CAS9 system in the Atlantic salmon gill (ASG)-10 cell line. Fish cell lines (ASG-10 and LG-1) developed within the Veterinary Institute of Norway were transfected using pmaxGFPTM plasmid and eSpCas9-GFP protein, on the NeonTM transfection system and NucleofectorTM 2b electroporation devices. Transfected cells were analyzed using a fluorescence microscope and flow cytometry. The best program identified was used for CRISPR-Cas9 transfection in the ASG-10 cell line. CYP1a was used as a target gene as it is important for detoxification, functional assays were available, and the gene is expressed in ASG-10. Two different Cas9 enzymes were used (GFP tagged and untagged) to make ribonucleoprotein (RNP) complexes. The analysis of gene editing from CRISPR-Cas9 transfection was performed using Cleavage assay and Sanger sequencing. From the analysis, edits were not detected, most likely due to low editing efficiency. The master project was successful in optimizing transfection protocols for plasmid and protein transfection in gill cell lines, as well as in establishing a CRISPR-Cas9 gene editing workflow.Norwegian veterinary institute (NVI)M-A

Sustainability Management and Business Cluster 7Sterke

Sustainability is the topic of discussion with the start of industrialization and advancement in human civilization. Sustainability is now topic of concern for various groups like government, NGOs, international organizations, financial institutions, business companies, academics, and consumers. The business companies are the center of attraction for economic activities which has a direct impact over environment and society. So, various stakeholders demand the sustainability performances report of the companies. There are various frameworks for sustainability measuring and reporting like UN SDGs, UN Global Compact, GRI, DSJI Index, CDP Index, etc. This thesis aims to find out how the companies inside the 7sterke business cluster are working for sustainability management by knowing their competency level and challenges faced by them. As well as this thesis also tried to find out how CEOs and managers understand and take sustainability in their business. The study relies on literature review and interview. Responses from companies are analyzed to understand the status quo. From the study, it is found that most of the companies are working for sustainability in certain forms, but they do not fully meet the TBL theory. Most of them are using ISO standards like ISO 14000, ISO 9000 and ISO 26000. There is not any company who is using standard sustainability reporting framework for sustainability measurement and reporting so transparency to all stakeholders is lacked. For many company's time, budget and financial stability of companies are challenged to implement sustainability programs. There are various similar examples and cases from which companies like in 7sterke can use to understand and work on sustainability. In this thesis also, examples from Orkla business group, Moelven ASA and TOMS shoe are discussed. This thesis has also given some recommendations which 7sterke can use for future research and work

Factors Affecting the Productivity of Coffee in Gulmi and Arghakhanchi Districts of Nepal

Coffee is one of the major potential cash crops with lucrative export value grown in mid-hills of Nepal. Nepalese coffee production has suffered long by low productivity. Research was conducted from February to May, 2019 to analyze the factors affecting the productivity of coffee in Arghakhanchi and Gulmi districts of Nepal. These two districts were, purposively selected for this study taking account of comparative advantage and past studies recommendations for coffee sector. Altogether, 100 coffee growing households 50 from each, Arghakhanchi and Gulmi, were sampled by using multistage sampling technique. A pre-tested semi-structured interview schedule was used to collect the primary information while secondary information was collected reviewing the relevant publications. Ordinary Least Square (OLS) regression model was used to determine the factors affecting the productivity of coffee. The study revealed that the number of active family members involved in coffee production (0.000), adoption of income diversification through intercropping (0.005), training (0.072) and technical assistance (0.021) had positive and significant effect on coffee productivity. Encouraging the household to have coffee production as their primary occupation, providing technical assistance on rational land utilization and intercropping and strengthening the skill and knowledge of farmers through trainings could significantly support in increasing the productivity of coffee

Determinants of gross income from carp production in Bara district, Nepal

A study was conducted from March to June 2020 to analyze the determinants of gross income from carp production in the Bara district of Nepal. Altogether, 90 carp producers, 45 each from Simraungadh and Pachrauta municipality in equal basis were sampled by using cluster sampling technique. Primary information was collected through a pre-tested semi-structured interview-based schedule while secondary information was collected reviewing the relevant publications. Data was entered in SPSS 25 and analyzed using STATA 12.1. The results  revealed that the cost of labor, cost of feed, assistances and services, and training had significant positive effect on gross income from carp prodcuiton. Furthermore, lack of quality inputs was identified as the most severe production problems whereas Dhalta to be given was recognized as the most severe marketing problems. Thus, encouraging the carp producer to manage the cost of labor and cost of feed deliberately, rationally providing the assistance and services and strengthening the skills and knowledge of producer through training could significantly increase gross income from carp production

Economic analysis and resource use efficiency of carrot production in Chitwan district, Nepal

A study was conducted from February to May 2020 to analyze the cost-benefit economics analysis and resource use efficiency of carrot production in Chitwan, Nepal. Altogether 70 households producing carrot were selected randomly and surveyed through the pre-tested semi-structured interview-based schedule. Data was entered and coded using SPSS 25 and analyzed using STATA 12.1. The study revealed that the total variable cost per hectare for carrot production was US $1803.1 and a benefit-cost ratio of 1.81 was estimated. Cobb-Douglas production function models the relationship between production output and production inputs . Production function analysis including seven explanatory variables, showed a significant positive effect of seed cost, tractor cost and human labor cost (P<0.01), fertilizer and manure cost (P<0.05) on gross return but herbicide cost, packaging cost, vitamin and micronutrients cost were found insignificant. The return to scale was found to be 1.42. According to estimated allocative efficiency indices, it is suggested to increase the seed, fertilizer and manure, tractor labor and  human labor costs by approximately 45%, 74%, 67%, and 79% respectively and reduce the herbicide and packaging costs approximately by 116% and 246% respectively. The adoption of modern technologies with adjustments to resource use should be suggested to maximize the productivity and profit from carrot production

Factors affecting the productivity of coffee in Gulmi and Arghakhanchi districts of Nepal

Coffee is one of the major potential cash crops with lucrative export value grown in mid-hills of Nepal. Nepalese coffee production has suffered long by low productivity. Research was conducted from February to May, 2019 to analyze the factors affecting the productivity of coffee in Arghakhanchi and Gulmi districts of Nepal. These two districts were, purposively selected for this study taking account of comparative advantage and past studies recommendations for coffee sector. Altogether, 100 coffee growing households 50 from each, Arghakhanchi and Gulmi, were sampled by using multistage sampling technique. A pre-tested semi-structured interview schedule was used to collect the primary information while secondary information was collected reviewing the relevant publications. Ordinary Least Square (OLS) regression model was used to determine the factors affecting the productivity of coffee. The study revealed that the number of active family members involved in coffee production (0.000), adoption of income diversification through intercropping (0.005), training (0.072) and technical assistance (0.021) had positive and significant effect on coffee productivity. Encouraging the household to have coffee production as their primary occupation, providing technical assistance on rational land utilization and intercropping and strengthening the skill and knowledge of farmers through trainings could significantly support in increasing the productivity of coffee

The infectious salmon anemia virus esterase prunes erythrocyte surfaces in infected Atlantic salmon and exposes terminal sialic acids to lectin recognition

Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease

Fostering effective and sustainable scientific collaboration and knowledge exchange: a workshop-based approach to establish a national ecological observatory network (NEON) domain-specific user group

The decision to establish a network of researchers centers on identifying shared research goals. Ecologically specific regions, such as the USA’s National Ecological Observatory Network’s (NEON’s) eco-climatic domains, are ideal locations by which to assemble researchers with a diverse range of expertise but focused on the same set of ecological challenges. The recently established Great Lakes User Group (GLUG) is NEON’s first domain specific ensemble of researchers, whose goal is to address scientific and technical issues specific to the Great Lakes Domain 5 (D05) by using NEON data to enable advancement of ecosystem science. Here, we report on GLUG’s kick off workshop, which comprised lightning talks, keynote presentations, breakout brainstorming sessions and field site visits. Together, these activities created an environment to foster and strengthen GLUG and NEON user engagement. The tangible outcomes of the workshop exceeded initial expectations and include plans for (i) two journal articles (in addition to this one), (ii) two potential funding proposals, (iii) an assignable assets request and (iv) development of classroom activities using NEON datasets. The success of this 2.5-day event was due to a combination of factors, including establishment of clear objectives, adopting engaging activities and providing opportunities for active participation and inclusive collaboration with diverse participants. Given the success of this approach we encourage others, wanting to organize similar groups of researchers, to adopt the workshop framework presented here which will strengthen existing collaborations and foster new ones, together with raising greater awareness and promotion of use of NEON datasets. Establishing domain specific user groups will help bridge the scale gap between site level data collection and addressing regional and larger ecological challenges

Unzipping flood vulnerability and functionality loss:tale of struggle for existence of riparian buildings

Floods pose significant risk to riparian buildings as evidenced during many historical events. Although structural resilience to tsunami flooding is well studied in the literature, high-velocity and debris-laden floods in steep terrains are not considered adequately so far. Historical floods in steep terrains necessitate the need for flood vulnerability analysis of buildings. To this end, we report vulnerability of riparian-reinforced concrete buildings using forensic damage interpretations and empirical/analytical vulnerability analyses. Furthermore, we propose the concept and implications of functionality loss due to flooding in residential reinforced concrete (RC) buildings using empirical data. Fragility functions using inundation depth and momentum flux are presented for RC buildings considering a recent flooding event in Nepal. The results show that flow velocity and sediment load, rather than hydrostatic load, govern the damages in riparian RC buildings. However, at larger inundation depth, hydrostatic force alone may collapse some of the RC buildings

Sustainability Management and Business Cluster 7Sterke

Sustainability is the topic of discussion with the start of industrialization and advancement in human civilization. Sustainability is now topic of concern for various groups like government, NGOs, international organizations, financial institutions, business companies, academics, and consumers. The business companies are the center of attraction for economic activities which has a direct impact over environment and society. So, various stakeholders demand the sustainability performances report of the companies. There are various frameworks for sustainability measuring and reporting like UN SDGs, UN Global Compact, GRI, DSJI Index, CDP Index, etc. This thesis aims to find out how the companies inside the 7sterke business cluster are working for sustainability management by knowing their competency level and challenges faced by them. As well as this thesis also tried to find out how CEOs and managers understand and take sustainability in their business. The study relies on literature review and interview. Responses from companies are analyzed to understand the status quo. From the study, it is found that most of the companies are working for sustainability in certain forms, but they do not fully meet the TBL theory. Most of them are using ISO standards like ISO 14000, ISO 9000 and ISO 26000. There is not any company who is using standard sustainability reporting framework for sustainability measurement and reporting so transparency to all stakeholders is lacked. For many company's time, budget and financial stability of companies are challenged to implement sustainability programs. There are various similar examples and cases from which companies like in 7sterke can use to understand and work on sustainability. In this thesis also, examples from Orkla business group, Moelven ASA and TOMS shoe are discussed. This thesis has also given some recommendations which 7sterke can use for future research and work