CHEMICAL MODIFICATION AND EVALUATION OF CELLS TOWARDS USE AS DELIVERY TOOLS

CHEMICAL MODIFICATION AND EVALUATION OF CELLS TOWARDS USE AS DELIVERY TOOLS SEPTEMBER 2023 BISHNU PRASAD JOSHI M.S., SRI SATHYA SAI INSTITUTE OF HIGHER LEARNING Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Michelle E. Farkas Endogenous cells are being studied for use in various applications, such as next generation therapeutics and drug delivery vehicles. This is on account of their biocompatibility, amenable distribution profiles, and in many instances, recruitment to and localization of diseased tissues. Multiple cell types have been employed, including macrophages, stem cells, red blood cells, and T cells. Most examples of cell-based delivery utilize phagocytosed entities as cargo. However, uniform, and timely loading and release of phagocytosed agents from the cellular vehicles remains a challenge. In this thesis, Idescribe the use and study of approaches that circumvent these limitations, while harnessing the beneficial characteristics of cells as targeted agents. Through collaborative work, we combined the inherent homing properties of macrophages with on-site manufacturing capabilities of bio-orthogonal nanozymes (NZs), by incorporating the latter into cells. We were able to show that the NZs can be internalized by macrophages without impacting their behavior, and while internalized, NZs can convert inactive pro-drugs to their active forms. In the latter half of this thesis, I describe studies involving cell surface modifications, which can be used to append imaging and/or chemotherapeutic agents for delivery applications. I used different types of mild bioconjugation chemistries at native and installed moieties on immortalized, model macrophages. The covalent surface modifications were assessed and their effects on cellular characteristics like viability, motility, chemotaxis, and phenotypic polarization are reported. Further, I showed that the modified cells can be used to evaluate macrophage-cancer interactions in both in vitro and in vivo models. Continuing this line of work, I expanded the studies to primary and immortalized macrophages and stem cells. I characterized the N-Hydroxysuccinimide (NHS) modification in terms of retention at the cell surface over time and determined effects on cellular characteristics like viability and migration. This work now sets the stage for further use of surface-modified cells as diagnostic tools and as delivery agents for therapeutics and molecular probes. In summary, this thesis explores the potential of cells for use in delivery and imaging applications beyond payload internalization, with a focus on facile chemical modifications at their surfaces. CHEMICAL MODIFICATION AND EVALUATION OF CELLS TOWARDS USE AS DELIVERY TOOLS SEPTEMBER 2023 BISHNU PRASAD JOSHI M.S., SRI SATHYA SAI INSTITUTE OF HIGHER LEARNING Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Michelle E. Farkas Endogenous cells are being studied for use in various applications, such as next generation therapeutics and drug delivery vehicles. This is on account of their biocompatibility, amenable distribution profiles, and in many instances, recruitment to and localization of diseased tissues. Multiple cell types have been employed, including macrophages, stem cells, red blood cells, and T cells. Most examples of cell-based delivery utilize phagocytosed entities as cargo. However, uniform, and timely loading and release of phagocytosed agents from the cellular vehicles remains a challenge. In this thesis, Idescribe the use and study of approaches that circumvent these limitations, while harnessing the beneficial characteristics of cells as targeted agents. Through collaborative work, we combined the inherent homing properties of macrophages with on-site manufacturing capabilities of bio-orthogonal nanozymes (NZs), by incorporating the latter into cells. We were able to show that the NZs can be internalized by macrophages without impacting their behavior, and while internalized, NZs can convert inactive pro-drugs to their active forms. In the latter half of this thesis, I describe studies involving cell surface modifications, which can be used to append imaging and/or chemotherapeutic agents for delivery applications. I used different types of mild bioconjugation chemistries at native and installed moieties on immortalized, model macrophages. The covalent surface modifications were assessed and their effects on cellular characteristics like viability, motility, chemotaxis, and phenotypic polarization are reported. Further, I showed that the modified cells can be used to evaluate macrophage-cancer interactions in both in vitro and in vivo models. Continuing this line of work, I expanded the studies [MF1] [BJ2] to primary and immortalized macrophages and stem cells. I characterized the N-Hydroxysuccinimide (NHS) modification in terms of retention at the cell surface over time and determined effects on cellular characteristics like viability and migration. This work now sets the stage for further use of surface-modified cells as diagnostic tools and as delivery agents for therapeutics and molecular probes. In summary, this thesis explores the potential of cells for use in delivery and imaging applications beyond payload internalization, with a focus on facile chemical modifications at their surfaces. [MF1]Because the amine items had to be removed, this is no longer the case. [BJ2]I clarified this was only NHS modification expanded to primary and immortalized macrophages and stem cells

Genetic variability, heritability, genetic advance and correlation among yield and yield components of rice (Oryza sativa L.)

This study was conducted during summer 2015 at Regional Agriculture Research Station, Dipayal, Doti, Nepal to estimate the genotypic and phenotypic variability, heritability, genetic advance and correlation on grain yield and yield associated traits using 26 advance genotypes of lowland irrigated rice. Analysis of variance revealed the existence of significant difference for days to flowering, maturity, plant height, panicle length, thousand grain weight and grain yield. High heritability was estimated for days to flowering (0.88), maturity (0.79), thousand grain weight (0.48) and plant height (0.43) suggesting these traits are under high genetic control. High phenotypic variation was observed for grain yield (24.87%), number of grains/panicle (22.45%), number of panicles/m2 (20.95%) and straw yield (20.75%) while grain yield had medium (12.02%) and remaining traits showed low genotypic coefficient of variation (<10%). High phenotypic coefficient of variation estimated as compared to genotypic coefficient of variation showed environmental influence on the expression of traits. Grain yield (11.98) and days to flowering (10.32) showed medium and remaining traits sowed low genotypic advance as percent of mean. High to low heritability with moderate to low genotypic advance as percent of mean suggested these traits were governed by non additive gene thus direct selection is not beneficial. Further improvements on yield potentiality and yield traits on these genotypes are suggested by creating variation and selection. Panicle length (r = 0.230), days to flowering (r = 0.247), effective tillers (r = 0.488) and straw yield (r = 0.846) manifested significant positive association with grain yield indicating that yield can be increased if selection applied in favor of those yield components

Entrepreneurial behavior of large cardamom growers: A case study in Lamjung district of Nepal

Study on entrepreneurial behavior of large cardamom growers in Lamjung District, Nepal was conducted from December 2017 to June 2018 in Marshyangdi Rural Municipality of Lamjung district. The Rural Municipality was selected purposely for the study due to the recent establishment of Cardamom Zone under the Prime Minister Agriculture Modernization Project in the Municipality focusing on large cardamom development. Altogether 80 large cardamom growers were selected randomly from 454 large cardamom growers of the study site. Data were collected through household surveys, focus group discussions, key informant interviews, personal observations, and other secondary sources. Results showed that a greater proportion of large cardamom growers were found to have medium level of innovativeness (45%), decision-making ability (51.2%), information-seeking ability (48.8%), risk orientation (46.2%), leadership ability (43.8%), achievement motivation (46.2%) and low management orientation (56.3%) which contributed to the overall medium entrepreneurial behavior (47.5%) of large cardamom growers in the study area. About 35 percent of farmers were belonging to the low entrepreneurial behavior and only a few numbers of farmers (17.5%) were under the high entrepreneurial behavior category. The high number of farmers with low and medium entrepreneurial behavior and a low number of farmers with high entrepreneurial behavior has resulted in poor commercialization of large cardamom in the study area. Educational status, landholding, extension participation, economic motivation, experience in large cardamom farming, and area of large cardamom cultivation were found to have a significant correlation with the entrepreneurial behavior of large cardamom farmers while age had a negative and significant effect. There is still scope to increase the number of large cardamom farmers with high entrepreneur behavior for commercializing large cardamom in the study area but programs should be designed and implemented accordingly

Influenza B virus: Need for heightened surveillance and epidemiologic case studies

Recent report of increased influenza B virus infection, particularly theclinical profiles and treatment challenges imposed like that of influenza A,underscores the importance of continuing influenza B virus surveillance.This is, especially in resource limited country, early detection of influenzavirus, its clinical presentation and complications would be vital in minimizingthe public heath burden imposed by this virus.Keywords: chronic obstructive pulmonary disease, influenza B, severe acutepulmonary infection

Agronomic performance and genotypic diversity for morphological traits among early maize genotypes

Detailed information on the genetic diversity between maize germplasm (Zea mays L.) is useful for their systematic and efficient use in breeding programs. Fourteen early maize genotypes were studied to assess their performance and genotypic diversity at Doti, Nepal in 2015. Days to tasseling, days to silking, plant height, ear height, ear length, ear diameter and grain yield were significant among genotypes. Genotype SO3TEY-PO-BM, COMPOL-NIOBP and ACROSS-99402 were found higher yielder with earlier maturity. Days to tasseling (0.85), days to silking (0.82), plant height (0.79), ear length (0.71) and ear diameter (0.66) were found highly heritable traits. Grain yield (0.39) and ear height (0.47) medium and remaining traits showed low heritability. High PCV was observed for grain yield (35.10%), number of plants/plot (34.46%), tesseling silking interval (26.85%), harvested ears/plot (24.45%) and husk cover rating (22.85%) where other traits showed medium to low PCV. Grain yield showed high GCV (21.96%), ear height and husk cover had medium and remaining traits showed low GCV (<10%). Plant height (r₌0.498), harvested plants/plot (r₌0.412), harvested ear/plot (r₌0.762), ear length (r₌0.472) and ear diameter (r₌0.470) showed significant positive correlation with grain yield. The yield can be improved if selection applied in favor of those yield components

Adenosquamous Carcinoma of Gallbladder with Unusual Prognosis: A Case Report

The adenosquamous carcinoma of the gallbladder is a rare variant accounting for only 1-4% of all primary gallbladder carcinoma. Regardless of the histological types, all gallbladder carcinomas have silent and rapid progression resulting in delayed diagnosis and poor prognosis. Even with medical and/or surgical interventions, the median survival of patients with adenosquamous carcinoma, one of the histological variants, is less than a year. However, we present a case of adenosquamous carcinoma with an unusually better prognosis. A 70-year-old female patient, after being diagnosed with gallbladder carcinoma was suggested for surgical resection but was lost to follow-up since then. Two years later, the patient presented and was managed with extended cholecystectomy. The slow progression and non-recurrence of the tumour during follow-up for two years after the surgery indicates a better prognosis in this case

Whole-Genome Sequence of Sungri/96 Vaccine Strain of Peste des Petits Ruminants Virus

We report the complete genome sequence of the Sungri/96 vaccine strain of peste des petits ruminants virus (PPRV). The whole-genome nucleotide sequence has 89 to 99% identity with the available PPRV genome sequences in the NCBI database. This study helps to understand the epidemiological and molecular characteristics of the Sungri/96 strain

Variation in grain zinc and iron concentrations, grain yield and associated traits of biofortified bread wheat genotypes in Nepal

Wheat (Triticum aestivum L.) is one of the major staples in Nepal providing the bulk of food calories and at least 30% of Fe and Zn intake and 20% of dietary energy and protein consumption; thus, it is essential to improve its nutritional quality. To select high-yielding genotypes with elevated grain zinc and iron concentration, the sixth, seventh, eighth, and ninth HarvestPlus Yield Trials (HPYTs) were conducted across diverse locations in Nepal for four consecutive years: 2015–16, 2016–17, 2017–18, and 2018–19, using 47 biofortified and 3 non-biofortified CIMMYT-bred, bread wheat genotypes: Baj#1, Kachu#1, and WK1204 (local check). Genotypic and spatial variations were found in agro-morphological traits; grain yield and its components; and the grain zinc and iron concentration of tested genotypes. Grain zinc concentration was highest in Khumaltar and lowest in Kabre. Likewise, grain iron concentration was highest in Doti and lowest in Surkhet. Most of the biofortified genotypes were superior for grain yield and for grain zinc and iron concentration to the non-biofortified checks. Combined analyses across environments showed moderate to high heritability for both Zn (0.48–0.81) and Fe (0.46–0.79) except a low heritability for Fe observed for 7th HPYT (0.15). Grain yield was positively correlated with the number of tillers per m2, while negatively correlated with days to heading and maturity, grain iron, grain weight per spike, and thousand grain weight. The grain zinc and iron concentration were positively correlated, suggesting that the simultaneous improvement of both micronutrients is possible through wheat breeding. Extensive testing of CIMMYT derived high Zn wheat lines in Nepal led to the release of five biofortified wheat varieties in 2020 with superior yield, better disease resistance, and 30–40% increased grain Zn and adaptable to a range of wheat growing regions in the country – from the hotter lowland, or Terai, regions to the dry mid- and high-elevation areas

Assessment of phosphorus loss risk from soil - a case study from Yuqiao reservoir local watershed in north China

The Yuqiao reservoir located in Tianjin, north China, is the drinking water source for nearly six million people in Tianjin central city. Over the past decade, water quality of the reservoir has become poor due to increasing eutrophication trend. A case study of Yuqiao reservoir local watershed has been conducted in order to study the spatial distribution and level of risk of P loss. In this case, a simplified indicator tool was used for the assessment of P loss, which was classified into two groups: source and transportation scheme

Evaluation of early maize genotypes for grain yield and agromorphological traits

The purpose of this study was to assess the variation on agro-morphological traits and grain yield. A set of 14 early maize genotypes were studied at research field of Regional Agricultural Research Station (RARS), Doti, Nepal in summer seasons of 2015 and 2016. The experiment was carried out in Randomized Complete Block Design (RCBD) with three replications in each year. The variation among genotypes was observed for grain yield and flowering. The genotype SO3TEY-PO-BM produced the highest grain yield (4.33 t/ha) in 2015 whereas Rajahar Local Variety produced the highest grain yield (2.52 t/ha) in 2016. The combined analysis over years showed that Farmer’s variety was found earlier in tasseling (36 days) and silking (39 days), followed by S97TEYGHAYB(3) in tasseling (45 days) and by S97TEYGHAYB(3) and Arun-4 in silking (48 days). EEYC1 produced the highest grain yield (3.17 t/ha), followed by COMPOL-NIBP (3.09 t/ha), SO3TEY-PO-BM (2.90 t/ha), S97TEYGHAYB(3) (2.78 t/ha) and Rajahar Local variety (2.77 t/ha), respectively. The information on variation for the agro-morphological traits among studied early maize genotypes will be helpful to plant breeders in constructing their breeding materials and implementing selection strategies