M.S. - Molecular Biosciences and Bioengineering

Permanent URI for this collectionhttps://hdl.handle.net/10125/2109

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Enhancing stress resilience of algae and fungi through their co cultivation and symbiosis
(University of Hawai'i at Manoa, 2025) Yuson, Julia L.; Du, Zhi-Yan; Molecular Biosciences and Bioengineering
Algae and fungi are ecologically vital yet belong to distinct kingdoms, each playing crucial roles in the environment. Algae, as autotrophic primary producers, drive photosynthesis, while fungi decompose organic matter, recycling essential nutrients. When co-cultivated, these organisms exhibit increased resilience to environmental stress, suggesting a mutualistic interaction that enhances their adaptive capacity. This study investigates the symbiotic relationship between the fungus Mortierella elongata AG77 and the unicellular green alga Chlamydomonas reinhardtii 21gr under extreme conditions mimicking ecological disruptions caused by climate change. By analyzing their physiological responses, genetic mechanisms, and adaptive strategies, we aim to uncover the functional benefits of this symbiosis. Given that similar partnerships, such as those in lichens, allow microalgae to survive harsh environments, understanding these interactions may provide insights into stress tolerance and adaptation. Our research seeks to harness this knowledge for applications in synthetic biology, biotechnology, and environmental sustainability. Enhancing algal-fungal co-cultivation could contribute to innovations in agriculture, bioengineering, and ecosystem restoration by improving stress resistance and resource efficiency. Ultimately, this work advances our understanding of mutualistic interactions while offering potential solutions for addressing global environmental challenges.
Clinical and pathologic phenotyping of mesotheliomas developing in carriers of germline BAP1 mutations
(University of Hawai'i at Manoa, 2025) Minaai, Michael; Carbone, Michele; Molecular Biosciences and Bioengineering
ABSTRACTIntroduction: Mesothelioma is frequent among carriers of inactivating heterozygous germline BAP1 mutations (BAP1+/-). We studied whether the natural history and the pathology of mesotheliomas in BAP1+/- carriers differed from sporadic, not-genetically related, mesotheliomas. Methods: During 1999-2024, we studied 47 families carrying BAP1+/- transmitted in a Mendelian fashion. We characterized these mutations, collected family history, clinical records, prepared family pedigrees and diagnosed their mesotheliomas. Results: We identified 34 different germline inactivating mutations. Among 238 BAP1+/- carriers aged 27-81, 84 were diagnosed with mesothelioma (35%), 1/84 had evidence of asbestos exposure. No mesothelioma was recorded among 123 siblings/relatives who did not inherit BAP1+/- p < 0.0001. The 84 BAP1+/- patients developed mesothelioma at a relatively young age; 45.2% developed multiple cancers. BAP1+/- patients had a florid, diffuse mesothelial hyperplasia often present in both pleural cavities, peritoneum and pericardium. Thoracoscopy and laparoscopy showed several multi-cavity ~1-3 mm whitish flat lesions, imaging was usually negative for cancer. Histology revealed epithelioid cells lacking BAP1 nuclear staining arranged in tubulo-papillary and trabecular architectures, focally invading sub-mesothelial adipose tissue. These findings may lead to the diagnosis of stage IV metastatic mesothelioma. However, we found that these tumors remain indolent for years and, at this early stage, patients do not require aggressive therapy. We refer to these tumors as “Low-grade-germline-mutant-BAP1-associated-mesotheliomas, L-BAM” to distinguish them from aggressive, therapy-resistant, sporadic mesotheliomas. For the 1/3 of patients who develop lesions visible by imaging, surgery and/or chemotherapy leads to survival of several years, some were cured. Deep invasion by mesothelioma cells with a solid architecture is rare: these cases have poor survival. Conclusions: Compared to sporadic mesotheliomas, mesotheliomas developing in BAP1+/- carriers are a different disease, biologically, histologically and clinically: these patients require a tailored clinical approach.
UPGRADING BLACK SOLDIER FLY LARVAE MEAL FOR AQUATIC FEEDS USING A SUSTAINABLE MICROBIAL PROCESS
(University of Hawai'i at Manoa, 2024) Ochoa, Daniel M.; Su, Wei Wen; Molecular Biosciences and Bioengineering
The current heavy reliance on fish meal (FM) in aquatic feed poses significant sustainability challenges. These ingredients are derived from wild stocks of marine forage fish and have traditionally been essential due to their complete nutritional profiles and wide availability. However, the increasing demand for seafood has driven up the cost of these feed components. With costs escalating due to increased demand and depleted fisheries, the urgency to find sustainable alternatives to FM as a primary protein source becomes more apparent. Over the past three decades, aquaculture has been the fastest-growing sector in global food production, further emphasizing the need for such alternatives. One promising source of proteins to replace costly and unsustainable FMs is the larvae of black soldier flies (Hermetia illucens; BSFL). This insect has received significant attention because its adults are not pests, and very dense larvae biomass can be produced on a large scale using organic wastes with relative ease. The main problem with using these highly nutritious insects is that their fat and chitin contents are as high as 58% and 9%, respectively. These properties negatively affect meal digestibility, trigger undesirable immune responses, cause liver damage, and alter microbiota in the fish; this prevents the incorporation of high proportions of the BSFLMs in aquaculture feeds. This study focuses on developing a sustainable microbial defatting process, instead of relying on costly mechanical and harmful chemical solvent extraction, to remove excess fat from the BSFLM. The solution to this problem is a simple solid-state fermentation process (SSF) process with the use of a filamentous fungus, Aspergillus oryzae, also known as “koji”. It is hypothesized that the enormous enzyme-producing capacity of this edible microbe can be exploited to upgrade BSFLM’s nutritional value in a straightforward and scalable process. Our study showed that koji can grow using BSFL fat as the sole carbon source in a minimal salt medium in liquid culture. We further demonstrated that SSF of BSFLMs with koji fungus is feasible, and it leads to significantly reduced overall fat content and increased protein content in the koji-fermented BSFLM. In this study, we validated and quantified the extent to which fat levels can be reduced and identified the optimal conditions for this low-cost microbial defatting process. Additionally, a simple SSF bioreactor system was developed to monitor and control the environment in which these fermentations were conducted. The system consists of autoclavable 1-liter polypropylene jars loaded with BSFLMs and inoculated with koji spores. They were incubated within an industrial proofer retrofitted to control temperature and humidity level of the fungal culture during SSF. An IR-based carbon dioxide gas sensor was incorporated into the fermentation jar to allow online monitoring of culture CO2 evolution rate, which was evaluated in this study and found to be suitable for indirect prediction of fungal growth during SSF of the BSFLM. Over 3 kg of fermented BSFLMs were successfully produced using the SSF system for use in a 12-week feed trial with tilapia hybrids (O. niloticus X O. mossambicus). From the tilapia feeding trial, the fermented (microbially defatted) BSFLM was found to be able to replace up to half of the fishmeal in feed formulations, potentially lowering production costs in feed manufacturing, and opening opportunities for the local production of a high-quality protein ingredient. In conclusion, the innovative approach of utilizing SSF with koji to defat and enhance the nutritional value of BSFLM presents a promising solution to the challenges posed by the heavy reliance on FM in aquatic feed.
Evaluating the Role of EXOC5 in Early Folliculogenesis
(University of Hawai'i at Manoa, 2024) Young, Shaina Fei; Ward, Steven; Molecular Biosciences and Bioengineering
Folliculogenesis is the process through which female germ cells (oocytes) develop into viable eggs. This process is reliant on extensive cell- cell communication between oocytes and their surrounding granulosa cells. EXOC5 plays multifaced roles in cell-cell communication, cell signaling, and autophagy. Each wave of folliculogenesis begins when a small subset of the pool of primordial follicles (oocytes with their granulosa cells) are activated to become primary follicles, which then progress to mature follicles to produce viable eggs.Our laboratory has previously determined that EXOC5 is required for folliculogenesis using a Cre-lox system in which ZP3-Cre was used to flox out the Exoc5 only in primary oocytes of primary follicles, to create Zp3-Exoc5-cKO mice. Histological examination of ovaries from these mice revealed that the first wave of folliculogenesis appeared normal at 20 days post-partum (DPP), but all subsequent, adult waves were blocked. This suggested that EXOC5 was not required for the first wave of folliculogenesis but was required for all subsequent waves. However, because the Zp3-Cre is only activated in primary follicles after folliculogenesis has been activated, we created a second conditional knockout mouse, Gdf9-Exoc5-cKO, using Gdf9-Cre, which is activated in the primordial follicle oocytes before folliculogenesis is activated. Histological examination from the ovaries of these mice indicated that in Gdf9-Exoc5-cKO mice, the first wave of folliculogenesis also appears normal, verifying that the first wave of folliculogenesis does not depend on EXOC5. In Zp3-Exoc5-cKO mice, there were no antral follicles present by 60 DPP but primary follicles were plentiful. In Gdf9-Exoc5-cKO mice, however, by 60 DPP, there are no follicles at all, suggesting a more rapid progression to complete infertility when Exoc5 is deleted early.
Modifying Hawaiian Drosophila laboratory diet with material from the wild: Phenotypic response to microbial and nutritional variation
(University of Hawai'i at Manoa, 2024) Konicek, Kelli R.; Yew, Joanne; Molecular Biosciences and Bioengineering
Invertebrate conservation and research are necessary to protect a large portion of Hawai`i’s nativefauna. Because fourteen species of Hawaiian Drosophila (Hawaiian picture-wing flies) are listed as endangered, with many more species in decline, understanding the biology and ecology of these animals is critical to their conservation. Research indicates that Hawaiian Drosophila exhibit phenotypic changes in reproduction and longevity based on their microbiome community assemblage. Lab-reared organisms maintain different microbiome community compositions than their wild counterparts, and Drosophila microbiomes are predominantly formed from diet. Therefore, understanding the phenotypic response of lab-reared flies to a wild-derived diet is necessary to establish robust populations of lab-reared Hawaiian Drosophila in their historical habitat range. Here, we supplement lab-reared Drosophila crucigera diet with rotting host plant to gauge the phenotypic effects of “wild” sources of nutrition. Flies exposed to a wild-derived diet did not exhibit increased fecundity compared to flies fed on a standard lab diet. However, flies who were exposed to frass from flies given a wild diet had higher fecundity than flies exposed to frass derived from a standard diet. Internal fly microbiome sequencing revealed that fungal diversity was impacted when flies were directly exposed to host plant substrate. Bacterial diversity was affected by inoculum that consisted of frass or a suspended solution of host plant microbes. Achieving effective diet supplementation for Hawaiian picture-wing flies in lab culture may ultimately improve rearing methods and conservation efforts to reintroduce endangered species to the wild.
Germline BAP1 mutation alter the tumor immune microenvironment and impact the progression of Malignant Mesothelioma (MM)
(University of Hawai'i at Manoa, 2024) Tyagi, Garima; Carbone, Michele Dr; Molecular Biosciences and Bioengineering
Asbestos causes malignant transformation of primary human mesothelial cells (HM), leading to mesothelioma. The primary inherited risk factor linked to mesothelioma is the dysfunction of BRCA1 Associated Protein 1 (BAP1). People with inherited BAP1 mutations face an increased risk of developing MM. However, to date, we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. Using a Bap1+/− mouse model, we found that, compared with their wild-type littermates, Bap1+/− mice exposed to low-dose asbestos fibres showed significant alterations of the peritoneal inflammatory response and lower levels of several chemokines and cytokines. Consistent with these data, Bap1+/ − mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos doses that rarely induced mesothelioma in wild-type mice. Additionally, our findings showed that tumors in Bap1+/- mice have more M2-type macrophages as compared to tumors in Bap1WT mice. This puzzled us even more since M2 macrophages promote immunosuppressive microenvironment and should have adverse effects on therapies, but we see the opposite response in BAP1 mutant patients. We speculated if asbestos had an effect on the observed results. We created a mouse model from mesothelioma-derived luciferase integrated cancer cell line (AN1) from Bap1+/- mice which were biallelically inactivated for BAP1 as found in 60% of BAP1 mutant MM patient tumors, to understand how BAP1 loss in mesothelioma impact tumour microenvironment without asbestos exposure. Here, I injected Bap1WT and Bap1+/- with AN1 (Bap1-/-) biallelic inactivated tumor cell line. Tumors were digested and tumor immune cells were collected. Harvested cells were immunoprofiled by flow cytometry. I did not find any significant differences in the immune cell population amongst the two genotypes i.e. Bap1+/- and Bap1WT. Additionally, I did not find any differences in the T cell population (CD8+, CD4+, their activation states and Tregs) and IFNY levels by flow cytometry. However, I found that tumor-associated macrophages (TAMs) in Bap1+/- showed reduced levels of MHCII expression as compared to Bap1WT. I did not find similar findings in other antigen-presenting cells i.e. dendritic cells, B cells and macrophages outside of the TME. Additionally, I found that tumor-promoting markers i.e. Trem2, CD9 and Ly6C were significantly reduced on TAMs in Bap1+/- as compared to Bap1WT mice. These findings imply that while alterations in BAP1 status i.e. partial or complete loss are associated with MM development, the relationship between BAP1 status and survival outcomes is more complex and possibly influenced by other factors or molecular pathways which need to be explored further.
Elucidating and Rewiring Regulation of Terpenoid Biosynthesis in Yarrowia lipolytica Utilizing Renewable Lipid Feedstocks
(University of Hawai'i at Manoa, 2024) Maruwan, Jessica; Su, Wei-Wen; Molecular Biosciences and Bioengineering
Terpenoids are a group of compounds with many important applications and oleaginous yeast Yarrowia lipolytica is a promising host for terpenoid production because of its highly active mevalonate pathway and high acetyl-CoA fluxes in lipid substrate. Moreover, it is generally recognized as safe (GRAS), and can metabolize renewable lipid feedstocks, in addition to common sugar feedstocks. Lipid is an important substrate as it is underutilized or used to produce low-value products. Our research aims to metabolically engineer Y. lipolytica to convert waste lipid feedstock into terpenes, specifically targeting β-carotene production. Two key aspects of metabolic engineering are emphasized: (1) Optimization of the auxin-inducible degron (AID) system for temporal regulation of protein in lipid substrates, and (2) Investigation into the NADPH supply in Y. lipolytica utilizing lipid substrates, especially its impact on β-carotene production. A unique AID conditional protein degradation system was developed, incorporating a CGD degron tag derived from mini-IAA7 inserted in the open loops of GFP, along with the F74A variant of Oryza sativa TIR1 F-box protein (OsTIR1(F74A)), and 5-Adamantyl-IAA as the inducer. Optimization strategies were employed to minimize leaky degradation by fine-tuning the expression level of the OsTIR1(F74A) F-box protein using the POLE1 medium-strength promoter, effectively improving degradation efficiency during the early culture phase. Metabolomic flux analysis and RNA sequencing suggest the significance of the pentosephosphate pathway and mannitol cycle as the main sources of cellular NADPH, an essential cofactor for β-carotene production. While the pentose-phosphate pathway remains important for NADPH and β-carotene production in lipid substrates, the importance of the mannitol cycle becomes prominent when the pentose-phosphate pathway is knocked out or when mannitol is supplemented to the lipid substrate. However, the correlation between NADPH levels and βcarotene production remains unclear, suggesting that other factors besides NADPH alone may influence β-carotene production.
ESTABLISHING EFFICIENT PROTOPLAST GENOME EDITING SYSTEMS USING CRISPR/CAS9 FOR MAIZE BREEDING AND IMPROVEMENT
(University of Hawai'i at Manoa, 2024) Higa, Lauren Akemi; Du, Zhi-Yan (Rock); Molecular Biosciences and Bioengineering
Amid the growing concerns over global food security, the use of gene editing technology in crop improvement is gaining traction as a promising solution. While this technology has been widely used to modify specific traits in crops, its potential to overcome breeding barriers remains largely unexplored. It is extremely challenging to crossbreed tropical maize and temperate maize under the long-day photoperiod of a temperate environment due to their differences in flowering time. This breeding barrier prevents the integration of tropical maize into temperate breeding programs, greatly limiting genetic diversity. Leveraging the power of the CRISPR/Cas9 system, this study is ultimately focused on three regulators of flowering time, ZmCCT9, ZmCCT10, and ZmRap2.7, to induce earlier flowering in tropical maize, aligning its timing with temperate maize. Since traditional plant transformation techniques are time-consuming and labor-intensive, this study has developed a maize protoplast CRISPR/Cas9 system to rapidly validate the activity of guide RNAs. Additionally, nine gRNAs targeting ZmCCT9, ZmCCT10, and ZmRap2.7 were designed, and their editing efficiencies were evaluated using the protoplast gene editing system.
EFFECTS OF HAWAIIAN-GROWN COFFEE ON SERUM LEPTIN LEVELS AMONG HEALTHY INDIVIDUALS
(University of Hawai'i at Manoa, 2021) Stillwell, Nyan Crandall; Nerurkar, Pratibha V.; Molecular Biosciences and Bioengineering
Coffee is one of the most widely consumed beverages in the world. New studies indicate that coffee consumption does provide some health benefits including protecting against cardiovascular disease, type II diabetes, Alzheimer’s disease, and obesity [1, 2]. Coffee also appears to improve cognitive function and decrease the risk of depression [2]. The mechanisms of how coffee provides these health benefits, however, are still unclear. Reductions in of oxidative stress and subclinical inflammation are thought to be possible mechanisms associated with the beneficial effects of coffee [3].In this study, we focused on leptin, an adipocytokine that plays an important role in metabolic regulation. Elevated levels of leptin have been associated with an increased risk of inflammation-related diseases like type 2 diabetes, hypertension, cardiovascular disease, and obesity [4]. In previous studies, consumption of coffee (Coffea arabica) has been shown to decrease serum levels of leptin [5]. The aim of this study is to determine if consumption of Hawaiian-grown coffee has any specific biological effects on serum leptin concentrations. Based on literature review, we hypothesize that short term consumption of Hawaii-grown coffee will reduce plasma leptin levels among healthy individuals. A randomized, blinded, crossover intervention study was conducted which included all ethnicities. Participants consumed 24oz. of Hawaiian-grown coffee per day for four weeks, preceded by one-week washout periods in which participants abstained from coffee or tea consumption. Blood samples were collected and analyzed for leptin concentrations. The results of the coffee interventions showed no significant changes to serum levels of leptin after 4 weeks of Hawaiian-grown coffee consumption for both varieties of coffee used in this study.
Development Of A Mesophyll Protoplast-based System For Gene Editing Of Papaya
(University of Hawaii at Manoa, 2022) Elias, Marc Joseph; Christopher, David A.; Molecular Biosciences and Bioengineering
Papaya (Carica papaya L.) is an economically important food crop grown in tropical and subtropical regions. The global gross agricultural production value for papaya reached $6.2 billion in 2020. Papaya and its byproducts are used in commercial production, cosmetics, and pharmaceutical industries, offering many health benefits. While papaya is important economically, it faces threats from microbial pathogens. Genetic engineering and traditional breeding methods have been used to produce papaya with improved resistance to some pathogens. However, few innate disease-resistance genes are present in the papaya genome, and novel gene introgression from closely related species has reported F1 sterility with low viability and vigor. CRISPR-Cas9 gene editing technology has enabled gene editing in papaya using the Agrobacterium-mediated transformation method. When introduced to the market, papaya developed using this transgenic method will face increased regulatory constraints. A non-transgenic DNA-free alternative is available using CRISPR-Cas9 ribonucleoprotein complexes. A high-yield protoplast isolation method, producing highly viable, durable protoplasts, was developed during this study. Papaya protoplasts were transfected with fluorescent reporter constructs using our papaya-optimized PEG-mediated transfection method. Transfected cells were shown to express and shuttle proteins to expected locations and organelles viably. A system for DNA-free gene editing in papaya was developed using PEG-mediated transfection to deliver CRISPR-Cas9 ribonucleoprotein complexes into papaya protoplasts. The albino phenotypic marker gene phytoene desaturase and the susceptibility gene MLO homolog mildew resistance locus O-like protein 6 were targeted during this study.
Chemical Approaches To Improve The Manufacturability Of Disulfide-rich Peptide Toxins – Engineering A Stable And Selective Peptide Blocker Of The KCa1.1 Channel
(University of Hawaii at Manoa, 2022) Delgado, Erick Medermel; Bingham, Jon-Paul; Biological Engineering
Scorpion venoms represent an immense source of bioactive peptides which are potent and selective effectors of sodium (Na+), potassium (K+), chloride (Cl-), and calcium (Ca2+) ion channels. Many of these compounds boast premium advantages which qualify them as attractive molecular tools and pharmaceutical leads: they penetrate tissue effectively while mitigating off-target toxicity, are capable of selectively binding specific ion channel subtypes, alter channel function in a variety of desired ways: inhibit, potentiate, change the kinetics, etc. Unfortunately, many scorpion venom peptides feature unstable functional groups, are prone to aggregation during synthesis or oxidation, and require specific disulfide connectivity, all of which can complicate manufacturing and ultimately reduce synthetic yield. Using solid-phase peptide synthesis, native chemical ligation (NCL), and orthogonal bioconjugation, an effective bioengineering approach was developed for a stable and selective peptide blocker of the KCa1.1, calcium-activated potassium channel. This novel 37 amino acid Iberiotoxin (IbTX) analog, azido-IbTX-W14F, incorporates a mid-sequence Val16Ala mutation to facilitate thioester mediated NCL, homologous substitutions of oxidation prone Met29 and Tyr14 residues, and an orthogonally conjugated azido-Lys19 residue capable of fluorescent bioconjugation with a phosphine fluorophore. Azido-IbTX-W14F retained native-like bioactivity according to electrophysiology studies, exhibited increased solution stability, and was demonstrated to be amenable to chemoselective fluorescent bioconjugation. The resulting fluorescent-IbTX analog has potential as a diagnostic tool or as a drug lead for disease states mediated by KCa1.1.
Flavonoid Metabolon Formation In Citrus Sinensis
(University of Hawaii at Manoa, 2022) Knittel, David; Owens, Daniel; Molecular Biosciences and Bioengineering
The flavonoid metabolon has been previously theorized and researched, but little work has been done on the mapping of later pathway and periphery enzymes. This research has demonstrated novel characterizations of interactions among glucosyltransferases (GTs) and the primary flavonoid pathway as well as created rapid assays for the characterization of protein-protein interactions of multiple classes of enzymes. Additionally, several assays have been created for the analysis of membrane bound P450 class enzymes, specifically Flavone Synthase II (FNSII). These procedures will allow for the rapid assaying of a wide variety of enzymes alongside partner groups of various experience levels. The putative flavonoid metabolon structure of Citrus sinensis differs from previously examined Arabidopsis thaliana and may provide insight into the stabilizing effects of periphery pathway enzymes. Genetic differences between blond and blood cultivars of sweet orange were also identified as potential binding sequences in GT enzymes.
Bioengineering of a Novel Peptide Sequence from the Venom of Conus obscurus
(University of Hawaii at Manoa, 2021) Wiere, Sean; Bingham, Jon-Paul; Molecular Biosciences and Bioengineering
The marine cone snail produces one of the fastest prey strikes in the animal kingdom with efficacious venom injection causing prey paralysis and death within seconds. Each snail produces hundreds of conotoxins and has been the source behind discovering and utilizing novel analgesic peptide therapeutics. In this study, we discover, isolate, and synthesize two α3/5 conotoxins derived from the milked venom of Conus obscurus: one novel (α-conotoxin ObI) and one previously found in the venom of Conus striatus (α-SI). We then generate five synthetic analogs, accompanying single and double mutations from the native α-conotoxin ObI. We integrate three post-translational modifications (PTMs) within analog development: N-terminal truncation, proline hydroxylation, and tryptophan bromination. α-Conotoxin ObI demonstrates nanomolar potency towards Poecilia reticulata (LD50) and the Homo sapiens muscle-type nAChR (EC50). Moreover, the analog α-ObI [P9K] displayed enhanced potency in both animal bioassays. The exhibited successful incorporation of 3 PTMs investigates the boundaries of peptide bioengineering in the generation of novel α-conotoxins.
Myostatin Inhibition Directly Influences DNA Methylation To Build Epigenetic Landscape Inducing Skeletal Muscle Hypertrophy
(University of Hawaii at Manoa, 2021) Vu, Huong Thanh; He, Yanghua; Molecular Biosciences and Bioengineering
Myostatin(MSTN) is a TGF β signaling ligand and a known inhibitor of skeletal muscle formation in adult s , which inactivation results in hypermuscular phenotype in animals, and has been vigorously researched for its applicability in agriculture and medicine. Some recent studies suggest that MSTN utilizes not only molecular signaling but also epigenetics modif ications to control the gene expression patterns of skeletal muscle. In this study, we developed a mouse line overexpressing MSTN prodomain (MSTN pro), an inhibitor of MSTN at birth to thoroughly study its effects on genomic DNA methylation using RRBS anal ysis contrasted with qPCR based gene expression of selected relevant genes. Surprisingly, DNA methyltransferases and demethylases were dysregulated at both global and local levels during fetal periods before transgene expression, and not after birth. Such dysregulation was hardly coincidental since identified DMRs are highly concentrated into pathways involved in skeletal muscle development. Post natal and post transgene expression skeletal muscle, on the other hand, had the DNA methylome indirectly altered through transcription factors and histone modifications, with minimal evidence of a direct relationship between MSTN and DNA methylation related enzymes. Overall, MSTN inhibition of early neonatal skeletal muscle resulted in the activation of TET3 demethy lase to ensure the smooth activation of Wnt and PI3K/Akt/mTOR pathways, both of which are direct targets inhibited by MSTN signaling. Additionally, the study revealed an unknown process during late fetal development in MSTN pro transgenic individuals which predisposes them to develop the hypermuscular phenotype later in life.
Anaerobic Co-digestion Of Coffee Pulp And Cattle Manure: Evaluation Of Process Stability
(University of Hawaii at Manoa, 2021) Karki, Renisha; Khanal, Samir K.; Molecular Biosciences and Bioengineering
About 530 million dry tons of agricultural biomass are produced annually in the U.S. (Almomani et al., 2019). Coffee is the second largest agricultural crop produced globally (9.2 million tons of coffee cherries per year), generating nearly 42% of coffee pulp (CP) of the total waste produced during coffee processing. Furthermore, approximately 900 million tons of cattle manure (CM) are reported to be generated in the U.S. annually (U.S. Department of Agriculture, S.C.S., 2018). Anaerobic digestion (AD) is a widely adopted technology for treating various organic waste streams; however, mono-digestion of CP is challenging due to the presence of toxic compounds such as caffeine, free phenols, and tannins. Hence, co-digestion of CP waste with CM could be a strategy to enhance digestibility and process stability through nutrient balance and dilution of toxic compounds. Biochemical methane potential (BMP) tests were conducted to select the optimum inoculum source and feedstock ratio for co-digestion. Nine different kinetic models (modified Gompertz, Chen and Hashimoto, first-order, transfer, cone, superimposed, modified Gompertz with second order equation, two-phase exponential, and multi-stage models) were evaluated to find the best-fit for the BMP experimental data. In the next phase, the optimum co-digestion ratio was compared with mono-digestion of feedstocks in semi-continuous studies at mesophilic conditions. The biomethane production of CP alone and co-digestion with CM was almost similar, however in terms of process stability, co-digestion yielded better results. The close monitoring of the start-up period and the use of co-substrate with high buffering capacity are highly recommended for efficient AD of CP.
Pathogenic Parkinson's Disease Mutations of LRRK2 Show Altered Golgi-Endosome Vesicle Trafficking
(University of Hawaii at Manoa, 2020) Nguyen, Thien Phuc Ngoc; James, Nicholas; Molecular Biosciences and Bioengineering
Mutations within the LRRK2 (Leucine Rich Repeat Kinase 2) gene represent the most common genetic alterations linked to sporadic and familial Parkinson’s disease (PD). The majority of these mutations localize within the enzymatic core of the LRRK2 protein, with biochemical characterization of these constructs shown to have altered GTPase and kinase activity. In other research findings, LRRK2 interacts with distinct membrane structures, including caveolin, the endosome, and the Golgi apparatus. The identification that LRRK2 kinase activity is targeting a subset of vesicle trafficking proteins (e.g., Rabs and Endophilin) has established a potential biological function towards vesicle maturation. However, there is a significant deficit of direct observation of LRRK2 being targeted to and interacting with membrane proteins. Therefore, this thesis focused on establishing a system wherein LRRK2 complex formation on vesicles could be tracked in live cells, thus providing an online readout of the LRRK2 function in real-time. Herein this work demonstrates that fluorescence fluctuation microscopy, specifically cross-correlation Raster Imaging Correlation Spectroscopy, can provide a quantitative measurement of LRRK2 interaction with Rab proteins within live cells. These results were exciting as they were nearly identical to values previous reports using traditional biochemicalmethods. Furthermore, the application of these live-cell methods helped differentiate LRRK2-Rab interactions and vesicles formation with cells expressing a PD variant of LRRK2. The most striking readout was our ability to isolate targeted trafficking of LRRK2 bound vesicles and time-dependent interaction with cellular structures. Our findings establish that nanoscale microscopy can provide a unique look into LRRK2 cellular activity, which is not accessible with other experimental techniques. Hence, this might open the pathway in LRRK2 mediated neurodegeneration towards a promising future in PD research.
Identification Of lncRNA Biomarkers For Lung Cancer Through Integrative Cross-platform Data Analysis
(University of Hawaii at Manoa, 2020) Zhao, Tianying; Deng, Youping; Molecular Biosciences and Bioengineering
This study was designed to identify lncRNA biomarker candidates using lung cancer data from RNA-Seq and microarray platforms separately. Lung cancer datasets were obtained from the Gene Expression Omnibus (GEO, n = 287) and The Cancer Genome Atlas (TCGA, n = 216) repositories, only common lncRNAs were used. Differentially expressed (DE) lncRNAs in tumor with respect to normal were selected from the Affymetrix and TCGA datasets. A training model consisting of the top 20 DE Affymetrix lncRNAs was used for validation in the TCGA and Agilent datasets. A second similar training model was generated using the TCGA dataset. First, a model using the top 20 DE lncRNAs from Affymetrix for training and validated using TCGA and Agilent, achieved high prediction accuracy for both training (98.5% AUC for Affymetrix) and validation (99.2% AUC for TCGA and 92.8% AUC for Agilent). A similar model using the top 20 DE lncRNAs from TCGA for training and validated using Affymetrix and Agilent, also achieved high prediction accuracy for both training (97.7% AUC for TCGA) and validation (96.5% AUC for Affymetrix and 80.9% AUC for Agilent). Eight overlapped lncRNAs were found to be potentially related with lung cancer diagnosis but not with prognosis.
Taro Vein Chlorosis Nucleorhabdovirus and Other Viruses of Taro in the Pacific
(University of Hawaii at Manoa, 2019) Loristo, Jarin; Melzer, Michael J.; Molecular Biosciences and Bioengineering
The taro virome has been documented to include a few currently known viral species. Among these, Taro vein chlorosis nucleorhabdovirus (TaVCV) is a virus that has been discovered in several different countries across the South Pacific, and, as of 2013, has started to infect taro in Hawaii. Efforts to detect TaVCV in infected taro across regional variants has remained a challenge, as the regional genomic diversity of this virus has been noted to reach as high as 21%. A previous study conducted on Hawaiian TaVCV isolates has determined a very low diversity among local variants, so a sampling method to take infected taro samples from Hawaii, Samoa, Guam, Palau and Vanuatu was conducted to determine the genomic sequences from each of these regions and perform a phylogenetic analysis of these variants. Next-generation sequencing (NGS) performed on double-stranded (ds)RNA found TaVCV genomes that diversified by up to 20% compared to the TaVCV sequence available from GenBank, derived from a Fijian isolate. Additionally, NGS also contributed to the discovery of a unique taro-associated totivirus, which has been fully sequenced and characterized.
Studies Defining the Role of Protein Disulfide Isomerase-9 in Pollen Biogenesis in Arabidopsis thaliana
(University of Hawaii at Manoa, 2019) Feldeverd, Elizabeth Michelle; Christopher, David A.; Molecular Biosciences and Bioengineering
Protein disulfide isomerases (PDIs) are disulfide bond catalysts that serve a breadth of important roles in eukaryotic growth and development. Arabidopsis thaliana has fourteen PDIs with variations on the canonical domain arrangement and subcellular location. Among them, PDI9 and its homolog PDI10 are part of the unfolded protein response (UPR) and have been shown to fold proteins via disulfide bonds. Using a PDI9-specific antiserum, PDI9 was found to be expressed in mature pollen. Here, the role of PDI9 in pollen biogenesis was characterized on molecular and cellular scales. First, using transfected leaf mesophyll protoplasts, PDI9 was shown to co-localize in the endoplasmic reticulum with two proteins (Leucine Rich Repeat-Extensin 8, LRX8, and ER Membrane Complex Subunit 7, EMC7) that are both expressed in pollen. Double knockout pdi9-pdi10 seedlings were used to demonstrate constitutive up-regulation of other PDIs and chaperones in non-UPR-stressed conditions relative to wild-type. Using an improved high-throughput Alexander staining method and scanning electron microscopy, PDI9 was found to play a crucial role in pollen development under prolonged heat stress. The single pdi9 and double pdi9-pdi10 knockout plants produce less viable pollen, dehisce fewer pollen grains, have impaired silique development, and exine formation is severely disrupted. Taken together, these data suggest that PDI9 mediates the development of healthy pollen under heat stress via its role in the UPR and its interactions with secretory substrates (LRX8, EMC7).
The Role Of The “abcc6 Pathway” And Dietary Pyrophosphate In Dystrophic Calcification
(University of Hawaii at Manoa, 2018-12) Julian, Charnelle Basilio; Le Saux, Olivier; Molecular Biosciences and Bioengineering
The Abcc6-/- mice display calcification symptoms similar to human PXE patients in addition to an acute and inducible dystrophic muscle calcification phenotype. We hypothesize 1) the lack of Abcc6 lowers ATP efflux and influences the expression of Enpp1 and Nt5e, thereby impacting plasma PPi levels and calcification susceptibility and 2) ABCC6 deficiency causes dystrophic calcification in cardiac tissues and skeletal muscle. The aims of this study were to 1) characterize the muscle calcification phenotype, 2) explore gene expression of the “Abcc6 pathway” and its molecular players, and 3) supplement PPi to counteract skeletal muscle calcification. Our inability to reproduce previous gastrocnemius calcification data for Aim 1 despite multiple trials and attempting protocol optimization led to the discovery of elevated levels of PPi in the rodent chow. Surprisingly, reversion to a low PPi diet did not restore the calcification phenotype and instead reduced calcium deposits in muscle tissues. The testing of an acceleration diet designed to enhance calcification was infructuous as it produced mineralization even in wild type mice. Our results demonstrated the impact that animal chow can have on phenotypic outcome. Moreover, our data may provide an explanation for the phenotype variability in PXE patients and further suggested that dietary intervention to slow the progression of the calcification phenotype of PXE in patients may be possible. Gene expression studies showed positive correlation between the Abcc6 status and Enpp1 and Nt5e mRNA levels in heart tissues. However, since we showed that PPi supplementation influences the expression of Enpp1, the presence of high levels of PPi in mouse chow probably negatively influenced our data. The Aim 3 experiments could not be completed as planned since we discovered elevated levels of PPi in the animal diet. However, results of specific Aim 1 provided some indications that indeed supplementation with PPi can effectively suppress dystrophic calcification in muscle tissues. Overall, the discovery of the elevated levels of PPi in the diet prevented us from achieving some of the goals of our specific aims and produced unanticipated but valuable data. Collectively, these studies have improved our understanding of the role of Abcc6 and PPi in the inhibition of soft-tissue calcification.

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