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- Creator:
- Overton, Michael
- Description:
- Viruses have played a central role in the evolution and ecology of cellular life since it first arose. The discovery of viral lineages that infect members of all three domain suggest that these lineages originated at the earliest stages of biological evolution. Research into these viruses is helping to elucidate the conditions under which life arose, and the dynamics that directed its early development. As well, investigations into viral molecular biology and ecological dynamics have propelled abundant progress in our understanding of living systems, including genetic inheritance, cellular signaling and trafficking, and organismal development. These aspects of viruses have motivated the current investigation into a novel, lysogenic variant of the archaeal virus Sulfolobus Turreted Icosahedral Virus (STIV), STIV3. Following previous work for STIV, a genetic system was developed for STIV3. This involved producing clones for five partitions of the viral genome. As well, an infectious clone was constructed using PCR and the Gibson assembly. Electroporating the infectious clone into pH shocked host cells produced free virions. Subsequent isolation of these virions in cell-free supernatant were applied to a fresh host culture, which, again, produced free virions. Cultures similarly infected with the original virus stock did produce a higher viral titer, but the timing of viral burst was highly coupled between the two. Bioinformatic investigations were also completed on STIV3, its sister variants STIV and STIV2, and metagenomic sequences collected from Yellowstone hot springs. 33 open reading frames were predicted for the 17,106bp STIV3 genome, all of which with homologs in at least one of the two other variants. Overall, the STIV3 genome resembled STIV more than STIV2 according to genetic distance, gene homology, and evolutionary signatures. dN/dS analysis of the turret-forming genes, A224, C384, and A586, revealed positive selection signatures that may be attributed to virus-host coevolution or host range expansion. The putative integrase gene B510 appears to be highly conserved among the STIV variants, particularly at the catalytic residues in the C-terminal. All other homologs exhibited high sequence similarity, indicating strong negative selection across most of the genome. Analyses were also carried out on metagenomic sequences, which found much higher sequence diversity in the environment than among STIV variants, even between STIV3 and STIV2, the latter of which was isolated from a hot spring in Iceland. Even with the small sample sets available, some population structuring was observed with PCA analysis. The work performed in this project provides a strong foundation for future investigations with this virus. A genetic system has been successfully constructed and partially validated. Future work can exploit this system to further our knowledge of virus biology, especially when combined with host molecular tools. As well, the STIV3 genetic system has the potential to further progress in biotechnology and bioengineering, particularly with the confirmed presence of a functioning integrase gene.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Biological Sciences
- Creator:
- Nworji, Kanayochukwu
- Description:
- Greenhouse crop cultivation using hydroponic techniques permits production of high-quality crops under controlled conditions that require less space, water, labor and time to harvest. In Nigeria, use of hydroponic cultivation is hampered by the availability of both capital and male labor in rural areas. This exploratory study set-out to determine whether a hydroponics system could be developed that is suitable to conditions there. A suspended net pot non-circulating method was selected as a proven system that minimized capital and technical requirements. Since the standard size of growing tanks is too large and too heavy to be manipulated by the female labor force in Nigeria, small tank sizes and large tank sizes were used to evaluate the economic viability of small tanks. Lettuce was selected as the crop due to its suitability for hydroponics cultivation and its importance commercially. Two trials were conducted, one during spring and one during winter. Also, the amount of light falling in different locations within the greenhouse was studied. The specific objectives were to determine: whether tank size affects leaf formation, size and shape; how tank location within the greenhouse affected the formation of leaves in both small and large tanks; and the economic feasibility of small tanks, due to expected nutrient temperature differences compared to big tanks. It was found that small production tanks had a significantly greater average fresh weight than the large tanks, in both studies. With regard to tank location, it was found this did not significantly affect the average fresh weight during the winter study but did during the spring study. Finally, with regard to economic feasibility, there was no significant quality or cost detriment in using smaller tanks. Overall, the use of the non-circulating net pot system with small tanks did not negatively impact crop production.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Plant Science
- Creator:
- Kent, Sarah
- Description:
- Gender plays a major role in an organism’s immune response to many pathogens. However, little is known about gender’s effect on Candida albicans (C. albicans) infections. C. albicans is a commensal organism that lives in the mucosa and skin of healthy individuals, however, C. ablicans can become an opportunistic pathogen in immunocompromised individuals. In our laboratory, it has been found that female mice are significantly more resistant to systemic C. albicans infections than male mice. Furthermore, castrated males are as resistant to the infection as female mice and that 5α-dihydroxytestosterone (the stable form of testosterone) supplementation of female mice decreases their ability to resist the yeast infection. Tumor necrosis factor-alpha (TNF-α), primarily secreted by macrophages, was one of the cytokines most affected by the sex hormone treatment. Thus, we suspect sex hormones play an important role in the immune response to C. albicans infection. It is possible that sex hormones (1) alter the growth rate of C. albicans and/or (2) that sex hormones alter immune responses to the yeast. To address (1), C. albicans was treated with different concentrations of β-estradiol (E2) or Testosterone (T), the yeast were plated on SAB agar, incubated at 37C and 24h later the yeast colonies were counted. To address (2), immortalized female (J774A.1) or male (RAW264.7) murine macrophages and Thiogylcollate-induced macrophages (TG-i M) derived from female and male c57BL/6 mice were treated with 1nM E2 or 10nM T for 24h and then challenged with C. albicans for various time points. Cytokines ((TNF-), interleukin-1 (IL-1), IL-6 and IL-10) and inducible nitric oxide synthase mRNA expression was determined via RT-qPCR. The concentration of TNF-, IL-2 and IL-6 protein secretion was quantified via Enzyme-linked Immunosorbent Assay. E2 inhibited TNF- protein secretion from J774A.1 after a 2h and 4h C. albicans challenge, however, E2 stimulated TNF- protein secretion from female derived TG-i M challenged for 24h with the yeast. Interestingly, while E2 had no effect on TNF- mRNA levels in yeast-challenged J774A.1 cells, this sex hormone suppressed TNF- mRNA levels in TG-i M cells. This results suggest that E2’s effects on TNF- mRNA produced from female derived macrophages is post-transcriptional and cell type dependent. E2 reduced IL-1 mRNA levels only in female derived TG-i M cells, but not in J77A.1 cells, supporting our hypothesis that E2’s effects are cell type dependent. E2 suppressed C. albicans-induced TNF- protein secretion from male derived TG-i M, but not from the immortalized male cell line RAW264.7. E2 had no effect on cytokine mRNA levels in male derived TG-i M, but E2 decreased TNF- mRNA levels while increasing IL-10 mRNA levels in RAW264.7 cells challenged with C. albicans for 2h. T did not affect the protein secretion of C. albicans-induced TNF- from female derived macrophages. However, T suppressed TNF- and IL-10 mRNA expression from J774A.1. T had no effect on cytokine mRNA levels in female derived TG-i M cells. T suppressed TNF- protein secretion from RAW264.7 cells, but stimulated the TNF- secretion from male derived TG-i M. These results suggest that E2, and to a lesser extent T, can alter C. albicans-induced cytokine production from macrophages derived from both sexes, and that these sex hormones act in a post-transcriptional manner.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Biological Sciences
- Creator:
- Lachner, Devin
- Description:
- The spacecraft assembly clean rooms where spacecraft are assembled represent a unique microbiological ecosystem. Stringent bioburden reduction such as controlled temperature, humidity, HEPA filtered air circulation, regular decontamination, and gowning procedures are implemented throughout the assembly and testing operations. The microorganisms that survive these cleaning procedures show a unique microbial diversity and are a complex blend of human and soil associated flora that are often spore-forming. This taxonomic characterization study is essential for understanding which microorganisms were present on the surface of the Phoenix Mars Lander after microbial reduction measures. Knowing the bacterial contaminants' identities and whether they form spores helps NASA to predict whether the microorganisms pose a forward contamination risk that could affect future planetary protection policy. A combination of MALDI-TOF MS and 16S rRNA sequencing was used to identify bacterial isolates collected from the Phoenix Mars Lander. There was a total of 637 isolates analyzed using MALDI-TOF MS, approximately 73% (320) of the 438 microorganisms identified to belong to spore-forming genera. For the samples that could not be identified using MALDI-TOF MS method, the 16S rRNA sequencing approach was implemented. Of the 405 isolates identified with 16S rRNA, there were 312 (77%) isolates that were identified as spore-forming genera. After combining the results from these two methods there were 626 isolates identified, with 460 (74%) of the isolates identified as a spore-forming genus. This bias for spore-forming isolates could be due to many of the samples being heat-shocked at 80◦C for 15 minutes when using the NASA standard assay (NSA) for bioburden detection. To better understand the relationship between the sample source and the prevalence of spore-forming bacteria, the unique microbiomes of three different locations on the Phoenix Mars Lander were compared. The three components analyzed were the Phoenix Mars Lander’s Fairing, MECA (Microscopy, Electrochemistry, and Conductivity Analyzer), and Robotic Arm. Since some of the sensitive electronics found on the Phoenix Mars Lander’s MECA and Robotic Arm have different sterilization procedures they could have a different frequency of spore-forming bacteria. A chi-square test of independence was performed to examine the relationship between the sample source and the presence of spore-forming bacteria. The relationship between these variables was found to be significant, and there have been fewer spore-forming isolates identified from the Fairing, MECA, and Robotic Arm, compared to the other locations. Further understanding of these complex interactions can provide insight for future Planetary Protection missions.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Biological Sciences
- Creator:
- Wong, Cynthia
- Description:
- Community gardens are a common land use and social activity in urban agriculture. Although there is plenty of research on the benefits and the necessity of community gardens, there is little research on the barriers that hinder the growth of community gardens. “Barriers” are defined in this paper as obstacles that prevent the development or progression of a community garden. Community gardens are important to urban agriculture and the social needs of a community, so why are we not seeing more gardens in urban communities? What are the challenges that cities and organizations face when trying to establish and maintain community gardens? Are community gardens a priority in urban communities? As we try to look closer into the barriers that hinder the growth of community gardens, semi-structured interviews with city employees (planners, community service managers, community development directors, etc.), garden owners, and non-profit organizations were conducted, transcribed and analyzed to examine which barriers are the most apparent. Interviewees have confirmed barriers that have been researched in other scholarly work such as the lack of land availability (Irvine et al., 1990; Angotti, 2015), funding (Twiss et al., 2003), social impacts (Kamper et al., 2018), NIMBYism (Lejava & Goonan, 2012), and policy and institutional barriers (Mikulec et al., 2013); but after researching cities in San Bernardino County and eastern Los Angeles County, findings suggest that barriers to community gardening are nonprofit challenges such as the lack of partnerships and collaboration, lack of dedication from staff and from volunteers, unstable budgets and staffing challenges, and community organizing barriers. Barriers also include institutional and policy barriers such as the lack of zoning code requirements, lack of government support, and more. Other barriers such as secure land tenure, language, application fees, communication with vacant lot owners, and break-ins are also playing a role in the growth of community gardens. Based on the prevalent barriers, it is recommended that community gardens located in the Inland Empire and in eastern L.A. County need to implement a larger community garden network in order to reach the inner-cities of L.A. County to create more partnerships and collaboration. There needs to be a standard in place to provide clarity of the grant process and make information available on grant availability for nonprofit organizations, including the need for dedicated budgets specifically for health-initiated projects, and community events to raise funds for community gardening. Local governments are also recommended to update their local policies to accommodate place-based initiatives, to prioritize the health of their residents, and to provide a list or directory of vacant lots to applicants who are interested in establishing a garden. It is also important to have a strong coordinating position to help bridge the gap between local organizers and City bureaucracy. It is with the hope that this research sheds light on how support from local governments can relieve most barriers to community gardening. Urban agriculture is a growing phenomenon and some cities have already incorporated these policies into their general plans and zoning codes; but a lot of local smaller cities are still behind. As city planners and researchers, it is important to reveal the challenges that are faced when trying to establish urban agriculture in order to close the gaps and better inform/create policies that are more inclusive to address the needs of the community.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Urban and Regional Planning
- Creator:
- Brannen, Eric
- Description:
- Encroachment at the wildland-urban interface (WUI) is a challenging issue in Southern California. The WUI is the geographic juncture at which the altered and natural environment meet. This connection is at the nexus of urban sprawl, where many issues arise. These issues include the introduction of exotic plants, subsidized species, and pressure on native food supplies. Nevertheless, people are attracted to homes where urban sprawl encroaches on the WUI. The model provides a means of investigation into the relationship between home prices and WUI proximity for the purpose of leveraging policy to combat sprawl. This study investigates whether homes that are closer to the WUI sell for a premium — causing the residential demand curve in the bid rent model to slope upwards at the edges of a metropolis. Specifically, the study areas of Topanga and Santa Clarita in Los Angeles County. A test for the hypothesis is created using a geographically weighted regression (GWR) coupled with hedonic analysis and travel time from trailhead parking lots to homes as a measure of WUI proximity. Results indicate that home prices are positively influenced by WUI proximity, although travel time is negatively influenced beyond a threshold of 17-minutes
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- John T. Lyle Center for Regenerative Studies
- Creator:
- Overton, Michael
- Description:
- Viruses have played a central role in the evolution and ecology of cellular life since it first arose. The discovery of viral lineages that infect members of all three domain suggest that these lineages originated at the earliest stages of biological evolution. Research into these viruses is helping to elucidate the conditions under which life arose, and the dynamics that directed its early development. As well, investigations into viral molecular biology and ecological dynamics have propelled abundant progress in our understanding of living systems, including genetic inheritance, cellular signaling and trafficking, and organismal development. These aspects of viruses have motivated the current investigation into a novel, lysogenic variant of the archaeal virus Sulfolobus Turreted Icosahedral Virus (STIV), STIV3. Following previous work for STIV, a genetic system was developed for STIV3. This involved producing clones for five partitions of the viral genome. As well, an infectious clone was constructed using PCR and the Gibson assembly. Electroporating the infectious clone into pH shocked host cells produced free virions. Subsequent isolation of these virions in cell-free supernatant were applied to a fresh host culture, which, again, produced free virions. Cultures similarly infected with the original virus stock did produce a higher viral titer, but the timing of viral burst was highly coupled between the two. Bioinformatic investigations were also completed on STIV3, its sister variants STIV and STIV2, and metagenomic sequences collected from Yellowstone hot springs. 33 open reading frames were predicted for the 17,106bp STIV3 genome, all of which with homologs in at least one of the two other variants. Overall, the STIV3 genome resembled STIV more than STIV2 according to genetic distance, gene homology, and evolutionary signatures. dN/dS analysis of the turret-forming genes, A224, C384, and A586, revealed positive selection signatures that may be attributed to virus-host coevolution or host range expansion. The putative integrase gene B510 appears to be highly conserved among the STIV variants, particularly at the catalytic residues in the C-terminal. All other homologs exhibited high sequence similarity, indicating strong negative selection across most of the genome. Analyses were also carried out on metagenomic sequences, which found much higher sequence diversity in the environment than among STIV variants, even between STIV3 and STIV2, the latter of which was isolated from a hot spring in Iceland. Even with the small sample sets available, some population structuring was observed with PCA analysis. The work performed in this project provides a strong foundation for future investigations with this virus. A genetic system has been successfully constructed and partially validated. Future work can exploit this system to further our knowledge of virus biology, especially when combined with host molecular tools. As well, the STIV3 genetic system has the potential to further progress in biotechnology and bioengineering, particularly with the confirmed presence of a functioning integrase gene.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Biological Sciences
- Creator:
- Nworji, Kanayochukwu
- Description:
- Greenhouse crop cultivation using hydroponic techniques permits production of high-quality crops under controlled conditions that require less space, water, labor and time to harvest. In Nigeria, use of hydroponic cultivation is hampered by the availability of both capital and male labor in rural areas. This exploratory study set-out to determine whether a hydroponics system could be developed that is suitable to conditions there. A suspended net pot non-circulating method was selected as a proven system that minimized capital and technical requirements. Since the standard size of growing tanks is too large and too heavy to be manipulated by the female labor force in Nigeria, small tank sizes and large tank sizes were used to evaluate the economic viability of small tanks. Lettuce was selected as the crop due to its suitability for hydroponics cultivation and its importance commercially. Two trials were conducted, one during spring and one during winter. Also, the amount of light falling in different locations within the greenhouse was studied. The specific objectives were to determine: whether tank size affects leaf formation, size and shape; how tank location within the greenhouse affected the formation of leaves in both small and large tanks; and the economic feasibility of small tanks, due to expected nutrient temperature differences compared to big tanks. It was found that small production tanks had a significantly greater average fresh weight than the large tanks, in both studies. With regard to tank location, it was found this did not significantly affect the average fresh weight during the winter study but did during the spring study. Finally, with regard to economic feasibility, there was no significant quality or cost detriment in using smaller tanks. Overall, the use of the non-circulating net pot system with small tanks did not negatively impact crop production.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Plant Science
- Creator:
- Lolarga, Jade
- Description:
- Metastasis occurs when cancer cells spread from their primary site to other tissues in the body, which is the main cause of death in most cancer patients. Metastasis is a multistep process and the initial step is the invasion of cancer cells to neighboring tissues. It is therefore important to understand the molecular mechanism underlying the regulation of cell invasion. TWIST1 is a transcription factor that has been previously identified as a key regulator of metastasis. BMI1 is a transcription target of TWIST1 and a subunit of Polycomb Repressive Complex 1 (PRC1) that possesses E3 ubiquitinating ligase activity. By mono-ubiquitinating histone H2A at Lysine 119 (K119), PRC1 can cause gene silencing. Our preliminary data showed that BMI1 works downstream of TWIST1 in promoting breast cancer cell invasion. On the other hand, USP16 is a deubiquitinase and is able to remove the ubiquitin from K119 of histone H2A. Our previous data has shown that Polo-like kinase 1 (PLK1) phosphorylates and activates USP16. We hypothesized that the PLK1-USP16 -mediated deubiquitination of histone H2A could inhibit the invasion of breast cancer cells by antagonizing TWIST1-BMI1-mediated ubiquitination at the same residue (K119) of histone H2A. To study cancer cell invasion regulated by these two molecular pathways, we adopted and optimized a three-dimensional (3D) cell culture system to analyze the invasiveness of cancer cells. This newly developed 3D tumor spheroid invasion assay provides data that more closely represents the in vivo behavior of cancer cells and is therefore rapidly gaining popularity among cancer researchers. In this study, we investigated the invasion of breast cancer cells regulated by TWIST1-BMI1 and PLK1- USP16 pathways in the 3D setting. The results supported our hypothesis that TWIST1-BMI1 pathway promotes cancer cell invasion, whereas PLK1-USP16 pathway inhibits the invasion. Furthermore, we showed that a harmala alkaloid called harmine could induce the degradation of TWIST1 and therefore inhibit breast cancer cell invasion in a dose-dependent manner. These results help us to better understand the regulation of metastasis and may contribute to the development of novel approaches for the treatment of metastatic breast cancer.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Biological Sciences
- Creator:
- Kent, Sarah
- Description:
- Gender plays a major role in an organism’s immune response to many pathogens. However, little is known about gender’s effect on Candida albicans (C. albicans) infections. C. albicans is a commensal organism that lives in the mucosa and skin of healthy individuals, however, C. ablicans can become an opportunistic pathogen in immunocompromised individuals. In our laboratory, it has been found that female mice are significantly more resistant to systemic C. albicans infections than male mice. Furthermore, castrated males are as resistant to the infection as female mice and that 5α-dihydroxytestosterone (the stable form of testosterone) supplementation of female mice decreases their ability to resist the yeast infection. Tumor necrosis factor-alpha (TNF-α), primarily secreted by macrophages, was one of the cytokines most affected by the sex hormone treatment. Thus, we suspect sex hormones play an important role in the immune response to C. albicans infection. It is possible that sex hormones (1) alter the growth rate of C. albicans and/or (2) that sex hormones alter immune responses to the yeast. To address (1), C. albicans was treated with different concentrations of β-estradiol (E2) or Testosterone (T), the yeast were plated on SAB agar, incubated at 37C and 24h later the yeast colonies were counted. To address (2), immortalized female (J774A.1) or male (RAW264.7) murine macrophages and Thiogylcollate-induced macrophages (TG-i M) derived from female and male c57BL/6 mice were treated with 1nM E2 or 10nM T for 24h and then challenged with C. albicans for various time points. Cytokines ((TNF-), interleukin-1 (IL-1), IL-6 and IL-10) and inducible nitric oxide synthase mRNA expression was determined via RT-qPCR. The concentration of TNF-, IL-2 and IL-6 protein secretion was quantified via Enzyme-linked Immunosorbent Assay. E2 inhibited TNF- protein secretion from J774A.1 after a 2h and 4h C. albicans challenge, however, E2 stimulated TNF- protein secretion from female derived TG-i M challenged for 24h with the yeast. Interestingly, while E2 had no effect on TNF- mRNA levels in yeast-challenged J774A.1 cells, this sex hormone suppressed TNF- mRNA levels in TG-i M cells. This results suggest that E2’s effects on TNF- mRNA produced from female derived macrophages is post-transcriptional and cell type dependent. E2 reduced IL-1 mRNA levels only in female derived TG-i M cells, but not in J77A.1 cells, supporting our hypothesis that E2’s effects are cell type dependent. E2 suppressed C. albicans-induced TNF- protein secretion from male derived TG-i M, but not from the immortalized male cell line RAW264.7. E2 had no effect on cytokine mRNA levels in male derived TG-i M, but E2 decreased TNF- mRNA levels while increasing IL-10 mRNA levels in RAW264.7 cells challenged with C. albicans for 2h. T did not affect the protein secretion of C. albicans-induced TNF- from female derived macrophages. However, T suppressed TNF- and IL-10 mRNA expression from J774A.1. T had no effect on cytokine mRNA levels in female derived TG-i M cells. T suppressed TNF- protein secretion from RAW264.7 cells, but stimulated the TNF- secretion from male derived TG-i M. These results suggest that E2, and to a lesser extent T, can alter C. albicans-induced cytokine production from macrophages derived from both sexes, and that these sex hormones act in a post-transcriptional manner.
- Resource Type:
- Thesis
- Campus Tesim:
- Pomona
- Department:
- Department of Biological Sciences