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Description
The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and high copy number in the protist genome. Recent studies have revealed the widespread occurrence of intragenomic (intra-individual) polymorphism in many protists, an understudied phenomenon which contradicts the assumed homogeneity of the 18S throughout an individual genome. This thesis quantifies and analyzes the level of intragenomic and intraspecific 18S sequence variability in three Trichonympha species (T. campanula, T. collaris, T. postcylindrica) from Zootermopsis termites. Single-cell DNA extractions, PCR, cloning, and sequencing were performed to obtain 18S rRNA sequence reads, which were then analyzed to determine levels of sequence divergence among individuals and among species. Intragenomic variability was encountered in all three species. However, excluding singleton mutations, sequence divergence was less than 1% in 53 of the 56 compared individuals. T. collaris exhibited the most substantial intragenomic variability, with sequence divergence ranging from 0 to 3.4%. Further studies with more clones per cell are needed to elucidate the true extent of intragenomic variability in Trichonympha.
ContributorsBobbett, Bradley (Author) / Gile, Gillian (Thesis director) / Liebig, Juergen (Committee member) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Adaptation of Camponotus floridanus’ Cuticular Hydrocarbon Profile under High Temperature Conditions
Description
Insects are small creatures highly susceptible to water loss. A major factor in the prevention of water loss through an insect’s cuticle are their cuticular hydrocarbons (CHC), a lipid layer consisting mostly of long-chain hydrocarbons. CHCs consist of different molecules called alkanes, alkenes, and methyl branched hydrocarbons which all have varying levels of hydrophobicity. Ants are a massively abundant family of insects with important roles in the ecosystem that also utilize CHCs. Camponotus floridanus isare athe native ant species of the Florida Keys which areis known to have variable environmental temperature. Being exposed to temperatures as high as 35 °C, these ants are expected to have mechanisms that allow them to adapt to their environment. It was hypothesized that CHCs may change in concentration or composition as a means to combat the changes in cuticular permeability due to the variable temperatures that the ants experience. We therefore used C. floridanus worker ants to learn more about CHC plasticity in insects when exposed to elevated temperatures. We found four CHC componentspeaks that showed a statistically significant increase in concentration when comparing the control to treatment colonies: 3,7 dimethyl C31, an underdetermined methyl branched C31, 3,7,11 trimethyl C31, and an undetermined tetramethylbranched C31. These significant changes in concentration occurred on longer chain hydrocarbons. Under further examination, it was found that there was a strong positive correlation between elution time and the differences in medians of peak area between control and treatment colonies. This shows that there was a shift in the CHC profile resulting in an increased concentration of longer chained methyl-branched hydrocarbons. It also suggests that branched hydrocarbons also play some role in the water proofing mechanism of C. floridanus.
ContributorsOn, Thomas (Co-author) / On, Tyler (Co-author) / Liebig, Juergen (Thesis director) / Harrison, Jon (Committee member) / Murdock, Tyler (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
When ants encounter liquid food, they have two options of transporting that food to their nests. The first is the social bucket method in which liquid is carried in the mandibles of the workers back to the nest. The second is trophallaxis in which liquid is imbibed and then transported inside the ant back to the nest. The liquid is then regurgitated to fellow nestmates. Ectatomma have been observed using the social bucket method of transport and were considered members of the Ponerine family. However, a new phylogeny created by Borowiec and Rabeling places Ectatomma near to Formecinae and Myrmicinae, both know for practicing trophallaxis. This seems to suggest either Ectatomma is able to utilize trophallaxis as well or that the evolutionary practice of trophallaxis is more plastic than previously believed. The ability of Ectatomma ruidum to utilize trophallaxis was examined in two experiments. The first experiment examined E. ruidum’s ability to practice worker to worker trophallaxis and the second examined E. ruidum’s ability to perform worker to larva trophallaxis. The results of both experiments indicated that E. ruidum cannot utilize trophallaxis but the larva of E. ruidum may be able to regurgitate to the workers. These results in turn seem to suggest that trophallaxis is a bit more plastic than originally thought.
ContributorsCunningham, Cassius Alexander (Author) / Pratt, Stephen (Thesis director) / Liebig, Juergen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cell viability is an important assessment in cell culture to characterize the health of the cell population and confirm if cells are alive. Morphology or end-line assays are used to determine cell viability of entire populations. Intracellular pO2 levels is indicative of cell health and metabolism that can be used as a factor to asses cell viability in an in-line assay. Siloxane based pO2 sensing nanoprobes present a modality to visualize intracellular pO2. Using fluorescent lifetime imaging microscopy (FLIM), pO2 levels can be mapped intracellular as a highly functional in-line assay for cell viability. FLIM is an imaging modality that reconstructs an image based of its fluorescent lifetime. Nanoprobes were synthesized in different manufacturing/storage conditions. The nanoprobes for both long- and short-term storage were characterized in a cell free environment testing for changes in fluorescent intensity, average and maximum nanoprobe diameter. The nanoprobes were validated in two different culture systems, 2D and microcarrier culture systems, for human derived neural progenitor cells (NPCs) and neurons. Long- and short-term storage nanoprobes were used to label different neuronal based culture systems to asses labeling efficiency through fluorescent microscopy and flow cytometry. NPCs and neurons in each culture system was tested to see if nanoprobe labeling effected cellular phenotype for traits such as: cell proliferation, gene expression, and calcium imaging. Long-term and short-term storage nanoprobes were successfully validated for both NPCs and neurons in all culture systems. Assessments of the pO2 sensing nanoprobes will be further developed to create a highly functional and efficient in-line test for cell viability.
ContributorsLeyasi, Salma (Author) / Brafman, David (Thesis director) / Kodibagkar, Vikram (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Callithrix penicillata, also known as the Black-tufted marmoset primarily lives in the Brazilian highlands and has had little research conducted on it. For this project I performed a genome curation on the newly assembled genome of this species. The scaffolds obtained by the Dovetail Genomics reads were organized and labeled into chromosomes using the 2014 Callithrix jacchus genome as a reference. Then, using that same genome as a reference, 13 of the chromosomes were reverse complimented to be continuous with the 2014 Callithrix jacchus genome. The N50 statistics of the assembly were calculated and found to be 124 Mb. Quality scores were run for the final genome using referee and visualized with a bar plot, with 99% of sites scoring above 0. Heterozygosity was also calculated and found to be 0.3%. Finally, the final version of the genome was visually compared to the 2017 Callithrix jacchus genome and the GRCh38 human genome. This genome was submitted to the NCBIs database to await further approval.
ContributorsJohnson, Joelle Genevieve (Author) / Cartwright, Reed (Thesis director) / Stone, Anne (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
Over 5.8 million people are currently living with Alzheimer’s disease (AD), with the sixth highest mortality rate in the United States. No known cure or substantially life-extending treatment exists. With the growing aging population these numbers are only expected to increase to about 13.8 million by the year 2050. Alzheimer’s is a multifactorial disease, giving rise to two main types: familial AD (FAD) and sporadic AD (SAD). Although there are different factors associated with each type of the disease, both FAD and SAD result in neuronal and synaptic loss and remain difficult to model in-vitro and treat overall.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.
ContributorsFrisch, Carlye Arin (Author) / Brafman, David (Thesis director) / Tian, Xiaojun (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Effectively modeling Alzheimer’s disease will lend to a more comprehensive
understanding of the disease pathology, more efficacious drug development and
regenerative medicine as a form of treatment. There are limitations with current
transgenic mouse models of Alzheimer’s disease and the study of post mortem brain tissue of Alzheimer’s diseases patients. Stem cell models can overcome the lack of clinical relevance and impracticality associated with current models. Ideally, the use of stem cell models provides the foundation to study the biochemical and physiological aspects of Alzheimer’s disease, but at the cellular level. Moreover, the future of drug development and disease modeling can be improved by developing a reproducible and well-characterized model of AD that can be scaled up to meet requirements for basic and translational applications. Characterization and analysis of a heterogenic neuronal culture developed from induced pluripotent stem cells calls for the understanding of single cell identity and cell viability. A method to analyze RNA following intracellular sorting was developed in order to analyze single cell identity of a heterogenic population
of human induced pluripotent stem cells and neural progenitor cells. The population was intracellularly stained and sorted for Oct4. RNA was isolated and analyzed with qPCR, which demonstrated expected expression profiles for Oct4+ and Oct4- cells. In addition, a protocol to label cells with pO2 sensing nanoprobes was developed to assess cell viability. Non-destructive nanoprobe up-take by neural progenitor cells was assessed with fluorescent imaging and flow cytometry. Nanoprobe labeled neurons were cultured long-term and continued to fluoresce at day 28. The proof of concept experiments demonstrated will be further expanded upon and utilized in developing a more clinically relevant and cost-effective model of Alzheimer’s disease with downstream applications
in drug development and regenerative medicine.
understanding of the disease pathology, more efficacious drug development and
regenerative medicine as a form of treatment. There are limitations with current
transgenic mouse models of Alzheimer’s disease and the study of post mortem brain tissue of Alzheimer’s diseases patients. Stem cell models can overcome the lack of clinical relevance and impracticality associated with current models. Ideally, the use of stem cell models provides the foundation to study the biochemical and physiological aspects of Alzheimer’s disease, but at the cellular level. Moreover, the future of drug development and disease modeling can be improved by developing a reproducible and well-characterized model of AD that can be scaled up to meet requirements for basic and translational applications. Characterization and analysis of a heterogenic neuronal culture developed from induced pluripotent stem cells calls for the understanding of single cell identity and cell viability. A method to analyze RNA following intracellular sorting was developed in order to analyze single cell identity of a heterogenic population
of human induced pluripotent stem cells and neural progenitor cells. The population was intracellularly stained and sorted for Oct4. RNA was isolated and analyzed with qPCR, which demonstrated expected expression profiles for Oct4+ and Oct4- cells. In addition, a protocol to label cells with pO2 sensing nanoprobes was developed to assess cell viability. Non-destructive nanoprobe up-take by neural progenitor cells was assessed with fluorescent imaging and flow cytometry. Nanoprobe labeled neurons were cultured long-term and continued to fluoresce at day 28. The proof of concept experiments demonstrated will be further expanded upon and utilized in developing a more clinically relevant and cost-effective model of Alzheimer’s disease with downstream applications
in drug development and regenerative medicine.
ContributorsKnittel, Jacob James (Author) / Brafman, David (Thesis director) / Salvatore, Oddo (Committee member) / School of Life Sciences (Contributor) / Dean, W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
An intimate view of the unique architecture of Harpegnathos saltwater nest using aluminum nest casts
Description
Abstract:
Given the incredible variety in ant nest architecture, this experiment sought to evaluate how the nest architecture of Harpegnathos saltator differs from other species’ nests. To achieve the ability to evaluate the structure of H. saltator nest, we created experimental colonies varying in size from 20, 40, 60, 80 workers of Harpegnathos saltator in five-gallon buckets of sand and then allowing the colonies to grow for four months and twelve days. To create the nest casts, we developed a charcoal kiln out of a galvanized trash can and used a ceramic crucible to hold the aluminum being melted. Using molten aluminum to create nest casts of each colony produced, we obtained three poorly developed nests and one decent nest. The decent nest cast, the 80 worker H. saltator nest, was lacking key features of H. saltator nests that have been excavated in the field. However, they do share many of the same structures such as the shaping of the chambers. The ability of the experimental colonies to excavate the soil provided in the buckets to them was likely halted by poor penetration of water into superficial layers of the soil, thus making the soil too difficult to excavate and form the structures that are key elements of the species nest architecture. Despite these key challenges which the colonies faced, the 80-worker colony showed extensive vertical development and did display features associated with natural H. saltator colonies. Thus, given the display of some key features associated with characteristics of the H. saltator nests excavated in the field, it can be said that with some modification to technique that this is a viable avenue for future study of nest architecture and colony structure.
Given the incredible variety in ant nest architecture, this experiment sought to evaluate how the nest architecture of Harpegnathos saltator differs from other species’ nests. To achieve the ability to evaluate the structure of H. saltator nest, we created experimental colonies varying in size from 20, 40, 60, 80 workers of Harpegnathos saltator in five-gallon buckets of sand and then allowing the colonies to grow for four months and twelve days. To create the nest casts, we developed a charcoal kiln out of a galvanized trash can and used a ceramic crucible to hold the aluminum being melted. Using molten aluminum to create nest casts of each colony produced, we obtained three poorly developed nests and one decent nest. The decent nest cast, the 80 worker H. saltator nest, was lacking key features of H. saltator nests that have been excavated in the field. However, they do share many of the same structures such as the shaping of the chambers. The ability of the experimental colonies to excavate the soil provided in the buckets to them was likely halted by poor penetration of water into superficial layers of the soil, thus making the soil too difficult to excavate and form the structures that are key elements of the species nest architecture. Despite these key challenges which the colonies faced, the 80-worker colony showed extensive vertical development and did display features associated with natural H. saltator colonies. Thus, given the display of some key features associated with characteristics of the H. saltator nests excavated in the field, it can be said that with some modification to technique that this is a viable avenue for future study of nest architecture and colony structure.
ContributorsAnderson, Clayton Edward (Author) / Liebig, Juergen (Thesis director) / Pratt, Stephen (Committee member) / School of Politics and Global Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
ContributorsLoveday, Tristan Andre (Author) / Wang, Xiao (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative brain disease that results from repetitive brain trauma causing brain structure, personality, behavioral, and cognitive changes. CTE is currently undiagnosable and untreatable in living patients. This thesis investigates research surrounding CTE and presents a comparative discussion of the advantages and disadvantages of current diagnostic methods used for other neurodegenerative diseases that may be useful for the diagnosis of CTE.
ContributorsBlair, Sierra (Co-author) / Blair, Taylor (Co-author) / Brafman, David (Thesis director) / Stabenfeldt, Sarah (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05