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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
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
The purpose of this thesis creative project was to create an educational video to present research findings on the increasingly important issue of human biospecimen preanalytic variables. When a human biospecimen, such as blood, urine, or tissue, is removed from the body, it is subjected to a plethora of variables that are not recorded or regulated in a vast majority of cases. Frequently, these samples arrive at the research or pathology lab with an unknown history, then undergo analysis for translational research purposes, or to guide clinical management decisions. Thus, compromised specimen quality caused by preanalytic variables has substantial, and potentially devastating, downstream effects. To identify the preanalytic variables with the greatest impact on blood and tissue specimen quality, 45 articles were gathered using PubMed and Google Scholar databases and cited. Based on the articles, the top five variables with the most detrimental effects were identified for both blood and tissue samples. Multiple sets of parameters ensuring specimen fitness were compared for each of the five variables for each specimen type. Then, specific parameters guaranteeing the fitness of the greatest number of analytes were verified. To present the research findings in greater detail, a paper was written that focused on identifying the top variables and key parameters to ensure analyte fitness. To present the overall issue in an easy-to-digest format, a storyboard and script were created as a guideline for a final video project. Ultimately, two alternate versions of the video were created to pertain to the audience of choice (one version for patients, one version for professionals). It is the hope that these videos will be used as educational tools to continue efforts to standardize and enforce human biospecimen preanalytic variable parameters. This is a necessary step to improve the accuracy of our biomedical research data and the healthcare of patients worldwide.
ContributorsAzcarate, Heather (Author) / Compton, Carolyn (Thesis director) / LaBaer, Joshua (Committee member) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor)
Created2018-12
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
Description
Cleavage and polyadenylation is a step in mRNA processing in which the 3’UTR is cleaved and a polyA tail is added to create a final mature transcript. This process relies on RNA sequence elements that guide a large multimeric protein complex named the Cleavage and Polyadenylation Complex to dock on the 3’UTR and execute the cleavage reaction. Interactions of the complex with the RNA and specific dynamics of complex recruitment and formation still remain largely uncharacterized. In our lab we have identified an Adenosine residue as the nucleotide most often present at the cleavage site, although it is unclear whether this specific element is a required instructor of cleavage and polyadenylation. To address whether the Adenosine residue is necessary and sufficient for the cleavage and polyadenylation reaction, we mutated this nucleotide at the cleavage site in three C. elegans protein coding genes, forcing the expression of these wt and mutant 3’UTRs, and studied how the cleavage and polyadenylation machinery process these genes in vivo. We found that interrupting the wt sequence elements found at the cleavage site interferes with the cleavage and polyadenylation reaction, suggesting that the sequence close to the end of the transcript plays a role in modulating the site of the RNA cleavage. This activity is also gene-specific. Genes such as ges-1 showed little disruption in the cleavage of the transcript, with similar location occurring in both the wt and mutant 3’UTRs. On the other hand, mutation of the cleavage site in genes such as Y106G6H.9 caused the activation of new cryptic cleavage sites within the transcript. Taken together, my experiments suggest that the sequence elements at the cleavage site somehow participate in the reaction to guide the cleavage reaction to occur at an exact site. This work will help to better understand the mechanisms of transcription termination in vivo and will push forward research aimed to study post-transcriptional gene regulation in eukaryotes.
ContributorsSteber, Hannah Suzanne (Author) / Mangone, Marco (Thesis director) / Harris, Robin (Committee member) / LaBaer, Joshua (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Complex animal societies consist of a plethora of interactions between members. To successfully thrive they must be able to recognize members and their kin, and to understand how they do this we need sufficient and reliable methods of testing. Eusocial insects are especially good at recognizing their nestmates, but the exact mechanism or how well they can discriminate is unknown. Ants achieve nestmate recognition by identifying varying proportions of cuticular hydrocarbons. Previous studies have shown ants can be trained to discriminate between pairs of hydrocarbons. This study aims to compare two methodologies previously shown to demonstrate odor learning to identify which one is the most promising to use for future odor learning experiments. The two methods tested were adapted from Sharma et al. (2015) and Guerrieri and d’Ettorre (2010). The results showed that the Guerrieri method demonstrated learning better and was more reliable and faster than the Sharma method. The Guerrieri method should be used in future experiments regarding odor learning discrimination
ContributorsDavis, Cole (Author) / Liebig, Juergen (Thesis director) / Stephen, Pratt (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
For colonies of ponerine ant species, sterility regulation after a founding queen's death is not totally achieved in the worker caste, and the possibility of sexual reproduction is opened to workers. The persisting survival of these colonies is dependent on capturing the optimal reproductive ratio; yet, an informational gap bounds the mechanisms detailing the selection of new reproductives and the suppression of ovarian development in rejected reproductives. We investigated the mechanisms of worker policing, one of the primary methods of ovarian suppression, through continuous video observation for a period of five days at the start of colony instability. Observations suggest policing in H. saltator is performed by a majority of a colony, including potential reproductives, and requires multiple events to fully discourage ovarian growth.
ContributorsChien, Jeffrey (Co-author) / Barat Ali, Fatima (Co-author) / Kang, Yun (Thesis director) / Liebig, Juergen (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12