Matching Items (98)
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
Spaceflight and spaceflight analogue culture enhance the virulence and pathogenesis-related stress resistance of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). This is an alarming finding as it suggests that astronauts may have an increased risk of infection during spaceflight. This risk is further exacerbated as multiple studies indicate that spaceflight negatively impacts aspects of the immune system. In order to ensure astronaut safety during long term missions, it is important to study the phenotypic effects of the microgravity environment on a range of medically important microbial pathogens that might be encountered by the crew. This ground-based study uses the NASA-engineered Rotating Wall Vessel (RWV) bioreactor as a spaceflight analogue culture system to grow bacteria under low fluid shear forces relative to those encountered in microgravity, and interestingly, in the intestinal tract during infection. The culture environment in the RWV is commonly referred to as low shear modeled microgravity (LSMMG). In this study, we characterized the stationary phase stress response of the enteric pathogen, Salmonella enterica serovar Enteritidis (S. Enteritidis), to LSMMG culture. We showed that LSMMG enhanced the resistance of stationary phase cultures of S. Enteritidis to acid and thermal stressors, which differed from the LSSMG stationary phase response of the closely related pathovar, S. Typhimurium. Interestingly, LSMMG increased the ability of both S. Enteritidis and S. Typhimurium to adhere to, invade into, and survive within an in vitro 3-D intestinal co-culture model containing immune cells. Our results indicate that LSMMG regulates pathogenesis-related characteristics of S. Enteritidis in ways that may present an increased health risk to astronauts during spaceflight missions.
ContributorsKoroli, Sara (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, C. Mark (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (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
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
Description
The International Space Station (ISS) utilizes recycled water for consumption, cleaning and air humidity control. The Environmental Control and Life Support Systems (ECLSS) have been rigorously tested at the NASA Johnson Space Center. Despite the advanced engineering of the water recovery system, bacterial biofilms have been recovered from this potable water source. Microbial contamination of potable water poses a potential threat to crew members onboard the ISS. Because astronauts have been found to have compromised immune systems, bacterial strains that would not typically be considered a danger must be carefully studied to better understand the mechanisms enabling their survival, including polymicrobial interactions. The need for a more thorough understanding of the effect of spaceflight environment on polymicrobial interactions and potential impact on crew health and vehicle integrity is heightened since 1) several potential pathogens have been isolated from the ISS potable water system, 2) spaceflight has been shown to induce unexpected alterations in microbial responses, and 3) emergent phenotypes are often observed when multiple bacterial species are co- cultured together, as compared to pure cultures of single species. In order to address these concerns, suitable growth media are required that will not only support the isolation of these microbes but also the ability to distinguish between them when grown as mixed cultures. In this study, selective and/or differential media were developed for bacterial isolates collected from the ISS potable water supply. In addition to facilitating discrimination between bacteria, the ideal media for each strain was intended to have a 100% recovery rate compared to traditional R2A media. Antibiotic and reagent susceptibility and resistance tests were conducted for the purpose of developing each individual medium. To study a wide range of targets, 12 antibiotics were selected from seven major classes, including penicillin, cephalosporins, fluoroquinolones, aminoglycosides, glycopeptides/lipoglycopeptides, macrolides/lincosamides/streptogramins, tetracyclines, in addition to seven unclassified antibiotics and three reagents. Once developed, medium efficacy was determined by means of growth curve experiments. The development of these media is a critical step for further research into the mechanisms utilized by these strains to survive the harsh conditions of the ISS water system. Furthermore, with an understanding of the complex nature of these polymicrobial communities, specific contamination targeting and control can be conducted to reduce the risk to crew members. Understanding these microbial species and their susceptibilities has potential application for future NASA human explorations, including those to Mars.
ContributorsKing, Olivia Grace (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Sustainability (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
Blood donations today undergo extensive screening for transfusion transmitted infections (TTI) since the discovery of the first infectious agent in the early 1900s. Nucleic Acid Testing (NAT) is a serological test used widely in disease detection. NAT is known to rapidly and effectively detect pathogenic genomic material in blood by reducing the "window period" of infection. However, NAT produces false negative results for disease positive samples posing a risk of disease transmission. Therefore, NAT is used in conjunction with the Enzyme-Linked Immunosorbent Assay (ELISA) to mitigate these risks. However, the ELISA assay also poses the same risk as NAT. This study proposes immunosignaturing as an alternative serological test that may combat this risk and investigates whether it would be more effective than other standardized serological tests in disease detection. Immunosignaturing detects antibodies by utilizing a microarray of randomized peptide sequences. Immunosignaturing provides information about an individual's immune health from the pattern of reactivity of antibody-peptide binding. Unlike ELISA and NAT, immunosignaturing can be programmed to detect any disease and detect multiple diseases simultaneously. Using ELISA, NAT, and immunosignaturing, immune profiles of asymptomatic patients were constructed for 10 different classes of blood borne diseases. A pattern of infection was identified for each disease and the sensitivity and specificity of these assays were assessed relative to each other. Results indicate that immunosignaturing can be a viable diagnostic tool in blood testing. Immunosignatures demonstrated increased sensitivity and specificity compared to ELISA and NAT in discerning disease positive and negative samples within and across different classes of disease.
ContributorsSharma, Megumi (Author) / McFadden, Grant (Thesis director) / Nickerson, Cheryl (Committee member) / Green, Alex (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Alzheimer’s Disease (AD) affects over 5 million individuals in the U.S. and has a direct cost estimated in excess of $200 billion per year. Broadly speaking, there are two forms of AD—early-onset, familial AD (FAD) and late-onset-sporadic AD (SAD). Animal models of AD, which rely on the overexpression of FAD-related mutations, have provided important insights into the disease. However, these models do not display important disease-related pathologies and have been limited in their ability to model the complex genetics associated with SAD.
Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.
It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.
It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
ContributorsBounds, Lexi Rose (Author) / Brafman, David (Thesis director) / Wang, Xiao (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Genetic manipulation of human cell lines has widespread applications in biomedical research ranging from disease modeling to therapeutic development. Human cells are generally difficult to genetically engineer, but exogenous nucleic acids can be expressed by viral, chemical, or nonchemical means. Chemical transfections are simpler in practice than both viral and nonchemical delivery of genetic material, but often suffer from cytotoxicity and low efficiency. Novel aminoglycoside antibiotic-derived lipopolymers have been synthesized to mediate transgene delivery to human cells. These polymers are comprised of either paromomycin or apramycin crosslinked with glycerol diglycidylether and derivatized with stearoyl chloride in varying molar ratios. In this work, three previously identified target lipopolymers were screened against a library of human embryonic and induced pluripotent stem cell lines. Cells were transfected with a plasmid encoding green fluorescent protein (GFP) and expression was quantified with flow cytometry 48 hours after transfection. Transfection efficiency was evaluated between three distinct lipopolymers and four lipopolymer:DNA mass ratios. GFP expression was compared to that of cells transfected with commercially available chemical gene delivery reagent controls\u2014JetPEI, Lipofectamine, and Fugene\u2014at their recommended reagent:DNA ratios. Improved transgene expression in stem cell lines allows for improved research methods. Human stem cell-derived neurons that have been genetically manipulated to express phenotypic characteristics of aging can be utilized to model neurodegenerative diseases, elucidating information about these diseases that would be inaccessible in unmanipulated tissue.
ContributorsMehta, Frea (Author) / Brafman, David (Thesis director) / Rege, Kaushal (Committee member) / Chemical Engineering Program (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05