Browsing by Subject "Differentiation"

Now showing 1 - 7 of 7
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    Differentiation and patterning of cells originating in the zebrafish neuroectoderm
    (2013-05) Lund, Caroline E.
    Vertebrate nervous system development requires a complex series of events to transform a flat neuroepithelium into complex structures containing specialized cell types. The anterior neuroectoderm gives rise to the brain and is the origin for some of the first neurons that differentiate. It is also the origin of the cranial neural crest cells that form craniofacial features. This thesis focuses on the embryonic development of two tissues that arise from the zebrafish anterior neuroectoderm, the epithalamus, a region of the dorsal forebrain, and the mandible, the lower jaw. Early in development, the flat neural plate folds into a neural tube. The pineal gland, an organ involved with circadian rhythms, begins as two precursor domains at the lateral edges of the neural plate that converge into a single tissue when the neural tube closes. The pineal gland, along with the parapineal gland and habenula nuclei, form the epithalamus in the dorsal forebrain. In embryos with open neural tubes, the left and the right sides of the pineal and surrounding epithalamus are widely spaced. I found that despite this displacement, pineal cell types differentiate normally and initiate their rhythmic function. Conversely left-right asymmetry in the epithalamus was lost; both sides exhibited left-sided characteristics. Further, this loss of asymmetry in the epithalamus was correlated to severity in neural tube defects. Embryos with left isomerism had significantly wider pineal anlage domains than those with normal or reversed asymmetry. Cranial neural crest cells from the dorsal neural tube migrate to form craniofacial structures, including the cartilaginous precursor to the mandible, Meckel's cartilage. The bigtime (bti) mutant exhibits reductions in mandibular development. I found that although cranial neural crest cells localize normally to the lower jaw region in these mutants, they fail to differentiate into functional chondrocytes that secrete a sufficient amount of collagenous extracellular matrix.
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    Differentiation of human induced pluripotent stem cells to oligodendrocyte progenitor cells
    (2013-01) Subramaniam, Sandhya
    The similarity between induced Pluripotent Stem Cells (iPSCs) and Embryonic Stem (ES) cells motivated the use of the Keirstead protocol in the differentiation of iPSCs to Oligodendrocyte Progenitor Cells (OPCs). The overall concept of the protocol was successful in differentiating the iPSCs to OPCs with modification at each stage to better suit the survival of the aggregates made from iPSCs. The OPCs generated from the iPSCs were primarily confirmed using immunostaining for Olig2 and NG2. The OPCs produced using this protocol, were expanded on matrigel in Glial Restrictive Medium (GRM) supplemented with Epidermal Growth Factor (EGF) and passaged for further expansion.
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    Falcon Centrifugal Concentrator for Gravity Upgrading of Taconite Concentrate
    (University of Minnesota Duluth, 1999-10-08) Wu, Chuying
    The Falcon Centrifugal Concentrator, or Gravitational Concentrator, separates materials based on differentiation of specific gravity in the centrifugal field. The Falcon Concentrator has been successfully applied in recovering ultra-fine gold from tailings. Recent work on recovery of fine hematite in Quebec has shown limited success. This project was funded by the Permanent University Fund, Minnesota Technology Institute (MTI), and USX. The project was funded in two phases, first to study its feasibility; second to investigate its application in place of flotation process that could do away with flotation reagent. The first part of this report contains the initial operation data and analysis, and the second part was to report the tests on pre-classified material, which was parallel to the project of preclassification of flotation feed, also funded by MTI. As presented in Figure 1, Falcon Concentrator contains a rotor assembly which rotate up to 1800 rpm to create 300 G of force. The slurry fed through the tube to the bottom of the rotator where centrifugal force immediately separates solids and water. The solids are pinned to the cone wall forming a compact blanket. Because of the expanding configuration of the cone, the solids move progressively towards the large diameter and ultimately to discharge. During this upward and outward progression, the blanket of solids diminishes in thickness. Light particles are drawn into the pulp stream by the overlying layer of fastmoving water and pass out with the tailings. The heavy particles migrate underneath the layer will discharged by a pinch valve assembly at control interval. These setup allowed continues operation at through put of 30 L TPH with a C 1 0 model.
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    Generating cells for lung tissue engineering
    (2014-05) Turgut, Aylin
    Decellularization of essential organs such as the lung has become an integral part of regenerative medicine. As the availability of donors is very low reseeding of these decellularized organs with a patient's own cells is a potential therapy for those desperately in need. This way, the risks associated with allogeneic immune rejection are avoided. Some research groups have been successful in reseeding the lung with allogeneic differentiated cells. However, the barrier to presently overcome is to seed with stem cells and ensure these cells differentiate to all the desired cell types of the lung. Another obstacle is obtaining the desired number of cells for recellularization of large organs such as the lung. Scale-up methods using stirred vessel bioreactors with conditions similar to the physiological environment are a desirable alternative to conventional cell culture. In this study, I demonstrate large-scale cell culture in stirred flask bioreactors by facing the challenges of scale-up from 2D to 3D suspension culture. I also show the existence of exosomes in decellularized pig and mouse lung and identify the miRNAs (miRNAs) contained within them. MicroRNAs are becoming increasingly popular research tools as they are known to regulate many essential processes. Exosomes are enriched with miRNAs and can be shuttled between cells, thereby affecting target cell behavior. I utilized the exosomes from decellularized lungs in directed differentiation of induced pluripotent stem cells (iPSCs) to the definitive endoderm (DE) lineage and compared it with conventional differentiation methods. The exosomes had a profound effect on the morphology of the cells which will lead to further studies on exosome-directed differentiation procedures.
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    Student Oral Proficiency in Grade Three Spanish Immersion: Linguistic Diversity, Student Interaction and Differentiated Scaffolding
    (2015-07) Young, Amy
    Spanish dual-language immersion programs in the U.S. increasingly work with both English and Spanish home language students who bring a wide variety of linguistic resources. One important approach to adapting to this linguistic diversity in immersion schools may be to differentiate support for student language development during content instruction. This paper explores a collaborative design-based study on the efficacy of instructional practices that afford increased student oral proficiency development through language-focused differentiated instruction. The school site was a grade-three classroom in an urban two-way Spanish immersion school that included students from a variety of home language backgrounds. Students from one classroom (n=24) were assessed for oral proficiency using the Student Oral Proficiency Assessment from the Center for Applied Linguistics. Focal student language production was evaluated with attention to complexity measures at the beginning of and end of the semester-long study. Focal student interactional discourse, as well as teacher and student interviews were analyzed in relation to linguistic diversity and differentiation using constructs from interactional sociolinguistics including authentication, intertextuality and adequation/distinction. Findings suggest that 1) students increased their language complexity as measured by number of different words, mean length of utterance, and subordination. 2) Students appeared to increase their participation in relation to their engagement with the target language structures, their exposure to increasingly complex language, and their developing awareness of how language works. 3) The classroom teacher's perception of linguistic diversity shifted to become less focused on "native speakers" and his conceptualization of "attention to language" changed from a focus on form to an exploration of functional options to facilitate student interaction. Teacher-identified "socially sanctioned niches" became safe spaces for students to engage with language play that reinforced their developing bilingual identities as students negotiated their relationships and made sense of content. Design and implementation of language-focused differentiated instruction is likely to promote linguistic development to the extent that it can afford (or constrain) the local emergence of bilingual identities in connection with increased language awareness, exposure to a wider variety of complex shared texts from which to draw, and access to increased participation within academic learning experiences.
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    A Systems Approach to Studying the Differentiation of Stem Cells Towards Hepatocytes
    (2017-08) Chau, David
    The recent advancements in stem cell biology have allowed for new and exciting opportunities to use stem cells in clinical and industrial applications. Stem cells have the unique ability to self-renew and differentiate into any specialized cell type found in the body. Using certain mechanical and biochemical cues, stem cells can be directed to become any specific cell type, such as hepatocytes. A robust and efficient process for expansion and differentiation to generate large quantities of functional hepatocytes from stem cells will be essential to establishing a stem cell bioprocess in the future for therapeutic and industrial applications of hepatocytes. In this study, a differentiation protocol with soluble growth factors and cytokines was used to mimic the key signaling cues during embryonic development. However, most directed differentiation processes have run into issues with limited scalability and lack of functionality in the differentiated cells. In an effort to bring stem cell therapy closer to reality, our strategy was to use a systems-based approach to enhance the quality and yield of stem cell-derived hepatocytes. To achieve higher cell yield, we modified an existing differentiation protocol to incorporate a cell expansion stage to facilitate simultaneous differentiation and cell growth. Using transcriptome analysis and mass cytometry, we showed how the population of cells changed over time on both the transcript and protein level. Both analyses revealed that with the new expansion stage, we obtained a higher quantity of hepatocytes within the same time frame compared to the conventional method of differentiation. We then showed the capability to scale up our differentiation for larger scale cultures by adapting the expansion stage onto Cytodex 3 microcarriers. Using the same culture volume as a tissue plate culture, we demonstrated the ability to achieve up to a 5-fold increase in cell number with a final cell density in the range of 4-5x106 cells/ml. These strategies show that the demand for large quantities of hepatocytes can be met by translating the conventional method of differentiation to suspension microcarrier differentiation. Encouraged by our ability to yield higher cell density using microcarrier culture, we explored assessing the functional maturity of our stem cell-derived hepatocytes using transcriptome analysis. We showed that stem cell-derived hepatocytes are still clearly different when compared to primary hepatocytes at the transcriptome level. In addition to evaluating cells using transcriptome analysis, we wanted to be able to compare the current in-vitro processes to embryonic liver development to understand the genetic roadblocks. The transcriptome data from hESCs hepatocyte differentiation was integrated with mouse liver development using principal component analysis and batch corrections. This allowed us to create a unified developmental scale to compare samples from different species and in-vitro to in-vivo platforms. The meta-analysis revealed that stem cell-derived hepatocytes are equivalent to the functional maturity of developing cells at E15 in mouse development. From the transcriptome analysis, we observed many different genes in energy metabolism with dynamic behavior over the course of differentiation. We sought to understand the effect of changes in different metabolic genes and the impacts on metabolic transition during differentiation. We characterized the energy metabolism of hESCs and assessed the metabolic demand of cells at different stages of differentiation. hESCs and early differentiated cells exhibited a high glycolytic flux. transitioning towards an oxidative metabolism as the differentiation progressed. Furthermore, using confocal microscopy, we also characterized the activity and morphology of the mitochondria in the cells at different stages of differentiation. Using the consumption rates of different nutrients as an input to our metabolic flux model along with our transcriptome findings, we were able to gain a deeper understanding of the metabolic behavior of cells during differentiation. Our analysis revealed that cells consume lower amounts of glucose over the course of the differentiation but become more efficient at transporting pyruvate into the mitochondria leading to increased oxidative phosphorylation. However, our metabolic and transcriptome data revealed that our stem cell-derived hepatocytes are not capable of mature metabolic functions such as gluconeogenesis, supporting the immature phenotype that has been described in literature. Together, these studies reveal that stem cells can provide a renewable and scalable source of hepatocytes for therapeutic applications. These cells demonstrate some phenotypic and functional properties of primary hepatocytes but have some contrasting elements compared to their in-vivo counterparts that will need further genetic intervention to enhance their maturation before cellular therapy can become a reality. However, this work is invaluable as it contributes to the current status of the field and facilitates the translation of laboratory practices of stem cell culture into a scalable technology.
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    Terminal differentiation of symbiotic rhizobia in certain legume species and its implications for legume-rhizobia coevolution.
    (2010-08) Oono, Ryoko
    The symbiotic association between legume plants (Fabaceae) and nitrogen-fixing rhizobia is a classic system of cooperation, but with largely unexplored differences among species in life history traits. Rhizobia transform physiologically and morphologically into nitrogen-fixing bacteroids inside host nodules. The transformation is terminal (bacteroids are swollen and apparently nonreproductive) in some legume host species but not others, regardless of rhizobial genotype. The phylogenetic distribution of this host trait in the Papilionoideae subfamily of legumes suggests that the common ancestor of the papilionoids did not host terminally differentiated bacteroids and there appear to have been at least five independent origins of hosts imposing terminal differentiation on bacteroids. To consider possible advantages of this host trait, I compared the symbiotic efficiency of terminally and non-terminally differentiated bacteroids of a single rhizobial strain with dual-host capabilities. In the two available dual-host cases, I found greater fixation efficiency (N2 fixation per CO2 respiration) as well as plant return (host biomass) on investment per nodule mass in the hosts with terminal bacteroid differentiation than in those without. This suggests that host traits leading to terminal bacteroid differentiation may have been derived multiple times because of increased net symbiotic benefits to the host. Lastly, I tested whether legumes hosting terminally differentiated bacteroids impose sanctions, i.e. reduce benefits to the undifferentiated reproductive clonemates of less-mutualistic bacteroids in the same nodule. Host sanctions could maintain the evolutionary stability of the symbiosis despite "cheaters" - less-mutualistic rhizobia that potentially benefit from the fixation by other rhizobia sharing the same individual plant host. Legume roots were split so that half of each nodulated root system was exposed to nitrogen-free atmosphere (Ar:O2) to simulate cheating and the other half was in normal air (N2:O2). Rhizobial fitness (rhizobia per nodule) was compared between the two halves. A clear host sanctions effect in peas and alfalfa demonstrated that terminal differentiation of bacteroids does not compromise a legume host's ability to sanction. Differences in rhizobial life history suggest various rhizobial symbiotic traits for cooperation and cheating, perhaps leading to different mechanisms in different legume host species that maintain stability of the mutualism.