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1
Menckeberg, Celia Lara. "Identifying lineage relationships in human T cell populations." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/3211/.
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CD4\(^+\) and CD8\(^+\) T cell populations can be divided into subpopulations based on expression of surface markers CCR7 and CD45RA. The resulting populations are referred to as naive, central memory, effector memory and effector memory RA\(^+\) (EMRA). The aim of this study was to identify potential lineage relationships between these subpopulations for both CD4\(^+\) and CD8\(^+\) T cells through microarray analysis. The genes found to distinguish between these subpopulations include many molecules with known functions in T cell differentiation, including CCR7, CD45RA, granzymes, L-selectin and TNF receptors. Several genes from the tetraspanin family of proteins were found to be differentially expressed at mRNA and protein level; suggesting a possible role for these genes in CD4\(^+\) and CD8\(^+\) T cell activation, migration and lysosomal function. Other genes identified, such as LRRN3 and CXCR5 which were expressed highest on naive and CM T cells respectively, provide interesting gene targets to follow up on their function in these T cell populations. Microarray data was validated through Real Time PCR and suggests that both CD4\(^+\) and CD8\(^+\) T cells differentiate along a linear pathway of naive to central memory to effector memory. The transcriptional programmes responsible for these differentiation steps were distinct between CD4\(^+\) and CD8\(^+\) T cells, although additional elements were common to both subsets.
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Minton, Elizabeth Jane. "Infection of the monocytic cell lineage by human cytomegalovirus." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315286.
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Perrett, Rebecca Mary. "The human germ cell lineage : pluripotency, tumourigenesis and proliferation." Thesis, University of Southampton, 2008. https://eprints.soton.ac.uk/66010/.
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Na, Erqian. "Lineage- and stage-specific gene expression in human hemopoietic cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0007/MQ28779.pdf.
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Murad, Nadia Yousif. "Differentiation of human embryonic stem cells to the pancreatic lineage." Thesis, University of Sheffield, 2008. http://etheses.whiterose.ac.uk/6102/.
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Human embryonic stem (hES) cells have great therapeutic potential for the treatment of degenerative conditions such as Parkinson's disease, cardiac failure and type I diabetes. This potential is based on the ability of hES cells in vitro to self-renew and also differentiate to cells of all three germ layers; ectoderm, mesoderm and endoderm. Type I diabetes is due to an autoimmune disease destroying the insulin-secreting cells of the pancreas (β-cells) that regulate plasma glucose concentration. The pancreas develops from the endoderm lineage. 2. To find a cure for type I diabetes based on the use of hES, it is essential to understand the differentiation process of ES cells into the endodermal, β-cell lineage. The aim of this study was to investigate the generation of insulin-secreting cells using hES cells in vitro and to compare sue with those in the developing pancreas of the foetus.
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Rinaldi, Federica. "Connexin 43 influences lineage commitment of human neural progenitor cells." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556745.
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Gap junctions (GJs) are intercellular channels connecting the cytoplasm of adjacent cells. This type of connection is an efficient way of cellular communications in many tissues including the central nervous system. Connexins are the proteins that constitute mammalian GJs, and Connexin43 (Cx43) is the most abundant isoform expressed in body cells. Cx43 has been detected within immature neural populations, but only in astrocytes in the adult brain and investigations have shown that Cx43 channel and adhesive properties largely influence neuronal differentiation of mouse neural progenitor (NP) cells. To date the role of Cx43 in neuronal differentiation remains unexplored in human systems, hence our study aimed to investigate the Cx43 participation in human NP differentiation. We largely detected Cx43 protein within the immature neural populations showing that protein expression occurred by fibroblast growth factor (FGF _2) stimulation through the ERKlj2 pathway; FGF _2 withdrawal induced NP differentiation and a progressive loss in Cx43 expression. Cx43's role in neuronal differentiation was explored by cloning lentiviral vectors (LV) coding for Cx43 or anti-Cx43 shRNA constructs and the protein knockdown resulted in an increase in neurons and a decrease in astrocytes, suggesting a role for Cx43 in human stem cell differentiation and neuronal fate. GJs mediate intercellular communications of several mouse and human embryonic stem (ES) cell lines; in our investigation we also showed the presence of functional GJ channels in human ES cells, as well as Cx43 protein expression. Manipulation of ES cells was attempted using LVs and results indicated that the CMV promoter in ES cells is largely inactive. In summary I demonstrated the active role of Cx43 in mechanisms that govern neurogenesis and differentiation of neural progenitor cells, furthermore we highlighted the importance of the internal promoter in LV constructs for genetic manipulation of embryonic ES cells.
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Gsour, Amna. "Differentiation of human cell line towards a pancreatic endocrine lineage." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/differentiation-of-human-cell-line-towards-a-pancreatic-endocrine-lineage(0c2c21fe-724d-449f-804c-02741c89828c).html.
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Islet transplantations have been successful in restoring glucose homeostasis in patients with diabetes; however, the limited number of donor organs limits the success of this treatment. The lineage reprograming of different cell sources to beta cells potentially provides an unlimited supply of insulin-producing cells for regenerative therapy for patients with diabetes. The aim of this study was to investigate the ability to transdifferentiate two cell lines into an endocrine lineage. Insulin production in pancreatic beta cells can be increased using a small molecule, 3,5-disubstituted isoxazole, N-cyclopropyl-t-(thiophen-2-yl)isoxazole-3-carboxamide (isoxazole) but its effect on other cell types has not been reported. Here, we investigated the lineage reprogramming of PANC-1 pancreatic ductal cells to insulin producing cells by isoxazole treatment. Gene expression was performed using RT-PCR and qPCR for approximately 30 genes critical to beta cell development and function. In addition, quantitative proteomic profiling was performed using LC-MS by monitoring protein abundance in isoxazole-treated PANC-1 cells compared to time-matched controls. Isoxazole treatment stimulated PANC-1 cells to aggregate into islet-like clusters and gene expression analysis revealed induction of important developmental beta cell markers including NGN3, NEUROD1 and INSULIN. In addition, beta cell surface markers were also upregulated such as CD200, GPR50, TROP-2, GLUT2 and SLC30A8. Using LC-MS a catalogue of approximately 2400 identified proteins was generated; 257 proteins were differentially expressed in isoxazole-treated cells compared to DMSO-vehicle controls at p < 0.05. Amongst the proteins upregulated were molecules that regulate metabolic processes and cytoskeletal reorganisation. The expression of the majority of these proteins has not been previously reported or studied in the context of beta cell differentiation. Functional analysis of the relative protein changes was determined using Ingenuity Pathway Analysis, IPA, and gene ontology, GO, software, which revealed the regulation of several cellular canonical pathways including metabolic pathways, cell adhesion, remodelling of epithelial adherens junctions and actin cytoskeleton signalling. The effects of isoxazole were further studied in the A549 lung cancer cell line. Similar effects were observed, such as the induction of pro-endocrine markers NGN3 and NEUROD1 and endocrine-specific hormones INS and GCG. These results indicate that isoxazole has the capacity to transdifferentiate pancreatic and non-pancreatic cell origins into an endocrine lineage. This study reveals the powerful induction capacity of isoxazole in inducing cellular reprogramming events.
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Almuraikhi, Nihal. "Direct differentiation of human iPS cells towards the erythroid lineage." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/45641.
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Pluripotent stem cells including induced pluripotent stem (iPS) cells and embryonic stem (ES) cells are known for their distinctive property of indefinite self-renewal in an undifferentiated status, with the potential to differentiate into all types of cells. Current protocols used in the differentiation of human iPS cells and human ES cells towards erythropoiesis utilize two main approaches: (1) embryoid body (EB) formation, which influences heterogeneity of the produced population, and/or (2) co-culture with mouse stromal cells, where obstacles of purification of the cells rise, which makes the xeno-free culture requirement difficult to achieve, in addition to the cytokine supplements. Moreover, these protocols reported low efficiency in number and functionality, especially with human iPS cells, and required long culture times. One of the major challenges in erythroid cell production from human ES/iPS cells is achieving full maturation and enucleation of erythroid cells in serum-free and feeder-free condition, in order to ensure a completely xeno-free culture condition suitable for clinical applications. In this study, we have designed a novel protocol for direct differentiation of human iPS cells towards erythroid cells under serum-free conditions bypassing the EB formation step without requiring co-culture. Our protocol involves three steps: (1) hematopoietic/erythropoietic induction, followed by (2) erythroid differentiation, and finally, (3) erythroid maturation and enucleation. Differentiated cells were separated into normoxia and hypoxia conditions. As early as day 7 of culture, an early hematopoietic marker, CD34, was observed, followed by a high expression of CD45, which is a pan leukocyte marker in parallel to less expression of an early erythroid marker, CD71. Over the culture period, an increase in the expression of the late erythroid marker, CD235a, was monitored, which reached high levels by the end of the 28-day culture protocol. Further studies on functional and morphological analysis using CFU assay showed that the cell population on day 14 were able to form erythroid progenitor colonies, i.e. BFUEs. Immunocytochemical staining showed the presence of heme-containing proteins, which was later confirmed by globin expressions by qPCR. Interestingly, staining with new methylene blue confirmed reticulocyte morphology, which indicated that partial maturation was achieved. Hypoxia condition is a key regulator for erythropoiesis and haemoglobin formation, as indicated by the BFU-Es formed under hypoxic conditions, together with formation of adult-type haemoglobin, as shown in qPCR. Further studies on maturation of those cells are required in order to achieve fully mature and functional erythroid cells phenotype. This thesis thus presents a direct differentiation protocol toward erythroid cells using human iPS cells in serum-free and feeder-free system, bypassing EB-stage and resulting on high efficiency of erythroid cells formation within 4 weeks of culture, which include partial maturation and formation of adult-type haemoglobin.
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Nickel, Gabrielle Celeste. "Positive Selection in Transcription Factor Genes Along the Human Lineage." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1220370670.
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Diaz-Araya, Claudia M. (Claudia Marcela). "Microglia and leucocyte lineage cells of the developing human eye." Thesis, Faculty of Medicine, 1995. http://hdl.handle.net/2123/12650.
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Rajendran, senthilkumar. "STUDIES ON LINEAGE SHIFT RESPONSES OF HUMAN PERIPHERAL BLOOD MULTIPOTENT CELLS." Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3423582.
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Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. As embryonic stem cells pose a lot of ethical issues, adult stem cells, isolated from various sources like cord blood, bone marrow or adipose tissue, are being considered as a realistic option due to their well documented therapeutic potentials. In our lab, we have standardised a method to isolate fibroblastic multipotent stem cells (PBMCs) from human peripheral blood, that are able to sustain long term in vitro culture and differentiate towards adipogenic, chondrogenic and osteogenic lineage.
In this work, PBMCs were stimulated to obtain in vitro neuronal and myogenic-like cells. Moreover, their restorative potential in degenerative diseases of skeletal muscle and nervous tissue was evaluated using in vivo models. In order to test the neuronal differentiation potential, the cells were seeded (1x104) on gelatin coated dishes and cultured for 7 days in neurobasal medium with EGF and FGF followed by Retinoic acid and NGF for next 7 days. Myogenic induction was carried out using IGF and ascorbic acid for 14 days. At different time points, morphological studies were performed by SEM and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. PBMCs showed characteristic dendrite like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of PBMCs under stimulation with KCl 56 mM and the secretion of the neurotransmitter, noradrenalin, a precursor in the dopamine synthesis confirmed their ability to acquire a functional phenotype. When premarked by a cell tracker Qdot 800 and injected stearotactically into a rat brain, PBMCs showed to be migratory and proliferative as detected after 10 and 20 days of injection. No tumor mass was identified. The myogenic potential of PBMCs were confirmed by their ability to form syncitium like structures in in vitro culture and to express typical myogenic markers both at early and late phases of differentiation. PBMCs were showed to integrate within the host tissue and to take part in tissue repair as demonstrated in a bupivacaine induced muscle damage model.Il trapianto di cellule staminali è una strategia terapeutica che sta conoscendo uno sviluppo sempre maggiore come possibile approccio clinico per il trattamento delle malattie degenerative. Considerando i problemi di carattere etico sollevati dall’impiego delle cellule staminali embrionali, le cellule staminali adulte isolate da varie fonti (sangue cordonale, midollo osseo, tessuto adiposo﴿ rappresentano una realistica alternativa, in virtù della loro potenzialità rigenerativa ben documentata. Nel nostro laboratorio è stato standardizzato un metodo per isolare cellule staminali fibroblastoidi multipotenti (Peripheral Blood Multipotent Cells, PBMC) da sangue periferico umano, che possono essere espanse in vitro durante la coltura a lungo termine e sono in grado di differenziare in senso adipogenico, condrogenico e osteogenico. Nel lavoro di tesi del Dott. Senthilkumar Rajendran, le cellule PBMC sono state stimolate per l’ottenimento in vitro di cellule simil-neuronali e -muscolari. Inoltre è stato valutato il loro potenziale rigenerativo nel trattamento di malattie degenerative del muscolo scheletrico e del tessuto nervoso attraverso la sperimentazione in vivo su modelli animali. Al fine di testare il potenziale di differenziazione neuronale, le cellule sono state seminate (1x104) su coating di gelatina e coltivate per i primi 7 giorni in Neurobasal medium addizionato con EGF e FGF, e per i 7 giorni successivi in terreno basale contenente acido retinoico e NGF. L’induzione miogenica è stata effettuata utilizzando IGF e acido ascorbico per 14 giorni. Ad ogni time point, sono stati realizzati studi morfologici mediante SEM e analisi di espressione di specifici marcatori neuronali e miogenici mediante RT-PCR, citofluorimetria e western blot. Le cellule PBMC hanno mostrato una caratteristica morfologia simil-dendritica e l’espressione di specifici marcatori neuronali a livello sia di mRNA che di proteine. Lo studio del flusso del calcio dopo stimolazione con KCl 56 mM e l’attività di secrezione del neurotrasmettitore noradrenalina, precursore nella sintesi della dopamina, hanno confermato la capacità delle cellule PBMC di acquisire un fenotipo funzionale. Dopo marcatura con il tracker cellulare Qdot 800 e iniezione per stereotassi in un cervello di ratto, le PBMC hanno dimostrato un elevato potenziale migratorio e proliferativo dopo 10 e 20 giorni dall'impianto. Non è stata identificata alcuna massa tumorale. Il potenziale miogenico delle popolazioni isolate è stato confermato dalla loro capacità di formare strutture simil-sinciziali durante la coltura in vitro e di esprimere marcatori tipici della linea miogenica, sia a tempi precoci che nelle fasi tardive del differenziamento. Infine, testate in un modello animale di danno muscolare indotto con bupivacaina, le cellule PBMC sono state in grado di integrarsi all'interno del tessuto ospite e di prendere parte nella riparazione dei tessuti.
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Naxerova, Kamila. "Tracing human cancer evolution with hypermutable DNA." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11253.
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Metastasis is the main cause of cancer morbidity and mortality. Despite its clinical significance, several fundamental questions about the metastatic process in humans remain unsolved. Does metastasis occur early or late in cancer progression? Do metastases emanate directly from the primary tumor or give rise to each other? How does heterogeneity in the primary tumor relate to the genetic composition of secondary lesions? Addressing these questions in representative patient populations is crucial, but has been difficult so far. Here we present a simple, scalable PCR assay that enables the tracing of tumor lineage in patient tissue specimens. Our methodology relies on somatic variation in highly mutable polyguanine (poly-G) repeats located in non-coding genomic regions. We show that poly-G mutations are present in a variety of human cancers. Using colon carcinoma as an example, we demonstrate an association between patient age at diagnosis and tumor mutational burden, suggesting that poly-G variants accumulate during normal division in colonic stem cells. We further show that poorly differentiated colon carcinomas have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We collect multiple spatially separated samples from primary carcinomas and their metastases and use poly-G fingerprints to build well-supported phylogenetic trees that illuminate each patient's path of progression. Our results imply that levels of intra-tumor heterogeneity vary significantly among patients.
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Wright, Elli Alexander. "The differentiation of human embryonic stem cells towards a pancreatic endoderm lineage." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509868.
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Gifford, Casey. "Transcriptional and Epigenetic Dynamics Observed During Lineage Specification of Human Embryonic Stem Cells." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11228.
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Epigenetic regulation of gene expression is essential for faithful cellular specification during embryonic development. Directed differentiation of pluripotent human embryonic stem cells (hESCs), which maintain the ability to give rise to each cell type found within the human body, provides a tractable system to study both the epigenetic mechanisms that facilitate cellular transitions, and the transcription factors (TFs) that dictate these events. To understand molecular events associated with major lineage decisions, we performed comprehensive genomic profiling, including RNA-Sequencing, Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) for six histone modifications and whole genome bisulfite-sequencing (WGBS) to interrogate DNA methylation levels, on three populations derived through directed differentiation of hESCs. Expression profiling detected signatures that resembled the three embryonic germ layers, namely ectoderm, mesoderm and endoderm. Integration of ChIP-Seq and WGBS data revealed widespread remodeling, predominantly at intergenic regions. To understand the impact of TF binding on epigenetic remodeling, we then complemented the epigenetic information with binding profiles for the pluripotency TFs OCT4, SOX2 and NANOG (O/S/N) in hESCs, and FOXA2 in the endoderm population. O/S/N binding was identified near pluripotency genes as expected, as well as regions that exhibited lineage specific remodeling during differentiation and are linked to later stages of development. We also discerned a novel epigenetic trend, in which H3K27me3 was unexpectedly gained at regions of low CpG density that exhibit high levels of DNA methylation in hESCs. These events overlapped with FOXA2 binding sites in the dEN that lose DNA methylation. Notably, these events were detected near genes associated with later stages of development, such as AFP. We postulate that these FOXA2-associated epigenetic remodeling events lead to acquisition of a transient, facultative heterochromatic state necessary to foster efficient differentiation of subsequent stages. Integration of these data sets yielded an unprecedented perspective of the orchestrated transcriptional and epigenetic events that occur during cell state transitions. Future studies that compare epigenomic profiles of in vitro derived cell types to their primary counterparts may identify regulatory elements that are held in improper epigenetic states, and ultimately lead to improved differentiation protocols and the in vitro derivation of therapeutically relevant cell types.
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Scott, Charlotte M. A. "The function of human macrophage metalloelastase (MMP-12) in cells of monocytic lineage." Thesis, University of East Anglia, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273500.
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Soh, Boon Seng. "Optimization of Human Embryonic Stem Cells Culture and their Differentiation towards the Lung Lineage." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516176.
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Furuta, Rie. "Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo." Kyoto University, 2018. http://hdl.handle.net/2433/232110.
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Ang, Lay Teng. "Regulation of lineage specification of human embryonic stem cells by microRNAs and serum response factor." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648127.
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Rockhill, Carter Anderson. "Coaching Lineage: The Application of Network Theory to Power-5 Coaching Trees." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1594387207820944.
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Weynans, Kevin [Verfasser]. "Direct lineage programming - a tool to generate and analyze human cortical layer specific neurons / Kevin Weynans." Bonn : Universitäts- und Landesbibliothek Bonn, 2020. http://d-nb.info/123552437X/34.
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Kaushik, Suresh Kumar. "Genetic modification of human embryonic stem cells for lineage selection, derivation and analyses of human 3rd pharyngeal pouch epithelium like cells and its derivatives." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28724.
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Human pluripotent stem cells (hPSCs) such as, human embryonic stem cells (hES) and human induced pluripotent stem cells (hiPS) are a valuable resource to generate bespoke cell types for a number of therapeutic applications involving cell therapy, drug screening and disease modelling. The overarching goal of this project was to generate a set of transgenic tools by gene targeting and genetic modification of hESCs for applications in stem cell biology such as the in vitro isolation, analyses and derivation of lineage specific cell types. The transgenic tools generated in this study were designed and tested in particular for the human 3rd pharyngeal pouch epithelium (3PPE) like cells and its derivatives, namely the thymus and parathyroid, which are key organs involved in T-cell development and calcium homeostasis respectively. The forkhead transcription factor FOXN1 is considered a master regulator of the development of the thymic epithelium (TEC), the major functional component of the thymic stroma, which is intimately involved in T-cell differentiation. So, to facilitate the prospective isolation of FOXN1 expressing TECs, gene targeting was employed to place a fluorescent reporter and a lineage selection antibiotic resistance gene under the direct control of the endogenous FOXN1 promoter. To date, I have not been able to detect either the fluorescent reporter, or FOXN1 expression using published directed differentiation protocols, but only what can be deemed as precursors expressing the cytokeratin K5 and other markers associated with the development of the thymus and parthyroid from 3PPE. The lack of endogenous FOXN1 activation was observed in both the unmodified parent and the targeted FOXN1 knock-in human ES lines. Further, over-expression of FOXN1 cDNA during the differentiation protocol did not result in the activation of endogenous FOXN1. So, the results evinced in this study could be due to a number of reasons such as, technical issues associated with transference of the published protocols to the cell lines used in this study, differences in hESC lines, and effects of different hESC culture methods and practices. The homeobox gene HOXA3 is expressed in the 3PPE during development. So, a HOXA3 transgenic reporter hESC line could be an invaluable tool for prospective isolation of in vitro derived 3PPE like cells. The reporter was generated by Piggy Bac transposase mediated transposition of a HOXA3 containing Bacterial Artificial Chromsome (BAC) in the FOXN1 knock-in human ES line. To date, this is biggest reported cargo that has been successfully transposed in human ESCs. Moreover, this is the first lineage specific double reporter transgenic hESC line that has been reported for this lineage. This HOXA3 reporter line was then used to isolate and enrich for HOXA3 expressing 3PPE like cells with very high efficiencies during the directed differentiation of hESCs, thus demonstrating the key objective of this transgenic hESC line for this study. In a novel parallel approach, I have conceived, designed and generated transgenic hESCs lines capable of inducible and constitutive over-expression of key transcription factors involved in the development of 3PPE and its derivatives, the thymus and parathyroid. The objective of the said over-expression hESC lines was to interrogate if such a system could elicit morphological and gene expression changes in hESCs following over-expression. By testing the chosen panel of transcription factors in hESCs, I was able to detect cells expressing FOXN1 and GCMB, which are key markers of TECs and PTECs. Further, I have isolated an expandable population of cells expressing markers analogous to their in vivo counterpart found in the 3PPE of a developing mouse embryo around E9.0. The in vivo potency of these in vitro derived 3PPE like cells is yet to be ascertained. Nevertheless, transgenic constructs generated in this experiment could also be tested during future attempts at the differentiation of hESCs to TECs and PTECs, and also used as a basis for future studies involving the direct conversion of patient specific fibroblasts to 3PPE like cells and its derivatives. In summary, several transgenic tools developed in this project, namely the FOXN1 knock-in transgenic hESC line, FOXN1-HOXA3 double transgenic hESC line, over-expression 3PPE transgenes and hESC transgenic lines, and results from the deployment of these tools provide a foundation, from which protocols to generate functional TECs and PTECs can be refined and optimised. These transgenic hESC lines also provide a tractable model, which could be used to interrogate the development of human TECs and PTECs from human 3PPE, and identify hitherto unknown early events in their development in an in vitro reductionist setting.
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VanOudenhove, Jennifer J. "Mechanisms Regulating Early Mesendodermal Differentiation of Human Embryonic Stem Cells: A Dissertation." eScholarship@UMMS, 2016. http://escholarship.umassmed.edu/gsbs_diss/849.
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Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events.
To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation.
A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.
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MacKay, Maria-Danielle L. "Characterization of Medullary and Human Mesenchymal Stem Cell-Derived Adipocytes." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1232775772.
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Chain, Jennifer Lee. "Elucidating the mechanisms of the human [alphabeta] vs. [gammadelta] lineage decision and the details of [gammadelta] thymocyte development." Oklahoma City : [s.n.], 2005.
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蘇志偉 and Chi-wai So. "Studies on the mixed lineage leukemia gene and identification of a novel partner gene, EEN, in human leukemia." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1996. http://hub.hku.hk/bib/B31236145.
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Muyrers-Chen, Inhua Taveira. "Effects of mixed lineage leukaemia, the human homologue of trithorax, and its leukaemic fusion proteins in Drosophila melanogaster." Thesis, Open University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.395259.
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Krishna, Benjamin Anthony Cates. "Investigating and exploiting the latency-associated expression of the human cytomegalovirus gene US28 in early myeloid lineage cells." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267737.
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Human cytomegalovirus (HCMV) is a betaherpesvirus which establishes a lifelong persistent infection, underpinned by its ability to establish latent infection in early myeloid lineage cells, in the infected host. Although well controlled by a healthy immune system, HCMV causes pathological and life threatening disease in individuals with a compromised or immature immune response, which can come from primary HCMV infection or reactivation of latent infection. Although progress is being made in understanding the mechanisms by which HCMV maintains latency and reactivates, a better understanding is essential towards the aim of targeting and killing latently infected cells. In this thesis, I will present evidence that the HCMV-encoded chemokine receptor homologue US28, which is expressed during latent infection of CD14+ monocytes, is necessary for maintaining HCMV latency in these monocytes and, in the absence of US28 protein expression, HCMV undergoes lytic infection. US28 expression was found to attenuate cellular signalling pathways in latently infected cells; in particular, MAP kinase and NFκB. Interestingly, deletion of the US28 gene or inhibition of the US28 protein resulted in the expression of lytic antigens which allowed detection of infected monocytes by the immune system. This observation may lead to a potential new immunotherapeutic strategy against latent HCMV. Having demonstrated that US28 protein is expressed on the surface of latently infected monocytes, I tested whether a new fusion-toxin protein, called F49A-FTP, which binds US28 protein, could be used to target and kill latently infected cells. I developed a protocol for treating latently infected monocytes with F49A-FTP which resulted in a significant reduction in virus reactivation after monocyte differentiation to dendritic cells. I was also able to show that this treatment kills CD34+ progenitor cells, which were experimentally latently infected with HCMV, as well as latently infected monocytes from a healthy, seropositive blood donor. Finally, during my investigations into the role of US28 during HCMV latency, a mass spectrometry screen was performed to measure changes in cellular protein expression when US28 protein is expressed in isolation, in THP-1 monocyte-like cell line. This identified CTCF, a transcription factor which appears to be modified by US28 in THP-1 cells. I showed that CTCF has a repressive effect on the HCMV MIEP, and that CTCF likely plays a role in HCMV latency. In summary, this work provides insights into the role of US28 during HCMV latency, and proposes potential novel therapeutic strategies to kill latently infected cells.
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So, Chi-wai. "Studies on the mixed lineage leukemia gene and identification of a novel partner gene, EEN, in human leukemia /." Hong Kong : University of Hong Kong, 1996. http://sunzi.lib.hku.hk/hkuto/record.jsp?B18156885.
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Ikuno, Takeshi. "Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP." Kyoto University, 2017. http://hdl.handle.net/2433/227586.
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Giblin, Sean. "Investigating cell lineage specific biosynthesis of tenascin-C during inflammation." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:8c7306d8-53cf-4131-a134-f74885e37cc9.
Full textAbstract:
The extracellular matrix (ECM) is a complex network of molecules secreted by cells, which is essential for providing structural support and facilitating cell processes including adhesion, migration and survival. Tenascin-C is an immunomodulatory ECM protein that exhibits limited expression in healthy tissues, but is transiently elevated at sites of tissue injury, and is persistently expressed in chronic inflammatory diseases and tumours. Alternative splicing of 9 of tenascin-C's fibronectin type III-like domains (FnIII- A1, A2, A3, A4, B, AD2, AD1, C and D) generates enormous diversity in form; yielding 511 possible isoforms. Post-transcriptional modification of tenascin-C has been studied in cancer and during development where disease and tissue specific isoforms exhibit distinct adhesive, migratory and proliferative effects. However, little is known of how tenascin-C is expressed or alternatively spliced during inflammation. This study characterises inflammation and disease specific tenascin-C isoforms made by immune cells and fibroblasts, and investigates their functional relevance. Biosynthesis and alternative splicing of tenascin-C was examined using standard curve qPCR, ELISA, Western blot and confocal immunocytochemistry in resting and activated primary human immune cells, dermal fibroblasts, and in synovial fibroblasts isolated from healthy controls and from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Based on these data, three recombinant proteins comprising FnIII domains AD2-AD1, B-C-D and B-AD2-AD1-C-D were cloned, expressed and purified, and their impact on cell behaviour including adhesion, morphology and migration was assessed. Basal tenascin-C expression was lower in myeloid and lymphoid cells than fibroblasts, and was induced in all following inflammatory stimulation. Tenascin-C expression was elevated in disease with RA and OA synovial fibroblasts containing higher levels than healthy controls. Alternative splicing following cell activation was cell-type specific: all FnIII except AD2 and AD1 were upregulated in dendritic cells and macrophages, in T-cells all FnIII remained unchanged with FnIII A1 absent; and no change in splicing was observed in activated dermal fibroblasts. Normal and OA synovial fibroblasts exhibited similar tenascin-C splicing patterns, but FnIII B and D were specifically elevated in RA. Functional analysis revealed differences in the adhesion, morphology and migration of myeloid cells and dermal fibroblasts cultured on FnIII AD2-AD1, B-C-D, B-AD2-AD1-C-D and full length tenascin-C substrates; FnIII B-C-D promoted MDDC migration while B-AD2-AD1-C-D promoted fibroblast adhesion, compared to full length tenascin-C. For the first time, this study reveals differences in tenascin-C biosynthesis and alternative splicing by immune cells and fibroblasts following activation with inflammatory stimuli; and starts to reveal how alternative splicing of tenascin-C may influence the behaviours of both stromal and immune cells types during inflammation and in inflammatory diseases.
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Sudheer, Smita [Verfasser]. "Differential modulation of BMP signaling by Activin, Nodal and FGF pathways in lineage specification of human embryonic stem cells / Smita Sudheer." Berlin : Freie Universität Berlin, 2011. http://d-nb.info/1026344670/34.
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Mori, Kiyoshi. "Gene expression of the human prostaglandin E receptor EP[4] subtype : differential regulation in monocytoid and lymphoid lineage cells by phorbol ester." Kyoto University, 1997. http://hdl.handle.net/2433/160737.
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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第6737号
医博第1837号
新制||医||656(附属図書館)
UT51-97-H121
京都大学大学院医学研究科内科系専攻
(主査)教授 大熊 稔, 教授 伊藤 和彦, 教授 中尾 一和
学位規則第4条第1項該当
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Li, Fei. "PHARMACOLOGICAL MANIPULATION OF PROTEIN KINASE C MODULATES THE GROWTH AND LINEAGE COMMITMENT OF ENRICHED HUMAN MYELOID PROGENITOR CELLS INDUCED BY HEMATOPOIETIC GROWTH FACTORS." VCU Scholars Compass, 1992. https://scholarscompass.vcu.edu/etd/5139.
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The activity of protein kinase C (PK-C) has been implicated in regulating the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C activators, phorbol 12,13- dibutyrate, mezerein, and bryostatin 1, on the proliferation and lineage commitment of enriched CD34+ human myeloid progenitor cells stimulated by lL-3, GM-CSF, stem cell factor and the lL-3/GM-CSF hybrid cytokine plXY321. Coadministration of these PK-C activators with plateau concentrations of rlL-3 or rGM-CSF induced 100-150% increase in the number of day 14 CFU-GM.with a selective stimulation on neutrophil and macrophage lineages while inhibiting eosinophilic growth. plXY321 stimulated an equivalent number of CFU-GM, including a predominant eosinophilic component, when compared to the combination of saturating levels of GM-CSF and lL-3. Bryostatin 1, when coadministered with plXY321 (or with the combination of lL-3 and GM-CSF), selectively enhanced the growth of neutrophilic and monocytic lineages while inhibiting eosinophil development. The inhibition of eosinophil colonies by bryostatin 1 was not mimicked by the coadministration of rSCF, rG-CSF or rCSF-1 with plXY321. Furthermore, neutralizing antibodies to rG-CSF and rCSF-1 failed to block potentiation of neutrophil or macrophage colony formation stimulated by bryostatin 1 in conjunction with plXY321, suggesting that accessory cell effects are not solely responsible for this phenomenona. rSCF synergistically enhanced plXY321 induced colony formation by an average of 144% by selectively stimulating neutrophilic and eosinophilic growth. Coadministration of bryostatin 1 with rSCF and plXY321 further increased colony formation by an average of 81%. This combination selectively stimulated cells of the macrophage lineage, and inhibited eosinophil differentiation. However, bryostatin 1 inhibited erythroid (BFU-E) and erythroid/myeloid mixed (CFU-GEMM) colonies induced by plXY321 alone or in combination with rSCF. Together these results indicate that 1) PK-C activity is involved in the growth and lineage commitment of early and committed myeloid progenitor cells. 2) Pharmacologic manipulation of PK-C may regulate the growth and differentiation of those cells exposed to early hematopoietic growth factors. This study raises the possibility that pharmacologic intervention at PK-C,in conjunction with hematopoietic growth factors, might be useful in the ex vivo expansion of hematopoietic progenitor cells.
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Ferrer, i. Admetlla Anna. "Human genetic diversity in genes related to host-pathogen interactions." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7163.
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La tesi que teniu a les mans recull quatre treballs amb un objectiu comú; determinar si els patògens (virus, bacteris, paràsits.) han exercit pressions selectives sobre els genomes dels seus hostes (com per exemple els humans).Sabent que la detecció de l'empremta de la selecció permet identificar aquelles regions del genoma que han estat rellevants al llarg de l'evolució d'una espècie, ja que a nivell local és la variació funcional qui acaba essent objecte de la selecció, ens hem disposat a estudiar els possibles senyals de selecció en gens relacionats amb la interacció hoste-patògen. En concret, hem analitzat gens que codifiquen per: a) components del sistema immunitari innat i, b) enzims de glicosilació, la majoria dels quals s'inclouen en quatre de les principals rutes biosintètiques de glicans, en diferents poblacions humanes.
Com a conclusió principal; ambdós conjunts de gens mostren clars senyals de selecció. A més hem vist que segons el context biològic on és troben certs gens és veuen més afectats per l'acció de la selecció natural.
The present thesis includes four studies with a common objective: determining whether pathogens (virus, bacteria, parasites.) have exerted selective pressures on the genome of their hosts (for example, humans).
Detecting signatures of positive selection is a useful tool to identify functionally relevant genomic regions since selection locally shapes the functional variation. Based on this premise, we have studied the possible signatures of selection in genes related to host-pathogen interactions. Specifically, we have analyzed those genes encoding: a) components of the innate immunity response; and ii) glycosylation enzymes most of them involved in four major glycan biosynthesis pathways, in different human populations.
The main conclusion obtained from these studies is that both studied gene categories show clear signatures of selection. Moreover, we have determined that according to their biological context certain genes are more prone to the action of selection.
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Marguet, Florent. "Effets de l'alcoolisation prénatale sur le développement du système GABAergique et de la myélinisation chez l'humain Prenatal alcohol exposure is a leading cause of interneuronopathy in humans Oligodendrocyte lineage is severely affected in human alcohol-exposed fetuses." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMR075.
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L’exposition prénatale à l’alcool est une cause non génétique majeure d’anomalies structurales et fonctionnelles du système nerveux central dont la forme la plus sévère est le syndrome d’alcoolisation fœtale, et dont les effets perdurent tout au long de la vie, associant retard mental, troubles neurocognitifs et comportementaux. L’exposition prénatale à l’alcool est un enjeu de Santé Publique, car actuellement, la plupart des enfants ne sont pas précocement dépistés en l’absence des signes majeurs que sont la dysmorphie crânio-faciale caractéristique et le retard de croissance. Si de nombreuses études sur les anomalies neurodéveloppementales ont été réalisées chez l’animal et dont les résultats sont souvent contradictoires, très peu de données sont disponibles chez l’humain et concernent essentiellement l’enfant. Afin d’expliquer la symptomatologie de ces enfants, nous avons réalisé une étude de la mise en place des interneurones et de la glie de myélinisation, éléments essentiels intervenant dans la synchronisation des réseaux neuronaux. A partir d’une cohorte de 15 fœtus humains à tous les stades du développement et de deux cas post-nataux âgés de trois mois et deux ans exposés à l’alcool, nous avons étudié l’ontogenèse des interneurones GABAergiques et calrétininergiques ainsi que les caractéristiques de leur migration vasculaire dans le cortex. Nous avons identifié une interneuronopathie consistant en un retard majeur de génération dans les zones de production (éminences ganglionnaires) aux stades précoces de développement et une malposition des interneurones calrétininergiques au sein du cortex des fœtus alcoolisés aux stades plus tardifs comparativement à des contrôles appariés. Ce retard de génération touche également les précurseurs des oligodendrocytes exprimant le PDGFRα qui restent ensuite anormalement nombreux aux dépends des précurseurs et préoligodendrocytes exprimant Olig2. Ces derniers restent pratiquement absents dans le cortex des fœtus alcoolisés jusqu’en fin de la grossesse. L’interneuronopathie et le défaut de différentiation oligodendrogliale pourraient expliquer en partie les troubles neurologiques observés chez les enfants et adultes exposés in utero à l’alcool, la modulation de l’activité neuronale et la myélinisation étant indispensables à l’établissement des réseaux neuronaux et à la conduction des influx nerveuxPrenatal alcohol exposure is a major non-genetic cause of structural and functional abnormalities of the central nervous system, the most severe form of which is fetal alcohol syndrome, and the effects of which persist throughout life, associating mental retardation, neurocognitive and behavioural disorders. Prenatal alcohol exposure is a public health issue, since currently most children are not early detected in the absence of the major signs consisting of characteristic cranio-facial dysmorphism and growth retardation. While numerous studies on neurodevelopmental abnormalities have been carried out in animals and the results of which are often contradictory, very little data is available in humans and mainly concerns children. In order to explain the symptoms of these children, we have conducted an ontogenetic study of interneurons and oligodendrocyte lineage, two essential events involved in the synchronization of neural networks. Using a cohort of 15 human fetuses at all stages of development and two postnatal cases aged three months and two years exposed to alcohol in utero, we studied the development of GABAergic and calretinergic interneurons as well as the characteristics of their vascular migration within the cortex. We identified an interneuronopathy consisting of a major generation delay in the production areas (ganglionic eminences) at the early stages of development and a mispositioning of calretinergic interneurons within the cortex of fetuses exposed to alcohol at later stages compared with age matched controls. This delay in generation also affects precursors of oligodendrocytes expressing PDGFRα, which then remain abnormally numerous at the expense of precursors and pre-oligodendrocytes expressing Olig2. The latter are virtually absent in the cortex of fetuses exposed to alcohol until the end of pregnancy. Interneuronopathy and the lack of oligodendroglial differentiation could partly explain the neurological disabilities observed in children and adults exposed in utero to alcohol, modulation of neuronal activity and myelination being essential for the establishment of neural networks and conduction of nerve outputs
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Rapisarda, Valentina. "Mechanisms of epigenetic regulation in epidermal keratinocytes during skin development : role of p63 transcription factor in the establishment of lineage-specific gene expression programs in keratinocytes via regulation of nuclear envelope-associated genes and polycomb chromatin remodelling factors." Thesis, University of Bradford, 2014. http://hdl.handle.net/10454/7164.
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During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.
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BOLAMPERTI, SIMONA. "ESTROGEN- GROWTH HORMONE INTERACTION IN BONE CELLS OF THE OSTEOGENIC LINEAGE: GROWTH HORMONE ANABOLIC ACTIVITY ON HUMAN OSTEOBLASTS AND THEIR MESENCHYMAL PRECURSORS IS MODULATED BY 17ΒETA-ESTRADIOL THROUGH A POST RECEPTOR MECHANISM." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229429.
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In aging the loss of bone mass goes along with a decline of 17beta-estradiol (E2) and Growth Hormone (GH), which are known to be bone anabolic factors. Recent evidence demonstrated in several cell lines an interplay between E2 and GH at a post receptor level. I thus evaluated the possible cross-talk between these two hormones in human osteoblast cells (hOBs) in primary culture and in their mesenchymal precursors (hMSCs).
E2 (10-8M) given 60 min before GH (5ng/ml) enhanced both GH intracellular pathway in hOBs and the transcription of the evaluated GH target genes involved in osteoblast activity and matrix deposition, osteopontin (OPN), bone sialoprotein (BSP) and insulin like growth factor 2 (IGF2). E2 effects occurred by decreasing the protein levels of SOCS2, one of the main GH signaling inhibitors, through an increase in SOCS2 ubiquitination and consequent degradation. This effect was blunted by pre-treating the cells with the proteasome inhibitor MG132 (5µM). Interestingly this effect did not involve an E2 mediated genomic activity as Actinomycin D (5µM) pre-treatment did not prevent E2 modulation of SOCS2 levels (Bolamperti S. et al., 2013). Further experiments demonstrated that this short term effect on SOCS2 levels was maintained over time: after 3h of E2 treatment there was still a decrease in SOCS2 levels. Yet, at this time point, the effect occurred via an inhibition of the transcription of SOCS2 gene. The fact that E2 negative regulation of the GH inhibitor SOCS2 involves an initially rapid protein degradation maintained for a longer time by a decrease in its gene expression strengthens the importance and the physiological relevance of this modulation for osteoblast activity. I was therefore interested to investigate whether or not two SERMs often used in clinics, Tamoxifen (Tam) and Raloxifene (Ral), share the same effect on GH signaling as E2. Cells treated with Tam (10-10M) or Ral (10-8M) 60 min before GH showed a trend to increase STAT5 phosphorylation even though reaching statistical significance, despite an observed reduction of SOCS2 levels with the SERMs alone. After 3h treatment no modulation of SOCS2 transcription was detected with either Tam, or Ral. These data suggest that despite their general estrogen agonistic properties on bone, none of the two drugs displayed the same features as E2.
The combined effect of E2 and GH was evaluated also in mesenchymal stem cells (MSCs) obtained from human bones. The cells were first tested for plastic adherence, for differentiation capability towards adipocytes or osteoblasts, and for the positivity to CD73, CD105, and CD90 following an NIH protocol. In these osteoblast precursors, pretreatment of E2 60 min before GH, increased STAT5 phosphorylation induced by GH and decreased SOCS2 levels, as shown in hOBs. Considering the lack of information about GH action in stromal precursors, we evaluated GH action in the isolated hMSCs, focusing on its possible role in both osteogenesis and adipogenesis. The results showed that long term GH treatment (5ng/ml, 14 days, 3 times/week) increased early osteogenic genes and prevented adipogenesis in the isolated hMSCs. Given the important role of microRNAs in MSC commitment to osteoblastogenesis or adipogenesis, I analized if GH was able to increase miR-22 and miR-29c, considered amongst the main regulators of osteoblastogenesis or decrease miR-204, which is a regulator of adipogenesis.
GH was able to upregulate the transcription of miR-22 and miR-29c, without affecting miR-204. E2 per se inhibited their transcription and, in the combined treatment with GH, E2 pre-treatment was able to inhibit the stimulatory effect of GH on miR22 and miR29c. In conclusion the study has shown a relevant hormone to hormone interaction; hence E2 can locally modulate GH activity potentiating its cellular signaling in osteoblasts and in their stromal precursors. In osteoblasts the increase in the activity of the GH signal transducers reflects an increase in the transcription of GH responsive genes involved in the regulation of the deposition and the turnover of minerals and in the control of osteoblasts and osteoclasts metabolism (Gehron Robey and Boskey, 2006). In mesenchymal stromal cells the E2 potentiating effect of GH signaling was not accompanied by a positive effect on miRNA expressions. This could be due to the strong inhibitory effect that E2 per se exerts on the evaluated miRNAs. Thus it can be suggested that the combined treatment of hMSCs with E2 and GH involves different mechanisms of the two hormones on microRNAs.
The data from the present study suggest that circulating estrogen levels should be considered in the management of GH replacement therapy in GH deficiency. Since the E2-GH cross talk is not shared by SERMs, the positive effect of E2 on GH signaling should be taken into account while developing new estrogen receptor modulators molecules.
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Tam, Sze Man. "Construction of the "Hou lineage" in the New Territories of Hong Kong : lineage organization, popular religion and local politics /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?HUMA%202003%20TAM.
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Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2003.Includes bibliographical references (leaves 221-231). Also available in electronic version. Access restricted to campus users.
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Stelmanis, Valters. "Investigation of human embryonic stem cell differentiation towards endothelial lineages." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8053/.
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Cardiovascular disease represents a significant socio-economic burden and minimally invasive therapies that address the needs of patients suffering with peripheral arterial disease and critical limb ischemia are needed. Cell therapies have been proposed as an alternative to pharmacological and surgical treatments, yet, have demonstrated somewhat limited efficacy. However, the unlimited capacity for self-renewal, and the ability to differentiate into cell types from all three germ layers, including endothelial cell (EC) forming mesoderm, make human embryonic stem cells (hESC) and human induced pluripotency stem cells a promising source for well-defined differentiated cell populations with high angiogenic capacity. Numerous endothelial differentiation protocols have been published with high differentiation efficiencies achieved recently. However, most of these approaches are not optimised for clinical purposes due to the use of poorly defined, non cGMP compatible reagents, or require additional processing steps, such as cell sorting, complicating the clinical approval of these therapies. Therefore, here it was aimed to develop and optimise a clinically compatible hESC-EC differentiation protocol that avoids using poorly defined reagents, and yields high percentages of cells expressing EC markers without the use of cell sorting. A novel, serum free hESC-EC differentiation protocol was developed in the lab. The use of Pluronic F-127 well coating was demonstrated as a low cost alternative to low adherence wells. Furthermore, inhibition of TGFB signalling during hESC-EC differentiation to increase the differentiation efficiency was evaluated and did not reveal any additional benefits and thus was not included in the optimised protocol. The optimised protocol consists of embryoid body based mesodermal induction phase, followed by plating and monolayer culture for vascular specification. By day 7, approximately 30% of cells express endothelial markers CD31 and CD144. In addition, transient induction of mesodermal gene, followed by induction of endothelial progenitor and endothelial gene expression was demonstrated, following the expected gene expression patterns. It was proposed that high throughput screening using hESC lines carrying fluorescent reporter constructs could be used to optimise the differentiation protocol for increased efficiency, thus, avoiding the need of cell sorting prior to therapeutic use. Here, reporter constructs where ETV2, ROBO4 and CDH5 promoter sequence fragments were cloned upstream from florescent reporter sequences were generated and preliminary validation was attempted in NCI60, HUVEC and HSVEC cell lines, and during the hESC-EC differentiation. Reporter gene expression was not observed in any of the validation experiments, suggesting that these constructs were not functional. Similarly, previously published CDH5 and commercially sourced ETV2 reporter constructs were validated during the hESC-EC differentiation. Preliminary testing of these reporter constructs showed non-specific reporter gene expression, therefore, the work with reporter constructs work was not pursued further. Next, rational targeting of novel signalling pathways that may contribute to the hESC-EC differentiation was employed as an alternative approach for the optimisation of hESC-EC differentiation. Firstly, it was hypothesised that intracellular cAMP levels could be targeted pharmacologically to increase the differentiation efficiency, and to induce expression of arterial and arterial phenotype associated genes, which could reduce the need for cell sorting and deliver arterial cell populations with a superior angiogenic profile. Forskolin treatments induced increased intracellular cAMP levels during the hESC-EC differentiation, yet, this did not result in increased arterial or arterial phenotype associated gene expression. However, an increase in the percentage of cells expressing EC markers was observed in Forskolin treated differentiations, mainly mediated via an increase in the CD144low CD31+ cell population. Additionally, it was hypothesised that angiotensin II (Ang II) signalling may play a role in hESC-EC differentiation and may be exploited to increase the endothelial differentiation efficiency. Indeed, differential renin angiotensin system receptor expression was demonstrated during hESC-EC differentiation, supporting a role for Ang II signalling in endothelial development. However, no significant differences in the differentiation efficiency and total cell numbers were observed when Ang II and AT1R antagonist Losartan treatments, in combination or alone, were applied during the hESC-EC differentiation. In contrast, a significant reduction in total cell numbers and a trend of reduced differentiation efficiency was observed when AT2R antagonist PD-123319 was used in combination with Ang II. These observations highlight the negative effects of AT1R signalling during hESC-EC differentiation and show that signalling via AT2R counterbalances these effects. In summary, a novel endothelial differentiation protocol was developed and rational selection of signalling pathways for the optimisation of the hESC-EC differentiation was employed. Here, the role of cAMP and Ang II signalling during hESC-EC was demonstrated, highlighting the contribution of various signalling systems to endothelial differentiation. Both of these signalling systems can be easily manipulated in a clinically compliant manner, and therefore represent an attractive target during clinically compatible hESC-EC differentiation. Further research is needed to investigate the underlying mechanisms of the observed effects and to evaluate other, novel signalling pathways that may be targeted to enhance endothelial differentiation. The work described has highlighted the difficulties of establishing efficient, clinically compatible hESC-EC differentiation methods, which are needed to provide highly defined cell populations for future cell therapies and tissue engineering.
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Ronke, Claudius, Michael Dannemann, Michel Halbwax, Anne Fischer, Christin Helmschrodt, Mathias Brügel, Claudine André, et al. "Lineage-specific changes in biomarkers in great apes and humans." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-176077.
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Although human biomedical and physiological information is readily available, such information for great apes is limited. We analyzed clinical chemical biomarkers in serum samples from 277 wild- and captive-born great apes and from 312 healthy human volunteers
as well as from 20 rhesus macaques. For each individual, we determined a maximum of 33 markers of heart, liver, kidney, thyroid and pancreas function, hemoglobin and lipid metabolism and one marker of inflammation. We identified biomarkers that show differences between humans and the great apes in their average level or activity. Using the rhesus macaques as an outgroup, we identified human-specific differences in the levels of bilirubin, cholinesterase and lactate dehydrogenase, and bonobo-specific differences in the
level of apolipoprotein A-I. For the remaining twenty-nine biomarkers there was no evidence for lineage-specific differences. In fact, we find that many biomarkers show differences between individuals of the same species in different environments. Of the four lineagespecific
biomarkers, only bilirubin showed no differences between wild- and captive-born great apes. We show that the major factor explaining the human-specific difference in bilirubin levels may be genetic. There are human-specific changes in the sequence of the promoter and the protein-coding sequence of uridine diphosphoglucuronosyltransferase
1 (UGT1A1), the enzyme that transforms bilirubin and toxic plant compounds into water-soluble, excretable metabolites. Experimental evidence that UGT1A1 is down-regulated in the human liver suggests that changes in the promoter may be responsible for the human-specific increase in bilirubin. We speculate that since cooking reduces toxic plant compounds, consumption of cooked foods, which is specific to humans, may have resulted in relaxed constraint on UGT1A1 which has in turn led to higher serum levels of bilirubin in humans.
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Strand, Kurt B. "Identification of two distinct lineages of macaque gamma-2 herpesviruses /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/9309.
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Lin, Wey-Ran. "Tracing cell lineages in health and disease : experimental and human studies." Thesis, Queen Mary, University of London, 2010. http://qmro.qmul.ac.uk/xmlui/handle/123456789/556.
Full textAbstract:
This study aimed to investigate stem cell biology in the normal and diseased pancreas and liver employing robust methods for tracking stem cells and their progeny in both pre-clinical and human scenario. Bone marrow (BM) plasticity had been demonstrated in diseased organ remodelling. By detection of the Y chromosome in female mice receiving a sexmismatch BM transplantation, BM-derived cells were present in murine pancreas with cerulein-induced pancreatitis. BM-derived myofibroblasts functionally contributed to around 8% of the total population of myofibroblasts, the cells with a key fibrogenic role. Fibrocytes are circulating pro-fibrogenic cells; a decrease of BM-derived fibrocytes in blood and detection of these cells in areas of collagen deposition indicated they migrated to inflamed pancreas and played a role in extracellular matrix formation. IL-10 is an anti-inflammatory cytokine mainly secreted by BM; a lack of IL-10 increased the fibrosis, the inflammation and the numbers of BM-derived myofibroblasts suggesting a potential role of IL-10 in chronic pancreatitis. Mitochondrial DNA (mtDNA) mutations permit lineage tracing within human tissues. Cells having identical mtDNA mutations within a cytochrome c oxidase (CCO)- deficient area must be related having originated from a common founder cell, presumably a stem cell. I have demonstrated that regenerative nodules in cirrhotic liver are invariably clonal populations, and that these nodules often originate from progenitor cells from the abutting ductular reactions. An attempt to build a phylogenetic tree based on the accumulation of mutations in normal liver reinforced the belief that hepatic stem cells are located within the portal tract area and that their cell progeny migrate centrifugally from the portal tract region. The same techniques were applied to the pancreas, but many areas of CCO deficiency could be ascribed to autolysis, while the 3 discovery of identical mtDNA base changes within and outwith CCO-deficient patches suggested these were genetic polymorphisms, previously unreported.
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Celik, Huseyin. "Linear And Nonlinear Analysis Of Human Postural Sway." Master's thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/12609927/index.pdf.
Full textAbstract:
Human upright posture exhibits an everlasting oscillatory behavior of complex nature, called as human postural sway. Variations in the position of the Center-of-Pressure (CoP) were used to describe the human postural sway. In this studyCoP data, which has experimentally been collected from 28 different subjects (14 males and 14 females with their ages ranging from 6 to 84), who were divided into 4 groups according to their ages has been analyzed. The data collection from each of the subjects was performed in 5 successive trials, each of which has lasted for 180-seconds long. Linear analysis methods such as the variance/standard deviation, Fast Fourié
r Transformation, and Power Spectral Density estimates were applied to the detrended CoP signal of human postural sway. Also the Run test and Ensemble averages methods were used to search for stationarity and ergodicity of the CoP signal respectively. Furthermore, in order to reveal the nonlinear characteristics of the human postural sway, its dynamics were reconstructed in m-dimensional state space from the CoPx signals. Then, the correlation dimension (D2) estimates from the embedded dynamics were calculated. Additionally, the statistical and dynamical measures computed were checked against any significant changes, which may occur during aging. The results of the study suggested that human postural sway is a stationary process when 180-second long biped quiet stance data is considered. In addition, it exhibits variable dynamical structure complex in nature (112 deterministic chaos versus 28 stochastic time series of human postural sway) for five successive trials of 28 different subjects. Moreover, we found that groups were significantly different in the correlation dimension (D2) measure (p&
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0.0003). Finally, the behavior of the experimental CoPx signals was checked against two types of linear processes by using surrogate data method. The shuffled CoPx signals (Surrogate I) suggested that temporal order of CoPx is important
however, phase-randomization (Surrogate II) did not change the behavioral characteristics of the CoPx signal.
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Alaqel, Abdullah. "The directed differentiation of human embryonic stem cells to lung cell lineages." Thesis, University of Bath, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760955.
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Human embryonic stem cells (hESCs) show significant therapeutic potential in treating degenerative disorders. This is in part because of their ability to produce a limitless supply of starting cells and their potential to differentiate into more than 200 different cell types. The aim of the current research was to generate a robust stage wise protocol for the differentiation of hESCs to respiratory epithelial cells. The epithelial cells could then be used either for transplantation studies or, as an in vitro model for drug toxicity testing. In order to achieve this goal, we must identify the key steps in lung development and apply these to the differentiation protocol. In this study, we maintained Shef3 hESCs in their undifferentiated pluripotent state to expand the cells prior to the differentiated towards the definitive endoderm (DE) lineage. I used a two-stage protocol based on culture with a novel glycogen synthase kinase-3 (GSK-3) inhibitor (termed 1m), along with Activin-A. We confirmed the status of the cells by a combination of immunostaining and PCR. We showed loss of the pluripotency markers (Sox2 and Oct3/4) and gain of DE markers (Sox17, FoxA2 and CXCR4). After the induction of DE from hESCs, we then treated the cells with transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) pathway inhibitors (SB431542 and Noggin respectively). This combinatorial treatment resulted in the differentiation into the anterior foregut endoderm (AFE) lineage based on expression of Pax9 and FoxA2 plus the up-regulation of Sox2. Further differentiation of AFE derivatives into more mature epithelial cells, termed lung progenitor cells (LPCs), was achieved following the treatment of AFE cells with a cocktail of trophic factors (BMP4, EGF, bFGF, FGF10, KGF and Wnt3a) yielded a population of NKX2.1-positive and FoxA2-positive cells that potentially corresponded to the lung lineage. Finally, prolonged treatment with FGF10 and FGF2 on LPC derived hESCs induced proximal (CC10, MUC5AC) and distal (SPB, SPC) airway epithelial cells. In addition, we also utilised the ectopic expression of an adenovirus expressing NKX2.1 to promote lung maturation. In conclusion, we have generated a protocol for the differentiation of hESCs into mature lung-like cells. The generation of these cells in vitro could potentially lead to a better in vitro model for toxicity testing and the development of novel therapies for promoting regeneration of lungs in patients with severe lung disorders.
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Yew, Chun Keat. "Human movement energy harvesting : a non-linear electromagnetic approach." Thesis, University of Hull, 2015. http://hydra.hull.ac.uk/resources/hull:15151.
Full textAbstract:
Energy harvesting is one of the methods that currently engage actively in energy “recycling”. Of the many energy sources that carry the potential to have energy harvested and recycled, humans are seen as a potential source of energy. High amounts of energy are wasted from daily activities that humans do, if only a portion of the wasted energy can be harvested and reused with the aim of improving the quality of life of the user. To do that, the accelerations of selected movements are recorded from sensors attached to four different locations of the body. Human movements operate on a low and wide frequency scale, nonlinear energy harvesting techniques is seen as a suitable technique to be applied. Nonlinear energy harvesting techniques are expected to increase the bandwidth of operation of the energy harvester. The electromagnetic method of transduction is also selected (using two opposing magnets) to be paired with the nonlinear energy harvesting techniques to evaluate the potential of energy harvesting from human movements. The pick-up coil to be used will be placed at a novel location within the energy harvester prototype. Through simulations and experiments, frequency responses obtained did show an increase in bandwidth which agrees with literature from nonlinear energy harvesting techniques. Phase portraits are also used to provide a more in depth understanding on the movements from the cantilever under linear and nonlinear dynamics. Result comparisons were made between the simulation model and the experimental prototype to verify the agreement between the two. Additionally, results obtained also showed that the resonant frequency of the system was reduced when operating under the nonlinear regime. These attribute favour energy harvesting though human movements. Finally, the novel placement of the pick-up coil within the nonlinear electromagnetic energy harvester had the desired effect. Similar power outputs were achieved even though the separation distances between the two opposing magnets were varied.
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Blaum, Bärbel. "Glycosaminoglycan-protein interactions and human complement factor H." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3868.
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Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal cell surfaces and within extracellular matrices. GAGs usually occur as parts of proteoglycans and often accomplish their biological functions through their interactions with proteins. GAG oligosaccharides for this work were produced via enzymatic digest of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide species obtained from this digest were characterised using 1H and 13C NMR spectroscopy. Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement system, a major component of human innate immunity. Acting as a cofactor to factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells from auto immune attack. This study focused on the interaction of factor H with GAGs, which are thought to be among the markers allowing factor H to distinguish between self and non self surfaces. Binding studies of two heparin-binding sites in fH are presented. These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8 and fH~19-20 were produced recombinantly in various isotope forms. The techniques used to study the protein-GAG interactions in this work encompass NMR spectroscopy, mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking. Several genetic studies suggest that a common polymorphism in the heparin-binding module fH~7, Y402H, plays a role in the development of age-related macular degeneration (AMD). The work presented here included preparation and backbone resonance assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments. Further NMR experiments were employed to investigate the role of the lysine and arginine sidechains of fH~7 in GAG binding. These studies were combined with the preparation and characterisation of a covalently cross linked GAG-protein complex using NMR and mass spectrometry. A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation between the disease and the heparin binding properties of the aHUS mutants was observed. The mutant proteins were also characterised with respect to their ability to compete with full-length fH in a physiological complement assay. Simultaneous binding of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using NMR. NMR experiments were also conducted with NK1, which comprises the two N-terminal heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin, HBD2, were performed to assess the role of GAGs in HBD2 self-association.
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Kasahara, Tomoko. "A modular differentiation system maps multiple human kidney lineages from pluripotent stem cells." Kyoto University, 2020. http://hdl.handle.net/2433/259016.
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Georgiev, Roumen H. "Reconstruction of three dimensional coordinates of multiple targets using linear sensors." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/3236.
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Serrano, Vicente Isabel. "Human Action Recognition Based on Linear and Non-linear Dimensionality Reduction using PCA and ISOMAP." Thesis, KTH, Reglerteknik, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-107500.
Full textAbstract:
Understanding and interpreting dynamic human actions is an important area of research in the field of computer vision and robotics. In robotics, it is closely related to task programming. Traditionally, robot task programming has required an experienced programmer and tedious work. By contrast, Programming by Demonstration is an intuitive method that allows to program a robot in a very flexible way. The programmer demonstrates or shows how a particular task is performed and the robot learns in an efficient and natural manner how to imitate or reproduce the human actions. Here, we develop a general policy for learning the relevant features of a demonstrated activity and we restrict our study to imitation of object manipulation activities. A Nest of Birds magnetic tracker is used for activity recognition and two different dimensionality reduction techniques are applied. The first one uses linear dimensionality reduction in order to find the underlying structure of the data. Particularly, Principal Component Analysis (PCA) is used to learn a set of principal components (PCs) to characterize the data. The main problem using PCA is that linear PCs cannot represent the non-linear nature of human motion. The second method uses a non-linear dimensionality reduction technique. Specifically, spatio-temporal Isomap is applied to uncover the intrinsic non-linear geometry of the data, and it is captured through computing the geodesic manifold distances between all pairs of data points. For classification purposes, both PCA and ST-Isomap can be viewed as a preprocessing step. When the dimensionality of the input data is so high that becomes intractable, most classification methods will suffer and even fail in their goals due to their sensitivity to the input data dimensionality. Fortunately, high dimensional data often represent phenomena that are intrinsically low dimensional. Thus, the problem of high dimensional data classification can be solved by first mapping the original data into a lower dimensional space using a dimensionality reduction method such as PCA or ST-Isomap and then applying K-nearest neighbors (K-NN), radial basis functions (RBF) or any other classification method to classify of the query sequence. In the first stage of our work, PCA combined with k-means clustering is applied. In the second stage of our work, spatio temporal Isomap (ST-Isomap) combined with Shepard’s interpolation is applied. For classification purposes, simple Euclidean distances are used. The experimental evaluation shows that a linear dimensionality reduction technique can not find the intrinsic structure of human motions due to their non-linear nature. In contrary, a non-linear one, such as spatio-temporal Isomap is able to uncover a low dimensional space in which the data lies facilitating the classification step in a much better way than PCA.
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Liu, Guodong McMillan Leonard. "A data-driven, piecewise linear approach to modeling human motions." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,781.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Computer Science." Discipline: Computer Science; Department/School: Computer Science.