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Dissertations / Theses on the topic 'Isogenes'
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1
Fan, Rong. "The roles of 1-aminocyclopropane-1-carboxylate synthase isogenes in the flower and fruit development in tomatoes." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/b40203906.
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Fan, Rong, and 樊榮. "The roles of 1-aminocyclopropane-1-carboxylate synthase isogenes in the flower and fruit development in tomatoes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40203906.
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Eriksson, Peter. "Identification of the two GPD isogenes of saccharomyces cerevisiae and characterization of their response to hyper-osmotic stress." Göteborg : Chalmers Reproservice, 1996. http://catalog.hathitrust.org/api/volumes/oclc/38202006.html.
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Ansell, Ricky. "Redox and osmoregulation in Saccharomyces cerevisiae the role of the two isogenes encoding NAD-dependent glycerol 3-phosphate dehydrogenase /." Göteborg : [Institute of Cell and Molecular Biology, Dept. of General and Marine Microbiology, Lundberg Laboratory, Göteborg University], 1997. http://catalog.hathitrust.org/api/volumes/oclc/38985539.html.
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Chassagne, Catherine. "Regulation de l'expression des isogenes codant la chaine lourde de la myosine et l'alpha-actine dans le coeur de rat normal et hemodynamiquement surcharge." Paris 5, 1993. http://www.theses.fr/1993PA05S006.
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Dans le coeur, la fonction contractile est assuree par les myocytes grace en particulier a la chaine lourde de la myosine (mhc) et a l'alpha-actine, constituants respectifs des filaments epais et fins du sarcomere. Ces proteines presentent chacune deux isoformes, l'alpha- et la beta-mhc, et les actines squelettique et cardiaque, produits de l'expression differentielle de leurs familles multigeniques. Chez le rat, l'expression de ces familles varie au cours de l'ontogenese et de l'hypertrophie cardiaque experimentale, et apparait non synchrone dans l'hypertrophie. Afin de mieux comprendre cette regulation, nos travaux ont eu un double objectif : 1) determiner s'il y a aussi une dissociation entre l'expression des ces deux familles au cours du developpement ontogenique et de la senescence et 2) determiner le niveau de regulation de l'expression de chacun des isogenes alpha-actine et mhc dans le cur adulte normal et hypertrophie. Nous avons d'abord analyse les mecanismes du controle de leur expression dans le cur normal grace a la mise au point de deux techniques, l'une mesurant in vitro sur des noyaux isoles de myocytes l'elongation des arnm deja inities in vivo, et l'autre l'accumulation des arnm alpha- et beta-mhc. Au cours du developpement, les changements d'expression des familles multigeniques de la mhc et de l'actine ne sont pas synchrones, confirmant l'hypothese qu'il n'y a pas de schema coordonne de regulation genique entre ces deux familles. En phase stationnaire, a l'age de trois semaines, les mecanismes qui controlent l'expression des isogenes alpha- actine et mhc sont essentiellement transcriptionnels. Dans les stades precoces de l'hypertrophie cardiaque (induite par une surcharge de pression), ces changements d'expression sont ventricule-dependants, et resultent d'une modulation differente de l'expression des isogenes au sein de chaque paire : antithetique pour les iso-mhc et unique pour l'actine alpha-squelettique. Ces modulations differentes d'expression semblent intervenir non pas pendant, mais apres la transcription.
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Stolbunov, Anton. "Cryptographic Schemes Based on Isogenies." Doctoral thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for telematikk, 2012. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-16560.
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Tsukazaki, Kiminori. "Explicit isogenies of elliptic curves." Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/57568/.
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Let E be an elliptic curve defined over a field K. The main topic of this thesis is to present a method for the explicit computation of all separable K- rational l-isogenies of E and isogenous curves for small primes l. The key tool for this explicit computation is that the modular curve X0(l) parametrises l- isogenies of elliptic curves. In [3], Cremona and Watkins give explicit isogeny formulae for l 2 f2; 3; 5; 7; 13g, where the modular curve X0(l) has genus 0. Their formula allow us to compute l-isogenies of E by simply substituting its j-invariant and twisting parameter into the formulae. We extend the work of Cremona and Watkins to the cases l 2 f11; 17; 19; 23; 29; 31; 41; 47; 59; 71g, where the genus of X0(l) is greater than 0 but the modular curve X+ 0 (l) has genus 0.
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Strittmatter, Axel. "Transcriptional Regulation and Differentiation in Saccharomyces and Aspergillus: jlbA, RPS26, and ARO3/4." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=970349076.
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Nichifor, Alexandra. "Iwasawa theory for elliptic curves with cyclic isogenies /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/5816.
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Mailhot, James Michael. "Selmer groups for elliptic curves with isogenies of prime degree /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/5801.
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Ikoma, Hideaki. "The Faltings-Moriwaki modular height and isogenies of elliptic curves." 京都大学 (Kyoto University), 2008. http://hdl.handle.net/2433/124367.
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Syvertsson, Simon. "Bistable differentiation in an isogenic cell population." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/3002.
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Single-cell organisms such as bacteria have traditionally been regarded as discrete units, which in turn has been reflected by the bulk-level methods used to study them. A growing culture of the bacterium Bacillus subtilis will exhibit a range of heterogeneous genetic developmental programmes such as motility, competence, and finally sporulation. As a popular choice for production of compounds in bioreactors, the bistable behaviours of B. subtilis may be undesirable traits, as they divert resources from their intended activity of synthesising a product. This thesis investigates a novel observation that expression of a ribosomal subunit gene (rpsD) is elevated in the non-motile state of B. subtilis, using unstable GFP reporter constructs. The implications of using a proteolytically unstable protein as a reporter are also investigated with regard to the effect of protein degradation rates on the reporter construct, as well as presenting evidence for modulation of ClpXP activity in a pnpA background. Investigation of the motile/non-motile heterogeneous phenotype of B. subtilis posed a challenge for automated analysis pipelines. This thesis addresses this problem by developing and testing microscopy analysis pipelines designed to circumvent the traditional requirement for physically separated objects in a phase contrast channel, and instead using nucleoid or membrane stains to identify cells in a microscopy image. Other factors impacting the activity of a proteolytically unstable PrpsD reporter construct were investigated, including the rate of degradation of the reporter, and integration locus of the reporter construct. To assess the impact of locus positioning, a genetic tool was also created to survey changes in noise and overall expression levels from two homogeneously expressed promoters across different positions on the chromosome.
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Martindale, Chloe. "Isogeny graphs, modular polynomials, and applications." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0086/document.
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Dans ma thèse j'etude les variétés abéliennes ordinaires définies avec multiplication réelle maximale. Je définis des polynômes modulaires dans ce situation et je donne un algorithme pour calculer sur les nombres complexes et pour les surfaces sur des corps finis. Je donne aussi un théorème de structure pour les graphs des isogénies dans ce contexte. Je donne une généralisation de Schoof-Elkies-Atkin aux courbes de genre 2 avec multiplication réelle maximale fixe en utilisant les polynômes modulairesMy thesis looks at ordinary abelian varieties defined with maximal real multiplication. I define modular polynomials in this setting and give an algorithm to compute them over the complex numbers, and for surfaces over finite fields. I also give a structure theorem for isogeny graphs in this setting. I give a generalisation of Schoof-Elkies-Atkin to genus 2 curves with fixed maximal real multiplication using the modular polynomials
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Sadovski, Jenny [Verfasser]. "Keratinozytenwachstumsfaktor reduziert das alveoläre Ödem in isogenen Lungentransplantaten der Ratte / Jenny Sadovski." Gießen : Universitätsbibliothek, 2014. http://d-nb.info/1068538740/34.
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Van, Eck Leon. "Aphid-induced transcriptional regulation in near-isogenic wheat." Diss., Pretoria : [s.n.], 2007. http://upetd.up.ac.za/thesis/available/etd-07152007-195017.
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Pirozi, Mônica Ribeiro. "Isogenic wheat lines in determining composition/functionality relationships /." Search for this dissertation online, 2003. http://wwwlib.umi.com/cr/ksu/main.
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Silva, Velón Javier. "Zero-knowledge proofs and isogeny-based cryptosystems." Doctoral thesis, Universitat Pompeu Fabra, 2021. http://hdl.handle.net/10803/671222.
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In this thesis, we present some public-key cryptographic schemes. This work is
divided in two halves. The rst half deals with zero-knowledge proofs in the
classical setting and under falsi able assumptions. In particular, we improve
upon the e ciency of an argument for linear equations, and we present a proof
of correct computation of a circuit that is of size logarithmic in the depth of
the circuit. In the second half, we introduce a signature scheme, an encryption
scheme and a trapdoor DDH scheme based on isogenies of supersingular elliptic
curves. The signature and encryption schemes are secure against quantum
adversaries.En aquesta tesi presentem alguns esquemes criptogràfics de clau pública. Aquest treball consta de dues parts. La primera meitat tracta de proves de coneixement nul en el context clàssic i basades en hipòtesis falsificables. En particular, millorem l’eficiència d’un argument de coneixement nul per a equacions lineals i presentem una prova de computació correcte d’un circuit que te una mida logarítmica en la profunditat del circuit. A la segona meitat, introduïm un esquema de signatures, un esquema de xifratge i un esquema DDH de trampa basat en lisogènies de corbes el·líptiques supersingulars. Els esquemes de signatura i xifrat són segurs contra adversaris quàntics.
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Langley, Roger Sean. "Metal binding by Pseudomonas aeruginosa PAO1 and isogenic lipopolysaccharide mutants." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ33244.pdf.
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Yan, Fengsheng. "Tate property and isogeny estimate for semi-abelian varieties /." [S.l.] : [s.n.], 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10798.
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Delfs, Christina [Verfasser], Andreas [Akademischer Betreuer] Stein, and Florian [Akademischer Betreuer] Hess. "Isogenies and endomorphism rings of abelian varieties of low dimension / Christina Delfs. Betreuer: Andreas Stein ; Florian Hess." Oldenburg : BIS der Universität Oldenburg, 2015. http://d-nb.info/1093683937/34.
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Delfs, Christina Verfasser], Andreas [Akademischer Betreuer] [Stein, and Florian [Akademischer Betreuer] Hess. "Isogenies and endomorphism rings of abelian varieties of low dimension / Christina Delfs. Betreuer: Andreas Stein ; Florian Hess." Oldenburg : BIS der Universität Oldenburg, 2015. http://nbn-resolving.de/urn:nbn:de:gbv:715-oops-28173.
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Knudsen, Gabriel Arther. "Investigating complex phenotypes: haplotype association mapping benzene pharmacokinetics in isogenic mouse strains." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/202998.
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A role for gene variants in regulating the pharmacokinetics of systemically available toxicants has not yet been established. A panel of 18 genetically-diverse inbred mouse strains was used to determine the range of total exposure kinetic parameters in blood and bone marrow following a single oral administration of benzene (100 μg/kg) to male and female mice. Large ranges in several pharmacokinetic parameters were found when data from blood and bone marrow were analyzed. AUC and CL_F pharmacokinetic parameters in blood and bone marrow pharmacokinetics were strikingly different as were these parameters in males and females. Final clearance (CL_F) was found to be the most statistically robust pharmacokinetic parameter as it accounted for exposure of the matrix (AUC) and normalized for dose variations among the strains. The CL_F values in blood and bone marrow used for haplotype association mapping showed 331 and 164 quantitative trait loci with statistical significance, respectively (male mice; -logP>4). Two loci were found to be shared between males and females QTL bone marrow data sets and one common locus was found for male blood and bone marrow data. No overlap was found among blood QTL in males and females (or between blood and bone marrow data from females). Protein and mRNA expression data for the primary benzene-metabolizing enzymes CYP2E1 and UGT1A6 showed very little strain-dependent variation. Strain dependent differences in mRNA levels of NQO1 and MPO were small but statistically significant, as were those for GAPDH and β2-microglobulin. These data demonstrated that polymorphisms with the greatest contribution toward overall variations in systemic exposures occurred in genes encoding for non-metabolic proteins. While exposure does not equate to toxicity, identification of the genes regulating distribution and clearance may be useful for investigating host susceptibility to toxic effects following benzene exposure. This research was supported in part by the NIEHS NTP Grant N01ES45529, NIEHS Toxicology and Toxicogenomics Training Grant (5T32ES007091-29), NIEHS/NTP Division of Intramural Research, and Southwest Environmental Science Center Grant P3ES06694.
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Oral, Münevver. "Insights into isogenic clonal fish line development using high-throughput sequencing technologies." Thesis, University of Stirling, 2016. http://hdl.handle.net/1893/24909.
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Isogenic clonal fish lines are a powerful resource for aquaculture-related research. Fully inbred individuals, clone founders, can be produced either through mitotic gynogenesis or androgenesis and a further generation from those propagates fully inbred clonal lines. Despite rapid generation, as opposed to successive generation of sibling mating as in mice, the production of such lines may be hampered due to (i) potential residual contribution from irradiated gametes associated with poorly optimised protocols, (ii) reduced survival of clone founders and (iii) spontaneous arisal of meiotic gynogenetics with varying degree of heterozygosity, contaminating fully homozygous progenies. This research set out to address challenges and gain insights into isogenic clonal fish lines development by using double-digest RADseq (ddRADseq) to generate large numbers of genetic markers covering the genome of interest. Analysis of potential contribution from irradiated sperm indicated successful uniparental inheritance in meiotic and mitotic gynogenetics European seabass. Exclusive transmission of maternal alleles was detected in G1 progeny of Atlantic salmon (with a duplicated genome), while G2 progenies presented varying levels of sire contribution suggesting sub-optimal UV irradiation which was undetected previously with 27 microsatellite markers. Identification of telomeric markers in European seabass, with higher recombination frequencies for efficient differentiation of meiotic and mitotic gynogenetics was successful, and a genetic linkage map was generated from this data. One clear case of a spontaneous meiotic gynogenetic fish was detected among 18 putative DH fish in European seabass, despite earlier screening for isogenicity using 11 microsatellite markers. An unidentified larval DNA restriction digestion inhibition mechanism observed in Nile tilapia prevented the construction of SNP-based genetic linkage map. In summary, this study provides strong evidence on efficacy of NGS technologies for the development and verification of isogenic clonal fish lines. Reliable establishment of isogenic clonal fish lines is critical for their utility as a research tool.
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Peters, Derek Tilghman. "Isogenic Human Pluripotent Stem Cell Models of Cardiovascular Disease-Associated Genetic Variation." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493401.
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A complex interplay of genetic and environmental factors underlies the development of common human diseases such as cardiovascular disease. Despite widespread use of existing medications, coronary heart disease (CHD), including myocardial infarction (MI), remains a significant cause of morbidity and mortality worldwide. The results of recent human genetic studies have provided unprecedented opportunities to elucidate the genes and molecular pathways that underlie CHD and risk factors like plasma lipid concentrations. Translating these findings into mechanistic insight promises to improve our understanding of disease pathogenesis and aid in the development of novel therapies. Precise functional characterization is required to bridge this gap; yet doing so can be challenging using traditional approaches.
The properties of human pluripotent stem cells (hPSCs) make them uniquely suited to address this challenge - they retain a normal human genome in culture, can be genetically modified, and can be differentiated into multiple cell types. The work presented in this thesis demonstrates the use of hPSCs and genome editing technology to generate human disease models in vitro. We developed a genome editing system optimized for hPSCs that can be used to efficiently generate hPSC lines with targeted genetic modifications. Modified and isogenic control hPSCs are then differentiated into a relevant cell type for phenotypic characterization. We applied this approach to investigate the functional effects of human genetic variation underlying plasma lipid concentrations.
We used TALEN genome editing to clarify the role of the SORT1 gene as a mediator of cellular metabolic processes. We targeted the ANGPTL3 gene using TALEN and CRISPR/Cas9 genome editing to create an in vitro model of a monogenic disorder, familial combined hypolipidemia, and investigated the putative role of ANGPTL3 in the regulation of low-density lipoprotein cholesterol metabolism. To facilitate the use of hPSC-models to study subtle phenotypes involving human liver, we developed a method for purifying hepatocyte-like cells following hPSC differentiation. Finally, we combined these approaches to model the tissue-specific effects of a common non-coding genetic variant in the chromosome 1p13 locus that is associated with risk of MI. This result demonstrates the feasibility of using hPSCs to characterize disease-associated common genetic variation.Medical Sciences
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Valera, Martín Javier. "Contribuciones a la cardinalidad de curvas elípticas y a los volcanes de isogenias." Doctoral thesis, Universitat de Lleida, 2017. http://hdl.handle.net/10803/457772.
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Avui en dia, un dels problemes matemàtics més utilitzats a l’hora de
dissenyar protocols criptogràfics és el problema del logaritme discret sobre
el grup de punts d’una corba el.líptica definida sobre un cos finit (ECDLP –
Elliptic Curve Discrete Logarithm Problem). No obstant, no totes les corbes
el.líptiques existents són vàlides per a aquest problema. Pel que se sap fins
ara, la validesa per al ECDLP d’una corba el.líptica E definida sobre un cos
finit Fq depèn del seu cardinal sobre Fq. Donat que el càlcul del cardinal de
E és un problema computacionalment costós, sembla raonable pensar que
si E és vàlida, puguem obtenir a partir d’ella altres corbes el.líptiques que
també ho siguin, és a dir, que també tinguin el seu mateix cardinal sobre
Fq. Per dur a terme aquesta tasca nom´es hem de calcular corbes el.líptiques
d–isògenes a E sobre Fq, és a dir, hem de calcular d–isogènies Fq–racionals.
Sigui ℓ un nombre primer tal que ℓ no divideix a q. El conjunt de totes
les classes d’isomorfia sobre Fq de corbes el.líptiques ordinàries amb un determinat
cardinal sobre Fq pot ser representat mitjan¸cant un graf dirigit on
els vèrtexs són les classes d’isomorfia i on els arcs representen ℓ–isogènies
Fq–racionals entre corbes el.líptiques dels vèrtexs. Cada component connexa
d’aquest digraf és un volcà de ℓ–isogènies o ℓ–volcà sobre Fq. Els vèrtex d’un ℓ–volcà es distribueixen per nivells. El nombre total de nivells menys
un és la seva altura. Calcular l’altura d’un ℓ–volcà pot millorar l’eficiència
de l’algoritme SEA, sent aquest algoritme el millor conegut fins ara per al
càlcul del cardinal d’una corba el.líptica. Altres aplicacions dels volcans de
ℓ–isogènies les trobem en el càlcul dels polinomis de classes de Hilbert o dels polinomis modulars. En totes elles cal recórrer els vèrtexs de ℓ–volcans.
En aquesta tesi, per una banda, donem nous mètodes per recórrer els
vèrtexs dels volcans de ℓ–isogènies. Per l’altra, coneguda la valoració ℓ–àdica
del cardinal de E sobre Fq, estudiem la valoració ℓ–àdica del cardinal de E
sobre una extensió de grau k de Fq. Coneguda l’estructura del subgrup de
ℓ–Sylow de E sobre Fq, també estudiem la del subgrup de ℓ–Sylow de E
sobre Fqk .Aunque uno de los problemas matemáticos más utilizados hoy en día en el diseño de protocolos criptográficos es el problema del logaritmo discreto sobre el grupo de puntos de una curva elíptica definida sobre un cuerpo finito (ECDLP – Elliptic Curve Discrete Logarithm Problem), no todas las curvas elípticas existentes son válidas para su uso en el. Por lo que se sabe hasta ahora, la validez para el ECDLP de una curva elíptica E definida sobre un cuerpo finito Fq depende de su cardinal sobre Fq. Como calcular el cardinal de E es un problema computacionalmente costoso, parece razonable pensar que si E es válida, podamos obtener a partir de ella otras curvas elípticas que también lo sean, es decir, que también tengan su mismo cardinal sobre Fq. Para ello lo único que tenemos que hacer es calcular curvas elípticas d–isógenas a E sobre Fq, es decir, debemos calcular d–isogenias Fq–racionales. Sea ℓ un número primo tal que ℓ no divide a q. El conjunto de todas las clases de isomorfía sobre Fq de curvas elípticas ordinarias con un determinado cardinal sobre Fq puede ser representado mediante un grafo dirigido cuyos vértices son las clases de isomorfía y cuyos arcos representan ℓ–isogenias Fq–racionales entre curvas elípticas de los vértices. Cada componente conexa de este digrafo es un volcán de ℓ–isogenias o ℓ–volcán sobre Fq. Los vértices de un ℓ–volcán se distribuyen por niveles. El número total de niveles menos uno es su altura. Calcular la altura de un ℓ–volcán puede mejorar la eficiencia del algoritmo SEA, siendo el SEA el mejor algoritmo conocido actualmente para calcular el cardinal de una curva elíptica. Otras 4 aplicaciones de los volcanes de ℓ–isogenias las encontramos en el cálculo de los polinomios de clases de Hilbert o los polinomios modulares. En todas ellas es preciso recorrer los vértices de ℓ–volcanes. En esta tesis, por un lado, damos nuevos métodos para recorrer los vértices de los volcanes de ℓ–isogenias. Por otro lado, conocida la valoración ℓ–ádica del cardinal de E sobre Fq, estudiamos la valoración ℓ–ádica del cardinal de E sobre una extensión de grado k de Fq. Conocida la estructura del subgrupo de ℓ–Sylow de E sobre Fq, también estudiamos la del subgrupo de ℓ–Sylow de E sobre Fqk .
One of the most used mathematical problems for the design of modern cryptographic protocols is the discrete logarithm problem over the group of points of an elliptic curve defined over a finite field (ECDLP). However, not all existing elliptic curves are valid for this problem. The validity for the ECDLP of an elliptic curve E defined over a finite field Fq depends on its cardinality over Fq. The computation of the group order of E is an expensive task. Therefore, if E has a “good” cardinality, it seems reasonable to obtain from E other elliptic curves with the same cardinality. For this task, we can compute some Fq–rational d–isogenies of E, where d is a positive integer. Let ℓ be a prime number such that ℓ does not divide q. The set of all Fq–isomorphism classes of ordinary elliptic curves with a given group order over Fq can be represented as a directed graph whose vertices are the Fq–isomorphism classes and whose arcs represent Fq–rational ℓ–isogenies. Each connex component of this graph is a volcano of ℓ–isogenies or ℓ–volcano over Fq. The vertices of a volcano of ℓ–isogenies can be stratified into levels. The number of levels minus one is called the height of the ℓ–volcano. The computation of this value can improve the SEA algorithm (the known best algorithm to compute the cardinality of an elliptic curve). Volcanoes of ℓ–isogenies have also been used to compute the Hilbert class polynomials or to compute the modular polynomials. In all these applications, it is necessary to go through the vertices of ℓ–volcanoes. In this thesis, on one hand, we give new methods to go through the vertices of the ℓ–volcanoes. On the other hand, assuming the knowledge of the ℓ–adic valuation of the cardinality of E over Fq, we study the ℓ–adic valuation of the cardinality of E over an extension of degree k over Fq. Assuming the structure of the ℓ–Sylow subgroup of E over Fq is known, we also study the structure of the ℓ–Sylow subgroup of E over Fqk .
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Ubina, Teresa Marie. "AN ISOGENIC STEM CELL MODEL OF ALZHEIMER'S DISEASE: DIRECT EXPRESSION OF AMYLOID-BETA." CSUSB ScholarWorks, 2017. https://scholarworks.lib.csusb.edu/etd/523.
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Alzheimer’s disease (AD), identified over 100 years ago and intensively studied since the 1970s, has no effective treatments or mechanistic understanding of the underlying neurodegenerative process. Most investigators believe accumulation or aggregation of amyloid beta (Ab) proteins plays a causative role. Aβ peptides (~39-43 residues) are generated by proteolysis of the transmembrane protein APP. One reason we know so little about AD is an incomplete understanding of the cellular mechanisms responsible for Ab proteotoxicity. Human ES and iPSC models of AD are recent additions to many other models used to investigate these mechanisms. AD, however is a chronic progressive condition of old age and cultured neurons may not live long enough to model what goes wrong in neurons from AD patients. In my research, I used hESCs which directly express Ab peptides thus avoiding the time it takes to process APP. One App allele in H9 hESCs was previously edited using TALEN. A homologous recombination cassette coding directly for a secretory form of either Ab1-42 or Ab1-40 and containing a stop codon, was inserted into the first exon of App upstream of the normal translational start site. I used multiple independently isolated clones of edited cells with 3 genotypes: App/App (unedited), App/Aβ1-40 and App/Aβ1-42. Expression of Ab from edited alleles was confirmed by qRT-PCR using primers specific for the edit. I first sought to establish if editing changed any aspects of neuronal differentiation in culture. All 3 genotypes have similar embryoid body (EB) development, and similar numbers and sizes of neuronal clusters (NC) up to 34 days after EB dissociation and neural differentiation. Immunostaining of neuronal markers, NeuN and DCX (doublecortin), likewise revealed no difference among edited and unedited cells, suggesting that the edits do not affect the ability of my stem cells to differentiate into neurons. I next measured accumulation of aggregated Ab using an aggregate specific antibody, 7A1a. Data at 34-days post EB dissociation indicates NCs in the Aβ1-42 edited cells accumulate significantly more aggregates relative to either unedited or Ab1-40 edited lines, a result consistent with the increased ability for Ab1-42 to form aggregates. Aβ aggregates also appear to be concentrated around fragmented nuclei within neuronal clusters suggesting that intracellular accumulation may play a key role in proteotoxicity. Additionally, I observed a significant decrease in the number of synapsin1 puncta, a marker of synapses, another feature of AD. I documented a nearly 3-fold greater neuronal cell death in both the Aβ1-40 and Aβ1-42 neurons at 70 days after differentiation. RNA sequencing data also shows independently isolated clones group together and show differential expression of genes related to memory and neuronal cell death. The early presence of Aβaggregation and subsequent cell death is in line with the chronic and progressive nature of AD and this is the first known model to exhibit a neurodegenerative phenotype. These isogenic cell lines thus appear to be useful to screen for therapeutics that may prevent or slow Ab1-42 dependent neurodegeneration and a tool to investigate Ab-dependent mechanisms with relevance to AD.
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Milio, Enea. "Calcul de polynômes modulaires en dimension 2." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0285/document.
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Les polynômes modulaires sont utilisés dans le calcul de graphes d’isogénies, le calcul des polynômes de classes ou le comptage du nombre de points d’une courbe elliptique, et sont donc fondamentaux pour la cryptographie basée sur les courbes elliptiques. Des polynômes analogues sur les surfaces abéliennes principalement polarisées ont été introduits par Régis Dupont en 2006, qui a également proposé un algorithme pour les calculer, et des résultats théoriques sur ces polynômes ont été donnés dans un article de Bröker–Lauter, en 2009. Mais les polynômes sont très gros et ils n’ont pu être calculés que pour l’exemple minimal p = 2. Dans cette thèse, nous poursuivons les travaux de Dupont et Bröker–Lauter en permettant de calculer des polynômes modulaires pour des invariants basés sur les thêta constantes, avec lesquels nous avons pu calculer les polynômes jusqu’à p = 7, tout en démontrant des propriétés de ces polynômes. Mais des exemples plus grands ne semblent pas envisageables. Ainsi, nous proposons une nouvelle définition des polynômes modulaires dans laquelle l’on se restreint aux surfaces abéliennes principalement polarisées qui ont multiplication réelle par l’ordre maximal d’un corps quadratique réel afin d’obtenir des polynômes plus petits. Nous présentons alors de nombreux exemples de polynômes et des résultats théoriquesModular polynomials on elliptic curves are a fundamental tool used for the computation of graph of isogenies, class polynomials or for point counting. Thus, they are fundamental for the elliptic curve cryptography. A generalization of these polynomials for principally polarized abelian surfaces has been introduced by Régis Dupont in 2006, who has also described an algorithm to compute them, while theoretical results can been found in an article of Bröker– Lauter of 2009. But these polynomials being really big, they have been computed only in the minimal case p = 2. In this thesis, we continue the work of Dupont and Bröker–Lauter by defining and giving theoretical results on modular polynomials with new invariants, based on theta constants. Using these invariants, we have been able to compute the polynomials until p = 7 but bigger examples look intractable. Thus we define a new kind of modular polynomials where we restrict on the surfaces having real multiplication by the maximal order of a real quadratic field. We present many examples and theoretical results
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Lee, Yi-Chen. "MENDELIZING QUANTITATIVE TRAIT LOCI THAT UNDERLIE RESISTANCE TO SOYBEAN SUDDEN DEATH SYNDROME." OpenSIUC, 2016. https://opensiuc.lib.siu.edu/theses/1999.
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Soybean (Glycine max [L.] Merr.) cultivars differ in their resistance to sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Breeding for improve SDS response has proven challenging, possible due to interactions among the 18 known loci for resistance. Four loci for resistance to SDS (cqRfs to cqRfs3) were found clustered within 20 cM of the rhg1 locus underlying resistance to soybean cyst nematode (SCN) on chromosome 18. Another locus on chromosome 20 (cqRfs5) was reported to interact with this cluster. The aims of this study were to compare the inheritance of resistance to SDS in a near isogenic line (NIL) population that was fixed for resistance to SCN but still segregated at 2 of the 4 loci (cqRfs1 and cqRfs) for resistance to SDS on chromosome 18; to examine the interaction with the locus on chromosome 20; and to identify candidate regions underlying quantitative trait loci (QTL). Used were a near isogenic line population derived from residual heterozygosity in an F5:7 recombinant inbred line EF60 1-40; SDS response data from 2 locations and years; four microsatellite markers and six thousand SNP markers. Polymorphic regions were found from 2,788 to 8,938 Kbp on chromosome 18 and 33,100 to 34,943 Kbp on chromosome 20. Both regions were significantly (0.005 < P > 0.0001) associated with resistance to SDS. A fine map was constructed that Mendelized the three loci. Substitution maps suggested the two loci on chromosome 18 were actually 3 loci (cqRfs, cqRfs1 and cqRfs19). Candidate genes for cqRfs19 were identified in a small region of the genome sequence of soybean. An epistatic interaction was inferred where the allele of loci on chromosome 18 determined the value of the locus on chromosome 20. It was concluded that SDS loci are both complex and interacting which may explain the slow progress in breeding for resistance to SDS.
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Etzold, Anna [Verfasser]. "Molekulargenetische und zellbiologische Charakterisierung primärer Fibroblasten einer Patientin mit konstitutiver BRCA1-Epimutation und multiplen Neoplasien im isogenen Zwillingsvergleich / Anna Etzold." Mainz : Universitätsbibliothek der Johannes Gutenberg-Universität Mainz, 2016. http://d-nb.info/1225685532/34.
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Abbott, Heather Elizabeth. "Comparisons of the factors influencing intrinsic radiosensitivity between two isogenic human ovarian carcinoma cell lines." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0021/MQ57080.pdf.
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Finnie, Sean McIlwain. "Wheat polar lipids : sources of variation among near-isogenic wheat lines with different endosperm hardness." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/1384.
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Kastl, Christin. "Metabolomic Discrimination of Near Isogenic Low and High Phytate Soybean [GLYCINE MAX (L.) MERR.] Lines." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/64195.
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Phytate is the major storage form of phosphorus in seeds of soybeans. Because phytate chelates mineral cations including calcium, iron, and zinc, these mixed salts are often excreted by non-ruminant animals such as humans, swine, poultry, and fish. While this causes iron and zinc deficiencies, phytate is also considered a water pollutant due to the excess phosphorus excreted in animal waste. These negative environmental and nutritional effects, create a need for low phytate soybeans. While several low phytate soybean lines have been developed, a major drawback is the reduced seedling emergence of these lines resulting in low yields. Therefore, understanding the genetic and molecular bases of the low emergence trait in relation to seed phytate content in major crops such as soybean is of great economic importance.
This PhD project worked towards the long term goal of developing low phytate soybean cultivars with good seedling emergence and high-yield. This dissertation focused on metabolomic differences between low and normal phytate lines and how these could relate to the low emergence phenotype. The genetic materials used here include four near isogenic lines that differ in mutations in two multi drug resistance-associated proteins (MRPs). Only the line with both mutations was low phytate.
The phytate levels, field- and lab-based emergence rates were determined for these lines, their parents and a control line through replicated field experiments for three consecutive years. The
emergence rates of the low phytate lines were not always reduced. This showed that the environment the seeds were produced in is highly important, especially when breeding and commercially growing low phytate lines.
A protocol was developed for successful metabolomic discrimination of these closely related soybean lines. The polar and non-polar metabolite profiles were determined using ultra performance liquid chromatography mass spectrometry and metabolomic differences between the low and normal phytate lines were identified. The low phytate double mutant did not contain C22 glucose terminated Group A soyasaponins and almost exclusively contained C22 xylose terminated Group A soyasaponins (A4, A5 and A6). Compared to the normal phytate lines, the low phytate soybean line showed a higher concentration of storage lipids (triacylglycerols and diacylglycerols) and certain phospholipids.Ph. D.
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Mahone, Gregory S. [Verfasser]. "Strategies for the design and analysis of introgression libraries and near-isogenic lines / Gregory S. Mahone." Gießen : Universitätsbibliothek, 2016. http://d-nb.info/108162745X/34.
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Zulfiqar, Shadaan [Verfasser], and Katja [Akademischer Betreuer] Nieweg. "APOE isoform-specific effects in isogenic human iPSC-derived neural cells / Shadaan Zulfiqar ; Betreuer: Katja Nieweg." Marburg : Philipps-Universität Marburg, 2020. http://d-nb.info/1213447143/34.
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Bobenko, Anna [Verfasser]. "Generierung eines humanen isogenen FlpIn -Expressions-Zelllinienpanels mit Hilfe von AAVS1-Zinkfinger-Nukleasen zur Untersuchung von Kandidatengenen bei kardiometabolischen Erkrankungen / Anna Bobenko." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2016. http://d-nb.info/1101455241/34.
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Wu, Shan. "The roles of OVATE and other elongation genes in regulating proximal-distal patterning of tomato fruit." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437586702.
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Gleißner, Christian [Verfasser], and Ingrid [Akademischer Betreuer] Bauer. "Threefolds Isogenous to a Product and Product quotient Threefolds with Canonical Singularities / Christian Gleißner ; Betreuer: Ingrid Bauer." Bayreuth : Universität Bayreuth, 2016. http://d-nb.info/1114270687/34.
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Mondal, Suchismita. "Use of near-isogenic wheat lines to determine glutenin and gliadin composition and funtionality in flour tortillas." Thesis, Texas A&M University, 2006. http://hdl.handle.net/1969.1/4390.
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The synthesis of high molecular weight (HMW) glutenin, low molecular weight glutenin and gliadin proteins are controlled by nine major loci present in wheat chromosomes. The loci Glu A1, Glu B1, Glu D1 and Gli A1, Gli B1, Gli D1 and Gli 2 and their allelic variants play important roles in determining the functional properties of wheat flour. This study focused on understanding the functionality of these protein subunits with respect to tortilla quality for use in developing varieties with ideal tortilla baking quality. Near-isogenic wheat lines in which one or more of these loci were absent or deleted were used in the study. These lines were analyzed using SSR primers to verify the chromosome deletions. A standard SDS PAGE gel and a Lab on Chip Capillary Electrophoresis method were used to confirm the protein composition of the deletion lines. Tortillas were prepared from each deletion line and the parent lines used to derive the deletion lines, and tortilla quality evaluations were analyzed. The analysis has revealed that elimination of certain HMW glutenins results in gain of function both for tortilla diameters and overall tortilla quality. The deletion line possessing 17+18 at Glu B1 and deletions in Glu A1 and Glu D1 had a gain of function in tortilla diameter, yet tortilla stability was compromised. The deletion line possessing Glu A1, Glu D1 (1,5+10) and a deletion in Glu B1 improved both the diameters and stability of the tortillas. Presence of subunits 5+10 is important for maintaining tortilla stability. Deletions in gliadin monomeric proteins also affected the tortilla diameters and stability.
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Marchosky, Ruben, Peter C. Ellsworth, Hal Moser, and T. J. Henneberry. "Bollgard® and Bollgard II® Efficacy in Near Isogenic Lines of 'DP50' Upland Cotton in Arizona." College of Agriculture, University of Arizona (Tucson, AZ), 2001. http://hdl.handle.net/10150/211299.
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The Cry1Ac gene (Bollgard®) is available in cotton either alone ('B') or in combination (Bollgard II®) with a second gene, Cry2Ab ('X'). We evaluated these two different transgenes, separately and together, in near isogenic lines of the upland cotton variety ‘DP50’. DP50B was previously transformed with the Cry2Ab gene to give rise to the experimental line 985BX which was then back-crossed to DP50 to produce near isogenic single gene variants, 985B and 985X. The lepidopteran target was pink bollworm (PBW), Pectinophora gossypiella (Saunders), which was evaluated in two field studies through a series of samples from artificially and naturally-infested bolls. In one study (NTO), three cotton lines (DP50, DP50B, 985BX) were evaluated under three spray regimes. In the second study (Isoline), five near isogenic lines (DP50, DP50B, 985B, 985X, 985BX) were evaluated under two spray regimes: fully sprayed and lepidopteran unsprayed. In lines containing only one transgene, Cry1Ac or Cry2Ab, bolls had consistently fewer PBWs than the non-Bt variety. Very few PBWs developed into large (3rd instar) larvae in these Bt varieties. The majority (NTO: 83%; Isoline: 94%) of PBWs recovered were dead first instar larvae. Less than 5% of the DP50B bolls in the NTO study were infested with feral large (≥3rd instar) larvae, and large larvae were present in less than 2% of naturally-infested bolls of single-gene lines in the Isoline study. PBW age and mortality distributions confirmed that the single transgenes were effective in stopping PBW development and killing young instars. Cry2Ab displayed a broader spectrum of efficacy as it was significantly more effective against citrus peelminer (Marmara spp.), an incidental lepidopteran present in high densities in the tests. The two-gene (Cry1Ac + Cry2Ab) line showed better (at least 10-fold) efficacy than the single-gene lines against PBW large larvae infestation. The PBW age distributions found in this variety consisted almost entirely (98%) of dead first instar larvae. Less than 0.6% of the bolls of the two-gene variety in the NTO study were infested with large (≥3rd instar) larvae, and there was no infestation by large larvae in any of the naturally-infested bolls in the Isoline study. Yields and other agronomic parameters of the two-gene and single-gene varieties were superior or similar to the null parent. Second pick yields of all Bt varieties were significantly higher than the recurrent parent non-Bt line, suggesting a high degree of efficacy against typically high PBW densities during the late season. Cotton lines with transgenes (Cry1Ac & Cry2Ab) separately and combined demonstrated a high degree of efficacy and agronomic performance for usage in Arizona against PBW. The ramifications of isogenic comparisons of PBW incidence on efficacy and resistance monitoring are discussed.
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Gemmill, Jamie Michael. "An evaluation of the apetalous character in winter oilseed rape (Brassica napus L.) using near-isogenic lines." Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398997.
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Kelly, Rhys Owen. "Molecular and physiological aspects of arbuscular mycorrhizal symbiosis with near-isogenic white clover : a gene expression study." Thesis, University of South Wales, 2004. https://pure.southwales.ac.uk/en/studentthesis/molecular-and-physiological-aspects-of-arbuscular-mycorrhizal-symbiosis-with-nearisogenic-white-clover-a-gene-expression-study(d43e48e0-fee7-44d0-8c9f-5d690fe185b2).html.
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White clover (Trifolium repens L.) is an important component of low input pasture systems and can form effective associations with both nitrogen-fixing Rhizobium and phosphate-scavenging arbuscular mycorrhizal fungi (AMF). Understanding the genetic control of plant-AM interactions will allow clover breeders to develop genetic markers for selection of phenotypes favourable to AM symbiosis. Clover is normally an out-breeding species. Near-isogenic lines (NILs) developed from inbred lines of clover are ideal candidates for genetic studies since important agronomic traits are fixed. Four closely-related NILs provided plant material with contrasting phenotypes when inoculated with AMF Glomus mosseae. Seed-grown and clonal plants of these closely-related NILs were used to study gene expression associated with AM colonization and functioning. In order to relate plant phenotype responses specifically to changes in phosphorus availability, and ultimately to gene expression, a nutrient flowing culture system was developed in which nutrient application was constant and could be controlled. The only consistent phenotypic response discovered was a decrease in root production in response to AMF colonisation. Differential display demonstrated differences in gene expression in both leaves and roots of clover with and without AMF. These genes were mainly down-regulated in AMF plants and three were identified from a range of NILs under varying P concentrations. More apparent differences in gene expression were found in roots than in leaves, and in low rather than high AMF colonising lines. In total, 45 sequences were identified and cloned; 30 from leaves and 15 from roots. Sixteen sequences had homology with known genes, including protochlorophyllide oxido reductase and leghaemoglobin. The majority corresponded to ESTs from model legumes and from studies such as colonisation with AMF or Rhizobium, and phosphate or nitrogen starvation experiments. Six sequences were not found on any database, indicating that these corresponded to undiscovered genes either expressed or suppressed in AM white clover.
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Perez, Marcos Francisco 1987. "Inter-individual physiological variation in the nematode Caenorhabditis elegans." Doctoral thesis, Universitat Pompeu Fabra, 2018. http://hdl.handle.net/10803/664089.
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For unknown reasons, genetically identical animals often differ substantially in their phenotypic traits, even in a controlled environment. Here I investigate the causes of inter-individual physiological variation using a model organism, the nematode Caenorhabditis elegans. Isogenic nematodes vary in their size at hatching, speed of development, growth rate, starvation resistance and fecundity. I show that much of this variation is due to the age of an individual’s mother, with young mothers producing progeny impaired for many traits. The underlying molecular mechanism for multiple traits is a progressive, age-dependent increase in the maternal provisioning of a lipoprotein complex, yolk/vitellogenin, to embryos. The production of sub-optimal progeny by young mothers likely reflects a trade-off between the competing fitness traits of a short generation time and progeny survival and fecundity. These results identify age-dependent changes in maternal provisioning to embryos as an important source of phenotypic variation throughout the life of an animal.Por causas desconocidas, animales idénticos genéticamente suelen variar sustancialmente en sus rasgos fenotípicos, aunque el ambiente en el que habiten sea el mismo. Aquí investigo las causas de variación fisiológica entre individuos utilizando un organismo modelo, el nematodo Caenorhabditis elegans. Nematodos isogénicos varían en su tamaño al nacer, en su velocidad de crecimiento y desarrollo, en su resistencia a la privación de alimento y en su fecundidad. Muestro que muchas de estas variaciones se deben a la edad de la madre del individuo, con las madres jóvenes engendrando a progenie peor en muchos respetos. Para muchos rasgos fenotípicos, el mecanismo molecular subyacente es un aumento progresivo con la edad de la madre de la provisión de un complejo lipoproteínico, yema/vitellogenin, a los embriones. Ecológicamente, es probable que la producción de progenie inferior por parte de las madres jóvenes esté compensada por la ventaja opuesta de tener un tiempo generacional corto. Los resultados presentados destacan los cambios en la provisión maternal de recursos a los embriones como una fuente significativa de variación fenotípica a lo largo de la vida de un animal.
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Brewer, Lauren Renee. "IR microspectroscopic imaging discriminates isogenic null waxy from parent wheats with lipid class profile supported by compositional analyses." Thesis, Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/1641.
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Bisson, Gaëtan. "Anneaux d'endomorphismes en cryptographie." Thesis, Vandoeuvre-les-Nancy, INPL, 2011. http://www.theses.fr/2011INPL047N/document.
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La cryptographie est indispensable aux réseaux de communication modernes afin de garantir la sécurité et l'intégrité des données y transitant. Récemment, des cryptosystèmes efficaces, sûr et riches ont été construits à partir de variétés abéliennes définies sur des corps finis. Cette thèse contribue à plusieurs aspects algorithmiques de ces variétés touchant à leurs anneaux d'endomorphismes. Cette structure joue un rôle capital pour construire des variétés abéliennesmunies de bonnes propriétés, comme des couplages, et nous montrons qu'un plus grand nombre de telles variétés peut être construit qu'on ne pourrait croire. Nous considérons aussi le problème inverse qu'est celui du calcul de l'anneau d'endomorphismes d'une variété abélienne donnée. Les meilleures méthodes connues ne pouvaient précédemment résoudre ce problème qu'en temps exponentiel ; ici, nous concevons plusieurs algorithmes de complexité sous-exponentielle le résolvant dans le cas ordinaire. Pour les courbes elliptiques, nous bornons rigoureusement la complexité de nos algorithmes sous l'hypothèse de Riemann étendue et démontrons qu'ils sont extrêmement efficaces en pratique. Comme sous-routine, nous développons notamment un algorithme sans mémoire pour résoudre une généralisation du problème du sac à dos. Nous généralisons aussi notre méthode aux variétés abélienne de dimension supérieure. Concrètement, nous développons une bibliothèque qui permet d'évaluer des isogénies entre variétés abéliennes ; cet outil nous permet d'appliquer une généralisation de notre méthode à des exemples jusqu'alors incalculablesModern communications heavily rely on cryptography to ensure data integrity and privacy. Over the past two decades, very efficient, secure, and featureful cryptographic schemes have been built on top of abelian varieties defined overfinite fields. This thesis contributes to several computational aspects of ordinary abelian varieties related to their endomorphism ring structure. This structure plays a crucial role in the construction of abelian varieties with desirable properties, such as pairings, and we show that more such varieties can be constructed than expected. We also address the inverse problem, that of computing the endomorphism ring of a prescribed abelian variety. Prior state-of-the-art methods could only solve this problem in exponential time, and we design several algorithms of subexponential complexityfor solving it in the ordinary case. For elliptic curves, we rigorously bound the complexity of our algorithms assuming solely the extended Riemann hypothesis, and demonstrate that they are very effective in practice. As a subroutine, we design in particular a memory-less algorithm to solve a generalization of the subset sum problem. We also generalize our method to higher-dimensional abelian varieties. Practically speaking, we develop a library enabling the computation of isogenies between abelian varieties; this building block enables us to apply a generalization of our algorithm to cases that were previously not computable
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Kneschke, Jan-Christoph [Verfasser]. "Vergleich von extrazellulären Proteinen Biofilm-positiver Staphylococcus epidermidis Stämme mit ihren isogenen sigma B Mutanten im Phänotyp, auf aktivitäts- und transkriptioneller Ebene / Jan-Christoph Kneschke." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2012. http://d-nb.info/1029110522/34.
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Al-Wesali, Mohammad Saad. "In vitro protein digestibility : a comparison of seeds from near isogenic pea lines and the role of trypsin inhibitors." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296360.
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Nishihara, Kana. "Identification of Genotoxic Compounds Using Isogenic DNA Repair Deficient DT40 Cell Lines on a Quantitative High Throughput Screening Platform." Kyoto University, 2016. http://hdl.handle.net/2433/215414.
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This is a pre-copyedited, author-produced PDF of an article accepted for publication in Mutagenesis following peer review. The definitive publisher-authenticated version is available online at:http://mutage.oxfordjournals.org/content/early/2015/08/03/mutage.gev055.full.Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第19588号
医博第4095号
新制||医||1014(附属図書館)
32624
京都大学大学院医学研究科医学専攻
(主査)教授 小泉 昭夫, 教授 渡邊 直樹, 教授 高田 穣
学位規則第4条第1項該当
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ALMEIDA, Micael de Jesus. "Disease correction and generation of isogenic induced pluripotent stem cell lines for diasease modelling in patients with DAND5 mutations." Master's thesis, Instituto de Higiene e Medicina Tropical, 2019. http://hdl.handle.net/10362/66374.
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As doenças cardiovasculares são a principal causa de morbidade e mortalidade, principalmente nos países desenvolvidos. Para melhorar as terapias já existentes para o miocárdio é necessário, em primeiro lugar, compreender claramente os mecanismos moleculares e as vias de sinalização envolvidas por trás da cardiomiogénese.
Utilizando um modelo de ratinho, foi possível observar o papel do DAND5 em dois processos embrionários distintos, um relacionado com o estabelecimento de LR e outro relacionado com a proliferação de cardiomiócitos. Além disso, em um projeto de sequenciamento do exoma, desenhado para identificar mutações associadas à assimetria esquerda-direita (LR) em humanos identificou-se uma mutação missense (c.455G>A) no exão 2 do gene DAND5 de dois doentes vivos independentes, apresentando características de assimetria LR e defeitos cardíacos associados. De acordo com a análise funcional in vitro, essa mutação leva a uma diminuição na função inibitória da proteína DAND5. No entanto, ainda há muito a aprender sobre a função de DAND5 durante a diferenciação em relação aos cardiomiócitos em humanos.
Com isso em mente, iPSCs humanas (hiPSCs) derivadas de diferentes pacientes com mutação em DAND5 foram estabelecidas. Mas, para estabelecer modelos para estudar a função deste gene e, em particular, a alteração c.455G> A, linhas isogénicas de células estaminais pluripotentes induzidas (iPSC) corrigidas em DAND5 devem ser geradas. Este é um controlo importante para superar o ruído fenotípico causado por fundos genéticos variáveis de linhas PSC não relacionadas. Assim como, a geração de linhagens isogénicas de iPSC DAND5 mutantes para esclarecer o efeito da completa perda de função de DAND5 durante a diferenciação em cardiomiócitos
Aqui, usando a edição de genoma mediada por CRISPR, fomos capazes de corrigir a mutação em DAND5 e gerar uma linha isogénica de hiPSC corrigida em DAND5, ou seja, é homozigótica para a base c.455G, confirmada por sequenciamento. Assim como de gerar uma isogénica hiPSC com o knockout de DAND5, após a utilização da edição do genoma mediada por CRISPR para inserir codões stop prematuros, confirmados por sequenciamento, tornando-se o gene DAND5 inoperante. A sua análise mostra então que esta linha celular mantém as propriedades hiPSC: morfologia tipo célula estaminal embrionária (ESC); análise de imunofluorescência (IF) confirmou a expressão do fator de transcrição e autorrenovação OCT4 e do marcador de superfície SSEA4, marcadores característicos de células estaminais pluripotentes; diferenciação in vitro baseada no corpo embrionário (EB) seguida de análise IF do marcador endodérmico alfafetoproteína (AFP), do marcador mesodérmico actina alfa do músculo liso (SMA) e o marcador ectodérmico tubulina βIII (TUBB3) confirmaram a pluripotencia das hiPSC e a sua capacidade de se diferenciar em todas as três camadas germinativas.
Essas linhas de hiPSC são agora ferramentas úteis para a modelação de doenças e para entender o papel desse gene em pacientes com mutações em DAND5, rastreio de drogas e medicina regenerativa. Especialmente para o estudo dos mecanismos moleculares relacionados com a proliferação de cardiomiócitos.Cardiovascular diseases are the leading cause of morbidity and mortality, mainly in developed countries. To improve already existing therapies for myocardium is firstly necessary to clearly understand the molecular mechanisms and signalling pathways involved behind cardiomyogenesis. Using a mouse model, it was possible to observe the role of DAND5 in two distinct embryonic processes, one related with the LR establishment and the other is related with the cardiomyocyte proliferation. Besides, on an exome sequencing project designed to identify mutations associated to left-right (LR) asymmetry in humans, it was identified a missense mutation (c.455G>A) in exon 2 of the DAND5 gene from two independent living patients presenting characteristic LR asymmetry-associated heart defects. According to functional analysis in vitro, this mutation leads to a decrease in the inhibitory function of the DAND5 protein. However, a lot remains to be learned about the function of DAND5 during differentiation towards cardiomyocytes in humans. With this in mind, human iPSCs (hiPSCs) derived from different patients with DAND5 mutation have been established. But, to establish models to study the function of this gene and, in particular, the c.455G>A alteration, isogenic DAND5-corrected induced pluripotent stem cell (iPSC) lines must be generated. This is an important control to overcome the phenotypic noise caused by variable genetic backgrounds of unrelated PSC lines. Even as, the generation of isogenic full-mutant DAND5 iPSC lines in order to clarify the effect of the complete DAND5 loss-of-function during differentiation towards cardiomyocytes Here, using CRISPR-mediated genome editing, we were capable to correct the defective DAND5 mutation and generate an isogenic DAND5-corrected hiPSC line, that means, is homozygous for the c.455G base, confirmed by sequencing. As well as generate an isogenic DAND5 knockout hiPSC, after using CRISPR-mediated genome editing to insert premature stop codons, confirmed by sequencing, made the DAND5 gene inoperative. Their analysis show then this cell line maintains the hiPSC properties: embryonic stem cell (ESC)-like morphology; immunofluorescence (IF) analysis confirmed the expression of self-renewal transcription factor OCT4 and the surface marker SSEA4, characteristic markers of pluripotent ESCs; in vitro embryoid body (EB)-based differentiation followed by IF analysis of the endodermal marker α-feto protein (AFP), the mesodermal marker smooth muscle actin (SMA) and the ectodermal marker βIII-tubulin (TUBB3) confirmed the pluripotency of hiPSCs and their ability to differentiate into all three germ layers. These hiPSC lines are now useful tools for disease modelling to understand the role of this gene in patients with DAND5 mutations, drug screening and regenerative medicine. Specially to study the molecular mechanisms related to the cardiomyocyte proliferation.
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Roth, Markus [Verfasser], Thorsten [Gutachter] Stühmer, Ralf C. [Gutachter] Bargou, and Justus [Gutachter] Müller. "Etablierung eines isogenen Zelllinienmodells zur Untersuchung der Bedeutung mono- und biallelischer TP53-Inaktivierungen beim Multiplen Myelom / Markus Roth ; Gutachter: Thorsten Stühmer, Ralf C. Bargou, Justus Müller." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1215033923/34.
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