Academic literature on the topic 'Tissue specific knock out'
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Journal articles on the topic "Tissue specific knock out"
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Rupec, Rudolf A., Stephanie Rämisch, Gerd Plewig, Klaus Pfeffer, and Gerald Messer. "Construction of a tissue-specific IκB-α knock-out mouse." Journal of Dermatological Science 16 (March 1998): S133. http://dx.doi.org/10.1016/s0923-1811(98)83792-4.
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Yoon, Donghoon, Bumjun Kim, Myunghi Kwon, and Josef T. Prchal. "Hematopoietic Specific GATA-1-Improved Cre Mouse for Erythroid-Specific Gene Modification." Blood 108, no. 11 (November 16, 2006): 1291. http://dx.doi.org/10.1182/blood.v108.11.1291.1291.
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Abstract Animal models of erythropoiesis related genes have been limited by the fact that some of these genes have non-erythroid expression and other functions in addition to erythropoiesis and thus their knock-out may be embryonic lethal. Tissue specific knock-out or knock-in mice models employing GATA-1-Cre and other constructs showed that these promoters are also active in non-hematopoietic tissues, i.e. GATA-1 has activity in early embryonic development and in neuronal tissue. Suzuki et al (Blood, 2002, 100; 2279) isolated the GATA-1 locus hematopoietic regulatory domain (GATA-1-HRD) and demonstrated that the expression of a transgene under its control is limited to the hematopoietic tissue. We generated a transgenic mouse expressing an improved Cre (iCre) under GATA-1-HRD promoter control. This mouse was crossbred with ROSA 26 mouse and the progeny was examined for tissue specificity of iCre expression using beta-galactosidase staining. Brain, spleen, kidney, heart, thymus, liver, lung and ovary were examined for whole organ LacZ staining. All tested organs were negative except kidney and spleen where some positivity was observed. Subsequently, we prepared tissue sections from kidney, spleen and bone marrow and stained with LacZ and anti-beta-galactosidase antibody. Only the bone marrow EpoR expressing cells were positive; the kidney and the spleen cells were negative. Although Suzuki et al previously showed expression of the GATA-1-HRD driven erythropoietin receptor in spleen using RT-PCR, we were not able to find iCre expression in the splenic cells using these approaches. We demonstrate that our transgenic mouse (GATA-1-HRD-iCre) showed a restricted iCre expression in hematopoietic tissue that differs from previous studies of other hematopoiesis specific cre mouse. We conclude that this mouse model should be useful in studies of function of erythroid specific genes.
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Dubyak, George R. "Knock-Out Mice Reveal Tissue-Specific Roles of P2Y Receptor Subtypes in Different Epithelia." Molecular Pharmacology 63, no. 4 (April 1, 2003): 773–76. http://dx.doi.org/10.1124/mol.63.4.773.
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Li, Mira, Sameer Agnihotri, Mark Wilson, Julie Metcalf, Olivia Singh, Shirin Karimi, Kelly Burrell, et al. "TMOD-02. DIFFUSE GLIOMATOSIS IN MOUSE MODEL OF GFAP TISSUE SPECIFIC KNOCK IN OF EGFRvIII AND KNOCK OUT OF p19 ARF." Neuro-Oncology 20, suppl_6 (November 2018): vi268. http://dx.doi.org/10.1093/neuonc/noy148.1115.
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Garcia-Arcos, Itsaso, Yaeko Hiyama, Konstantinos Drosatos, Kalyani G. Bharadwaj, Yunying Hu, Ni Huiping Son, Sheila M. O'Byrne, et al. "Adipose-specific Lipoprotein Lipase Deficiency More Profoundly Affects Brown than White Fat Biology." Journal of Biological Chemistry 288, no. 20 (March 31, 2013): 14046–58. http://dx.doi.org/10.1074/jbc.m113.469270.
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Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice. ATLO LpL mRNA and activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicron TG uptake into BAT and lung. ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL in BAT modulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the “gatekeeper” for tissue lipid distribution.
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Shim, Kye Shik. "The Growth and Pubertal Development in Female Mice with Tissue-specific Knock out of Estrogen Receptor." Journal of Korean Society of Pediatric Endocrinology 16, no. 2 (2011): 67. http://dx.doi.org/10.6065/jkspe.2011.16.2.67.
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Chesney, Kari L., Hongsheng Men, Miriam A. Hankins, and Elizabeth C. Bryda. "The Atg16l1 gene: characterization of wild type, knock-in, and knock-out phenotypes in rats." Physiological Genomics 53, no. 6 (June 1, 2021): 269–81. http://dx.doi.org/10.1152/physiolgenomics.00114.2020.
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ATG16L1 is a ubiquitous autophagy gene responsible, in part, for formation of the double-membrane bound autophagosome that delivers unwanted cellular debris and intracellular pathogens to the lysosome for degradation. A single, nonsynonymous adenine to guanine polymorphism resulting in a threonine to alanine amino acid substitution (T300A) directly preceded by a caspase cleavage site (DxxD) causes an increased susceptibility to Crohn’s disease (CD) in humans. The mechanism behind this increased susceptibility is still being elucidated, however, the amino acid change caused by this point mutation results in increased ATG16L1 protein sensitivity to caspase 3-mediated cleavage. To generate novel rat strains carrying genetic alterations in the rat Atg16l1 gene, we first characterized the wild-type rat gene. We identified four alternative splice variants with tissue-specific expression. Using CRISPR-Cas9 genome editing technology, we developed a knock-in rat model for the human ATG16L1 T300A CD risk polymorphism, as well as a knock-out rat model to evaluate the role of Atg16l1 in autophagy as well as its potential effect on CD susceptibility. These are the first reported rat strains with alterations of the Atg16l1 gene. Consistent with studies of the effects of human ATG16L1 polymorphisms, models exhibit morphological abnormalities in both Paneth and goblet cells, but do not develop spontaneous intestinal permeability or inflammatory bowel disease. Analysis of the gut microbiota does not show inherent differences in bacterial composition between wild-type and genetically modified animals. These Atg16l1 strains are valuable new animal models for the study of both autophagy and CD susceptibility.
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Man Law, Ivy Ka, Carl Rankin, and Charalabos Pothoulakis. "14 COLONIC EPITHELIAL CELL-SPECIFIC AFTIPHILIN KNOCKDOWN REGULATES EPITHELIAL BARRIER FUNCTION THROUGH MYOSIN LIGHT CHAIN-ASSOCIATED ACTIN ORGANIZATION IN VITRO AND INTESTINAL LENGTH IN VIVO." Inflammatory Bowel Diseases 26, Supplement_1 (January 2020): S28. http://dx.doi.org/10.1093/ibd/zaa010.067.
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Abstract Background and Aims Colonic epithelial integrity is often compromised during colonic inflammation and Inflammatory Bowel Disease. Aftiphilin (AFTPH) is a downstream target of microRNA-133a and its expression is reduced in colonic tissues of wild type mice from experimental colitis models and colonic biopsies from patients with ulcerative colitis. We have previously shown that AFTPH is involved in regulating intestinal epithelial barrier function and actin organization in human colonic epithelial cells in vitro (DDW 2016). On the other hand, our results suggested that global aftiphilin knock-out is embryonic lethal in mouse models (DDW 2019). Here, we further examined the role of AFTPH in regulating actin organization in vitro and characterize the colonic epithelial cell-specific aftiphilin knock-out mice. Methods Human colonic epithelial NCM460 cells were transfected with si-RNA against AFTPH to achieve transient AFTPH gene-silencing. Stable AFTPH knock-down clones were generated by transducing Caco2-BBE cells with recombinant lentivirus carrying sh-AFTPH or control sh-RNA. To create intestinal epithelial cell-specific aftiphilin knock-out mice, Aftph flox/flox mice were cross-bred with B6.Cg-Tg(Vil1-cre)997Gum/J mice, which express Villin-driven Cre recombinase (Vil-Cre), to generate intestinal epithelial cell-specific aftiphilin knock-out mice (Aftph Vil-/Vil-). Protein expression of F- and G-actin and p70S6K were detected using Western blot. Tissues from various organs were collected with Aftph Vil-/Vil- and its wildtype counterparts at 12 weeks. Results Results from western blot analysis showed that F-/G-actin ratio in AFTPH gene-silenced NCM460 cells were 0.6±0.17 fold, when compared to the treatment control. In addition, AFTPH gene-silencing in human colonic epithelial cells activated p70S6K, a kinase that is involved in actin organization, when compared to treatment control (1.2±0.15 vs. 2.0±0.15, p=0.0354). Furthermore, transepithelial electric resistance (TER) of Caco2-BBE cells deficient in AFTPH is significantly lower than that of control cells (0.5±0.07 fold). Lastly, in vivo intestinal epithelial cell-specific Aftph knock-out increased the length of small intestine, when compared to that of wild type mice (30.7±0.33 vs. 34.8±0.97, p=0.02), while the tissue weight of spleen to body weight was reduced (0.30±0.011 vs. 0.26±0.006, p=0.0169). Summary and Conclusions Our results indicate that AFTPH directly regulates epithelial barrier function and actin organization through mediating F-/G-actin ratio in human colonic epithelial cells, possibly through p70S6K. Importantly, intestinal epithelial cell-specific knock-out in vivo increased intestinal length and reduced size of the spleen. Our results suggested that AFTPH is crucial in regulating colonic epithelial barrier function in vitro and intestinal homeostasis.
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Petrova, Tsvetana, Kyle Bennett, Sambit Nanda, Sam Strickson, Cheryl L. Scudamore, Alan R. Prescott, and Philip Cohen. "Why are the phenotypes of TRAF6 knock-in and TRAF6 knock-out mice so different?" PLOS ONE 17, no. 2 (February 14, 2022): e0263151. http://dx.doi.org/10.1371/journal.pone.0263151.
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The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4+ and CD8+ T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44high/CD62Llow expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2–3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.
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Salveridou, Eva, Steffen Mayerl, Sivaraj Mohana Sundaram, Boyka Markova, and Heike Heuer. "Tissue-Specific Function of Thyroid Hormone Transporters: New Insights from Mouse Models." Experimental and Clinical Endocrinology & Diabetes 128, no. 06/07 (November 13, 2019): 423–27. http://dx.doi.org/10.1055/a-1032-8328.
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AbstractThyroid hormone (TH) transporters are required for cellular transmembrane passage of TH and are thus mandatory for proper TH metabolism and action. Consequently, inactivating mutations in TH transporters such as MCT8 or OATP1C1 can cause tissue- specific changes in TH homeostasis. As the most prominent example, patients with MCT8 mutations exhibit elevated serum T3 levels, whereas their CNS appear to be in a TH deficient state. Here, we will briefly summarize recent studies of mice lacking Mct8 alone or in combination with the TH transporters Mct10 or Oatp1c1 that shed light on many aspects and pathogenic events underlying global MCT8 deficiency and also underscore the contribution of Mct10 and Oatp1c1 in tissue-specific TH transport processes. Moreover, development of conditional knock-out mice that allow a cell-specific inactivation of TH transporters in distinct tissues, disclosed cell-specific changes in TH signaling, thereby highlighting the pathophysiological significance of local control of TH action.
Dissertations / Theses on the topic "Tissue specific knock out"
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Staab, Christine [Verfasser], and Lars [Akademischer Betreuer] Nitschke. "Production of Recombinant Human Soluble CD83 in an Eukaryotic System and Generation of Tissue-Specific CD83 Knock-out Mice / Christine Staab. Betreuer: Lars Nitschke." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2011. http://d-nb.info/1015475329/34.
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Dali-Youcef, Nassim. "Generation of mouse models for SIRT genes conditional knock-outs : Phenogenomics of adipocyte-specific retinoblastoma deficient mice." Université Louis Pasteur (Strasbourg) (1971-2008), 2007. http://www.theses.fr/2007STR13155.
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Chez les mammifères, les protéines SIRT, appelées aussi sirtuines, appartiennent à la famille des histones désacétylases (HDAC) de classe III et ont été nommées ainsi pour leur homologie avec le gène de la levure Saccharomyces cerevisiae Sir2. Il est clairement établi que le gène Sir2 joue un rôle primordial dans l’extension de la durée de vie de la levure induite par la restriction calorique et que des copies supplémentaires de ce gène retardent le vieillissement chez la levure. Contrairement aux HDAC de classe I et II, les protéines SIRT nécessitent un cofacteur, le NAD+, pour désacetyler les histones ainsi que d’importants facteurs régulateurs de la transcription. Le chef de file des sirtuines, SIRT1, semble impliqué dans des pathologies variées telles le diabète, l’obésité, l’insuffisance cardiaque, le SIDA et les maladies neurodégénératives. Même si le rôle de SIRT1 n’a pas encore été clairement établi in vivo, il apparaît indissociable de la régulation de différents processus biologiques allant de l’apoptose à la différentiation adipocytaire et musculaire ainsi que dans le métabolisme glucidique en régulant la néoglucogenèse. La fonction des autres SIRT n’est pas encore élucidée. Le sujet de ma thèse de doctorat est d’essayer de comprendre les fonctions biologiques des gènes SIRT en les inactivant de façon spatio-temporelle par l’utilisation de la stratégie du « knock-out conditionnel » (inactivation conditionnelle) et le système Cre/Lox. Cette technologie permet d’inactiver de façon contrôlée le gène d’intérêt dans un organe donné d’une souris adulte afin d’éviter les problèmes liés aux anomalies que l’absence du gène pourrait provoquer au cours du développement de l’animal lors d’un « knock-out classique ». Pour parvenir à notre objectif, nous avons réalisé des constructions modifiées des différents gènes SIRT. La particularité de ces constructions consiste à introduire des séquences Lox autour du domaine du gène codant pour la partie catalytique de l’enzyme. Les Lox sont des séquences nucléotidiques de 34 paires de bases qui sont excisées par l’enzyme Cre-recombinase lors de l’inactivation du gène. Les vecteurs contenant les gènes SIRT modifiés ont été électroporés dans des cellules souches embryonnaires (ES) de souris 129/Sv afin de s’intégrer au génome par recombinaison homologue. Les clones positifs ont été injectés par la suite dans des blastocystes. Nous avons obtenu ainsi des souris transgéniques pour le gène d’intérêt. Ces souris doivent par la suite être croisées avec des souris exprimant une protéine de fusion Cre-ERT sous le contrôle d’un promoteur spécifique d’un organe donné. La protéine Cre-ERT est une fusion entre la Cre-recombinase et le récepteur aux oestrogènes ayant une affinité élevée pour le ligand tamoxifène. En cas d’injection de tamoxifène, il y a activation de la Cre qui induit l’inactivation du gène d’intérêt à un moment donné de la vie de la souris et permet d’étudier ainsi les effets biologiques qui en résultent. A l’heure actuelle, les constructions pour les gènes SIRT1-7 ont été réalisées. Nous disposons des souris transgéniques pour le gène SIRT2 « floxé » et les croisements sont en cours avec des souris transgéniques CMV-Cre-ERT2 (inactivation de SIRT2 dans tous les tissus) et Syn-Cre-ERT2 (inactivation de SIRT2 dans les cellules nerveuses). Les autres constructions ont été électroporées dans les cellules ES et sont en cours d’analyse. Partie II : Etude de la fonction du gène Rb dans la différentiation adipocytaire et le métabolisme énergétique. Le gène du rétinoblastome Rb est le premier gène suppresseur de tumeur identifié et a été largement étudié. Son rôle a bien été démontré dans différents processus biologiques tels le contrôle du cycle cellulaire, l’apoptose, la prolifération et la différenciation cellulaires. Récemment, des études in vitro ont montré que la protéine pRb avait un rôle important dans la différentiation adipocytaire et qu’un déficit en pRb provoque un changement dans le programme de différentiation d’adipocytes blancs de souris en adipocytes bruns. Dans notre étude, nous avons utilisé le système Cre-lox pour inactiver spécifiquement le gène Rb dans le tissu adipeux. Soumises à un régime riche en graisses, des souris déficientes en pRb restent maigres par rapport à leurs congénères qui voient leurs poids augmenter de manière significative. L’analyse histologique et en microscopie électronique du tissu adipeux blanc (WAT) des souris déficientes en pRb montre une transformation d’une partie du WAT en tissu adipeux brun (BAT) avec une augmentation significative du nombre de mitochondries suggérant une activité métabolique élevée. L’analyse moléculaire a démontré une augmentation de l’expression des gènes impliqués dans la dépense énergétique tels UCP-1, PGC-1a et Dio2. Ceci se traduit physiologiquement par une augmentation de la consommation en O2 et de la production de CO2 démontrant une élévation de la dépense énergétique chez les souris pRb-déficientes. Ces données confirment que l’absence de pRb provoque la conversion d’une partie du WAT en BAT, un tissu connu pour son rôle actif dans la dépense énergétique. Le gène Rb devient ainsi une cible thérapeutique potentielle dans le but d’induire une perte de poids chez les patients obèsesIn mammals, the sirtuin family of histone deacetylases (HDACs) family was named after their homology to the yeast Saccharomyces cerevisiae gene Sir2. In yeast, it has been shown that Sir2 mediates the effects of calorie restriction on the extension of lifespan and that high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins (SIRT1-7) are class III HDACs and require NAD+ as a cofactor to deacetylate substrates such as histones and transcription regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. SIRT1, the most studied sirtuin, seems to be implicated in several pathologies such as diabetes, obesity, heart failure and neurodegenerative disorders. The aim of this Ph. D. Thesis was to help understand the biological function of SIRT genes through their inactivation in mice in a spatial and temporal controlled manner, using the Cre/Lox technology. This system allows the controlled inactivation of a gene of interest in a given organ of an adult mouse to avoid abnormalities that could occur during the development when using a “classical knock-out”. We have generated genetically modified constructs for all SIRT genes by introducing 2 LoxP sequences flanking the catalytic domain of the enzyme. LoxP sites are sequences of 34 nucleotides that can be recognized and excised by the Cre-recombinase enzyme. Vectors containing the modified SIRT gene constructs were electroporated in embryonic stem cells (ES) of 129/Sv mice in order to be integrated in their genome by homologous recombination. Positif clones were then injected in blastocysts of C57BL/6J pseudopregnant mice. We obtained transgenic mice for the gene of interest. These mice will be crossed with mice expressing the Cre recombinase fused to a modified estrogen receptor with high affinity for the synthetic ligand tamoxifen, under the control of a cell specific promoter that targets Cre expression in a specific organ or cell type (promoter-Cre-ERT2). After tamoxifen injection, the Cre recombinase is activated and subsequently the SIRT gene of interest will be inactivated in a specific cell type (e. G. Adipocytes), whilst its activity remains in other cells, allowing the study of the biological effects that result from such gene inactivation. At present, we have completed the constructs for all SIRT(1-7) genes. We obtained mice with a floxed SIRT2 allele that were crossed with a CMV-Cre-ERT2 and Synapsin-Cre-ERT2 transgenic mice to generate cohorts of double transgenic mice expressing the floxed SIRT2 allele and the Cre-ERT2 either in all cell types or specifically in neurons. Constructs for other SIRT genes have been electroporated in ES cells and the generation of mice is underway. PART 2: Phenogenomics of adipocyte-specific retinoblastoma deficient miceThe role of the tumor suppressor retinoblastoma protein (pRb) has been firmly established in the control of cell cycle, apoptosis, and differentiation. Recently, it was demonstrated that lack of pRb promotes a switch from white to brown adipocyte differentiation in vitro. We used the Cre-Lox system to specifically inactivate pRb in adult adipose tissue. Under a high-fat diet, pRb-deficient (pRbad-/-) mice failed to gain weight because of increased energy expenditure. This protection against weight gain was caused by the activation of mitochondrial activity in white and brown fat as evidenced by histologic, electron microscopic, and gene expression studies. Moreover, pRb(-/-) mouse embryonic fibroblasts displayed higher proliferation and apoptosis rates than pRb(+/+) mouse embryonic fibroblasts, which could contribute to the altered white adipose tissue morphology. Taken together, our data support a direct role of pRb in adipocyte cell fate determination in vivo and suggest that pRb could serve as a potential therapeutic target to trigger mitochondrial activation in white adipose tissue and brown adipose tissue, favoring an increase in energy expenditure and subsequent weight loss
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Göngrich, Christina. "Metabolic alterations in connexin36 knock-out mice induce gender-specific changes in dentate gyrus function." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:16-opus-87371.
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De, Santis Flavia. "Genome editing to understand neural circuits formation : a novel CRISPR/Cas9-based strategy for conditional mutagenesis and functional study of the role of the meteorin gene family in zebrafish neurodevelopment." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066269/document.
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Depuis quelques années, le poisson zèbre (Danio rerio) est devenu un modèle de choix pour l'étude du système nerveux et de ses fonctions. Récemment, des technologies nouvelles d'édition du génome permettent la génération d'allèles mutés de manière constitutionnelle et l'étude fonctionnelle de gènes chez ce modèle vertébré. Néanmoins, certains loci nécessite une inactivation spatiotemporelle précise et contrôlée. La première partie de ma thèse décrit la mise au point d'une nouvelle stratégie de disruption génétique de manière tissu-spécifique, basée sur la technologie du CRISPR/Cas9 et du système UAS/Gal4. Cette technique permet l'introduction de mutations somatiques dans des tissus, des clones ou des cellules individuelles préalablement génétiquement marqués, rendant ainsi possible le suivi in vivo de l'effet de la mutation générée grâce au gène rapporteur. La seconde partie de ma thèse se centre sur l'étude fonctionnelle d'une famille des gènes, les meteorines, durant le développement du système nerveux et lors du ciblage axonale chez le poisson zèbre. Les Meteorines sont des protéines conservées chez les vertébrés qui ont été impliquées dans la prolifération, la différentiation des progéniteurs de neurones et notamment dans l'élongation axonale in vitro. Nous avons pu mettre en évidence que les meteorines sont exprimées le long de la ligne médiane du système nerveux chez les larves et au niveau du plancher de la partie postérieure du cerveau et de la moelle épinière. Par l'utilisation du CRISPR/Cas9, nous avons généré des lignées mutantes pour chaque gène meteorine et avons ainsi procédé à l'analyse de l'établissement des projections axonales dans ces lignées mutantesIn recent years, the zebrafish (Danio rerio) has emerged as a powerful model organism to study neuronal circuit development and function. To date, different genome editing technologies allow the generation of constitutive mutant alleles, permitting the study of gene loss-of-function in this vertebrate model. Nevertheless, to assess the role of certain loci it might be required a precise spatiotemporal control of gene inactivation. The rst part of my thesis describes a novel strategy for tissue-specific gene disruption based on the CRISPR/Cas9 and the Gal4/UAS systems. The described technique allows the induction of somatic mutations in genetically labeled tissues, cell clones or single cells, making it possible to follow the effect of gene disruption in vivo via reporter gene expression. The second part of the thesis focuses on the functional analysis of the role of the meteorin gene family during neuronal development and axonal targeting in zebra sh. Meteorin family is conserved among vertebrates and its members have been shown to be involved in neuronal progenitor proliferation and differentiation and axonal elongation, in vitro. We used the zebrafish nervous system as a model to dissect the role of Meteorins during embryonic development, focusing on their potential role as novel guidance molecules. Interestingly, we found that genes belonging to the meteorin family are expressed along the midline of the larval central nervous system and at the floor plate in the hindbrain and spinal cord. We generated CRISPR/Cas9 mutant lines carrying out-of-frame deletions in the coding sequence of each member of the zebrafish meteorin family and we performed a comprehensive analysis of the establishment of axonal projections in the mutants. Our data pointed out that metrns loss-of-function affects the earliest process of axonal development, demonstrating a crucial role in the process of axonal outgrowth for this new family of evolutionary conserved guidance molecules
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Storbeck, Markus [Verfasser], Brunhilde [Akademischer Betreuer] Wirth, and Elena [Akademischer Betreuer] Rugarli. "Characterization of Neuronal-Specific Tra2b Knock-Out Mice and Identification of Tra2b Splicing Targets / Markus Storbeck. Gutachter: Brunhilde Wirth ; Elena Rugarli." Köln : Universitäts- und Stadtbibliothek Köln, 2014. http://d-nb.info/1049523385/34.
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Andersson, Matilda. "To Knock the Eye Out of a Friend : Assessment of an Orthographic Reform Upon the English Language." Thesis, Högskolan i Halmstad, Sektionen för humaniora (HUM), 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:hh:diva-25371.
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This essay is a theoretical qualitative study, which examines the possibility for a spelling reform into English. The history of orthographical changes into British English, as well as Brown’s categorisation of spelling reforms, is reviewed. Four spelling reform proposals are analysed and compared. Additionally, the social discourses of Eira, which are relevant to a spelling reform, are analysed and discussed with regard to English. There is only speculation as to why no modern day spelling reform has been implemented in British English, but it is connected to its historical events, the social discourses and the implementation process. Spelling reform into English is theoretically feasible, but it depends on the implementation strategies and support from those who wish to perform such a change.
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Dutta, Sayantanee [Verfasser], and Stefan K. [Akademischer Betreuer] Bohlander. "CALM/AF10 leukemia : a tissue specific knock-in Mouse Model and Analysis of BMI1 as a Collaborator in Leukemogenesis / Sayantanee Dutta. Betreuer: Stefan K. Bohlander." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1060006162/34.
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Zhang, Han. "An Optimized Polymerase Chain Reaction to Verify the Presence or Absence of the Growth Hormone Receptor Gene." Ohio University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1366378898.
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Göngrich, Christina [Verfasser]. "Metabolic alterations in connexin36 knock-out mice induce gender-specific changes in dentate gyrus function / presented by Christina Göngrich." 2008. http://d-nb.info/991438728/34.
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Bolcun-Filas, Ewelina. "Expression and functional analysis of the germ cell specific genes ADAM 27 and Testase 2." Doctoral thesis, 2004. http://hdl.handle.net/11858/00-1735-0000-000D-F14F-4.
Full textBooks on the topic "Tissue specific knock out"
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Wood, Adrian J. Subcellular analysis of normal and pathological gastrointestinal tissue with specific reference to peroxisomes: A thesis submitted in partial fulfilment of the University of Wolverhampton for the degree of Doctor of Philosophy : this research programme was carried out in collaboration with the Royal Wolverhampton Hospitals Trust. Wolverhampton: University of Wolverhampton, 1994.
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Powell, Jenny. Normal skin function. Edited by Patrick Davey and David Sprigings. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780199568741.003.0243.
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In simplest terms, our skin is a layer that separates and protects us from the external environment. This assumes the skin is a passive covering to keep the insides safe and the outside out, and overlooks its enormous complexity. The skin is our largest organ and is constantly regenerating, but how efficiently it does so depends on a number of factors, some known, others unknown. It is an efficient mechanical barrier (designed for wear and repair), and a complex immunological membrane. It has a generous vascular, lymphatic, and nervous supply, all covering a considerable area. It has specialist structural and functional properties relating to specific areas, but also specialist cells within the layers of the skin. Most importantly, skin is the organ of display, an important part of social and sexual behaviour, immediately accessible to all, and often regarded as a barometer of the general state of health. Permanent scars inflicted on the skin may be a cause of great distress to the patient. Skin consists of a superficial layer, ‘the epidermis’ (concerned with producing protective keratin and a pigment called melanin), which adheres closely to the deeper layer, ‘the dermis’ (which provides the strength of the skin and houses the appendages), via the basement membrane. Loose connective tissue and fat underlie the dermis.
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Yurdakul, Sebahattin, Emire Seyahi, and Hasan Yazici. Behçet’s syndrome. Oxford University Press, 2013. http://dx.doi.org/10.1093/med/9780199642489.003.0135.
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Behçet's syndrome is a systemic inflammatory panvasculitis (affecting all sizes of vessels) of unknown aetiology. It is in vogue to include it among the systemic autoinflammatory conditions. Behçet's syndrome is more frequent along the ancient 'Silk Route' across Asia than it is in Western countries. The usual onset is the second or third decade, equally affecting either gender. However, young patients and male patients have more severe disease. Almost all patients have recurrent oral ulceration. Scar-forming genital ulcers, a variety of skin lesions including acneiform, erythema nodosum-like lesions, arthritis, potentially blinding panuveitis, thrombophlebitis, gastrointestinal disease, central nervous system (CNS) involvement, and life-threatening bleeding pulmonary artery aneurysms are seen. The pathergy phenomenon is a heightened tissue inflammatory response. The strongest genetic association is with HLA B51. There are immunological aberrations but not prominent enough to call it an autoimmune disease. Similarly, Behçet's syndrome does not fit easily into the broad concept of autoinflammatory diseases. The histopathology is also non-specific and the diagnosis is mainly clinical. Differentiation from Crohn's disease is very difficult. In more than one-half of the patients the disease burns out in time, thus only symptomatic therapy is indicated in some patients. However, eye involvement, pulmonary vascular disease, thrombophilic complications, CNS involvement, and gastrointestinal disease need prompt recognition and treatment. Brief courses of glucocorticosteroids along with immunosuppressives including the newer biologicals, interferon, and colchicine are commonly used. However, controlled clinical trials are not available for some of these medications especially when thrombophilia, CNS, and gastrointestinal disease are present.
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Skiba, Grzegorz. Fizjologiczne, żywieniowe i genetyczne uwarunkowania właściwości kości rosnących świń. The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 2020. http://dx.doi.org/10.22358/mono_gs_2020.
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Bones are multifunctional passive organs of movement that supports soft tissue and directly attached muscles. They also protect internal organs and are a reserve of calcium, phosphorus and magnesium. Each bone is covered with periosteum, and the adjacent bone surfaces are covered by articular cartilage. Histologically, the bone is an organ composed of many different tissues. The main component is bone tissue (cortical and spongy) composed of a set of bone cells and intercellular substance (mineral and organic), it also contains fat, hematopoietic (bone marrow) and cartilaginous tissue. Bones are a tissue that even in adult life retains the ability to change shape and structure depending on changes in their mechanical and hormonal environment, as well as self-renewal and repair capabilities. This process is called bone turnover. The basic processes of bone turnover are: • bone modeling (incessantly changes in bone shape during individual growth) following resorption and tissue formation at various locations (e.g. bone marrow formation) to increase mass and skeletal morphology. This process occurs in the bones of growing individuals and stops after reaching puberty • bone remodeling (processes involve in maintaining bone tissue by resorbing and replacing old bone tissue with new tissue in the same place, e.g. repairing micro fractures). It is a process involving the removal and internal remodeling of existing bone and is responsible for maintaining tissue mass and architecture of mature bones. Bone turnover is regulated by two types of transformation: • osteoclastogenesis, i.e. formation of cells responsible for bone resorption • osteoblastogenesis, i.e. formation of cells responsible for bone formation (bone matrix synthesis and mineralization) Bone maturity can be defined as the completion of basic structural development and mineralization leading to maximum mass and optimal mechanical strength. The highest rate of increase in pig bone mass is observed in the first twelve weeks after birth. This period of growth is considered crucial for optimizing the growth of the skeleton of pigs, because the degree of bone mineralization in later life stages (adulthood) depends largely on the amount of bone minerals accumulated in the early stages of their growth. The development of the technique allows to determine the condition of the skeletal system (or individual bones) in living animals by methods used in human medicine, or after their slaughter. For in vivo determination of bone properties, Abstract 10 double energy X-ray absorptiometry or computed tomography scanning techniques are used. Both methods allow the quantification of mineral content and bone mineral density. The most important property from a practical point of view is the bone’s bending strength, which is directly determined by the maximum bending force. The most important factors affecting bone strength are: • age (growth period), • gender and the associated hormonal balance, • genotype and modification of genes responsible for bone growth • chemical composition of the body (protein and fat content, and the proportion between these components), • physical activity and related bone load, • nutritional factors: – protein intake influencing synthesis of organic matrix of bone, – content of minerals in the feed (CA, P, Zn, Ca/P, Mg, Mn, Na, Cl, K, Cu ratio) influencing synthesis of the inorganic matrix of bone, – mineral/protein ratio in the diet (Ca/protein, P/protein, Zn/protein) – feed energy concentration, – energy source (content of saturated fatty acids - SFA, content of polyun saturated fatty acids - PUFA, in particular ALA, EPA, DPA, DHA), – feed additives, in particular: enzymes (e.g. phytase releasing of minerals bounded in phytin complexes), probiotics and prebiotics (e.g. inulin improving the function of the digestive tract by increasing absorption of nutrients), – vitamin content that regulate metabolism and biochemical changes occurring in bone tissue (e.g. vitamin D3, B6, C and K). This study was based on the results of research experiments from available literature, and studies on growing pigs carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences. The tests were performed in total on 300 pigs of Duroc, Pietrain, Puławska breeds, line 990 and hybrids (Great White × Duroc, Great White × Landrace), PIC pigs, slaughtered at different body weight during the growth period from 15 to 130 kg. Bones for biomechanical tests were collected after slaughter from each pig. Their length, mass and volume were determined. Based on these measurements, the specific weight (density, g/cm3) was calculated. Then each bone was cut in the middle of the shaft and the outer and inner diameters were measured both horizontally and vertically. Based on these measurements, the following indicators were calculated: • cortical thickness, • cortical surface, • cortical index. Abstract 11 Bone strength was tested by a three-point bending test. The obtained data enabled the determination of: • bending force (the magnitude of the maximum force at which disintegration and disruption of bone structure occurs), • strength (the amount of maximum force needed to break/crack of bone), • stiffness (quotient of the force acting on the bone and the amount of displacement occurring under the influence of this force). Investigation of changes in physical and biomechanical features of bones during growth was performed on pigs of the synthetic 990 line growing from 15 to 130 kg body weight. The animals were slaughtered successively at a body weight of 15, 30, 40, 50, 70, 90, 110 and 130 kg. After slaughter, the following bones were separated from the right half-carcass: humerus, 3rd and 4th metatarsal bone, femur, tibia and fibula as well as 3rd and 4th metatarsal bone. The features of bones were determined using methods described in the methodology. Describing bone growth with the Gompertz equation, it was found that the earliest slowdown of bone growth curve was observed for metacarpal and metatarsal bones. This means that these bones matured the most quickly. The established data also indicate that the rib is the slowest maturing bone. The femur, humerus, tibia and fibula were between the values of these features for the metatarsal, metacarpal and rib bones. The rate of increase in bone mass and length differed significantly between the examined bones, but in all cases it was lower (coefficient b <1) than the growth rate of the whole body of the animal. The fastest growth rate was estimated for the rib mass (coefficient b = 0.93). Among the long bones, the humerus (coefficient b = 0.81) was characterized by the fastest rate of weight gain, however femur the smallest (coefficient b = 0.71). The lowest rate of bone mass increase was observed in the foot bones, with the metacarpal bones having a slightly higher value of coefficient b than the metatarsal bones (0.67 vs 0.62). The third bone had a lower growth rate than the fourth bone, regardless of whether they were metatarsal or metacarpal. The value of the bending force increased as the animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. The rate of change in the value of this indicator increased at a similar rate as the body weight changes of the animals in the case of the fibula and the fourth metacarpal bone (b value = 0.98), and more slowly in the case of the metatarsal bone, the third metacarpal bone, and the tibia bone (values of the b ratio 0.81–0.85), and the slowest femur, humerus and rib (value of b = 0.60–0.66). Bone stiffness increased as animals grew. Regardless of the growth point tested, the highest values were observed for the humerus, tibia and femur, smaller for the metatarsal and metacarpal bone, and the lowest for the fibula and rib. Abstract 12 The rate of change in the value of this indicator changed at a faster rate than the increase in weight of pigs in the case of metacarpal and metatarsal bones (coefficient b = 1.01–1.22), slightly slower in the case of fibula (coefficient b = 0.92), definitely slower in the case of the tibia (b = 0.73), ribs (b = 0.66), femur (b = 0.59) and humerus (b = 0.50). Bone strength increased as animals grew. Regardless of the growth point tested, bone strength was as follows femur > tibia > humerus > 4 metacarpal> 3 metacarpal> 3 metatarsal > 4 metatarsal > rib> fibula. The rate of increase in strength of all examined bones was greater than the rate of weight gain of pigs (value of the coefficient b = 2.04–3.26). As the animals grew, the bone density increased. However, the growth rate of this indicator for the majority of bones was slower than the rate of weight gain (the value of the coefficient b ranged from 0.37 – humerus to 0.84 – fibula). The exception was the rib, whose density increased at a similar pace increasing the body weight of animals (value of the coefficient b = 0.97). The study on the influence of the breed and the feeding intensity on bone characteristics (physical and biomechanical) was performed on pigs of the breeds Duroc, Pietrain, and synthetic 990 during a growth period of 15 to 70 kg body weight. Animals were fed ad libitum or dosed system. After slaughter at a body weight of 70 kg, three bones were taken from the right half-carcass: femur, three metatarsal, and three metacarpal and subjected to the determinations described in the methodology. The weight of bones of animals fed aa libitum was significantly lower than in pigs fed restrictively All bones of Duroc breed were significantly heavier and longer than Pietrain and 990 pig bones. The average values of bending force for the examined bones took the following order: III metatarsal bone (63.5 kg) <III metacarpal bone (77.9 kg) <femur (271.5 kg). The feeding system and breed of pigs had no significant effect on the value of this indicator. The average values of the bones strength took the following order: III metatarsal bone (92.6 kg) <III metacarpal (107.2 kg) <femur (353.1 kg). Feeding intensity and breed of animals had no significant effect on the value of this feature of the bones tested. The average bone density took the following order: femur (1.23 g/cm3) <III metatarsal bone (1.26 g/cm3) <III metacarpal bone (1.34 g / cm3). The density of bones of animals fed aa libitum was higher (P<0.01) than in animals fed with a dosing system. The density of examined bones within the breeds took the following order: Pietrain race> line 990> Duroc race. The differences between the “extreme” breeds were: 7.2% (III metatarsal bone), 8.3% (III metacarpal bone), 8.4% (femur). Abstract 13 The average bone stiffness took the following order: III metatarsal bone (35.1 kg/mm) <III metacarpus (41.5 kg/mm) <femur (60.5 kg/mm). This indicator did not differ between the groups of pigs fed at different intensity, except for the metacarpal bone, which was more stiffer in pigs fed aa libitum (P<0.05). The femur of animals fed ad libitum showed a tendency (P<0.09) to be more stiffer and a force of 4.5 kg required for its displacement by 1 mm. Breed differences in stiffness were found for the femur (P <0.05) and III metacarpal bone (P <0.05). For femur, the highest value of this indicator was found in Pietrain pigs (64.5 kg/mm), lower in pigs of 990 line (61.6 kg/mm) and the lowest in Duroc pigs (55.3 kg/mm). In turn, the 3rd metacarpal bone of Duroc and Pietrain pigs had similar stiffness (39.0 and 40.0 kg/mm respectively) and was smaller than that of line 990 pigs (45.4 kg/mm). The thickness of the cortical bone layer took the following order: III metatarsal bone (2.25 mm) <III metacarpal bone (2.41 mm) <femur (5.12 mm). The feeding system did not affect this indicator. Breed differences (P <0.05) for this trait were found only for the femur bone: Duroc (5.42 mm)> line 990 (5.13 mm)> Pietrain (4.81 mm). The cross sectional area of the examined bones was arranged in the following order: III metatarsal bone (84 mm2) <III metacarpal bone (90 mm2) <femur (286 mm2). The feeding system had no effect on the value of this bone trait, with the exception of the femur, which in animals fed the dosing system was 4.7% higher (P<0.05) than in pigs fed ad libitum. Breed differences (P<0.01) in the coross sectional area were found only in femur and III metatarsal bone. The value of this indicator was the highest in Duroc pigs, lower in 990 animals and the lowest in Pietrain pigs. The cortical index of individual bones was in the following order: III metatarsal bone (31.86) <III metacarpal bone (33.86) <femur (44.75). However, its value did not significantly depend on the intensity of feeding or the breed of pigs.
Book chapters on the topic "Tissue specific knock out"
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Plateroti, Michelina, Cristina Angelin-Duclos, Frederic Flamant, and Jacques Samarut. "Tissues Specific Action of Thyroid Hormones: Insights from Knock out Animal Models." In Syndromes of Hormone Resistance on the Hypothalamic-Pituitary-Thyroid Axis, 13–33. Boston, MA: Springer US, 2004. http://dx.doi.org/10.1007/978-1-4020-7852-1_2.
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Akhmedov, Dmitry, Nicholas S. Kirkby, Jane A. Mitchell, and Rebecca Berdeaux. "Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice." In Methods in Molecular Biology, 361–76. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9121-1_21.
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Aoki, Koji, and Makoto M. Taketo. "Tissue-Specific Transgenic, Conditional Knockout and Knock-In Mice of Genes in the Canonical Wnt Signaling Pathway." In Methods in Molecular Biology, 307–31. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-249-6_24.
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Paro, Renato, Ueli Grossniklaus, Raffaella Santoro, and Anton Wutz. "Regeneration and Reprogramming." In Introduction to Epigenetics, 135–49. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-68670-3_7.
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AbstractDuring regenerative processes, cells are required to restructure parts of a damaged or worn-out organ and tissue. Here, you will become acquainted with the strategies that organisms developed to provide the material for tissue and organ repair. On the one hand, somatic cells can become dedifferentiated to increase their developmental potential and produce the plasticity required to replace the entire cellular complexity of a damaged part. On the other hand, organisms retain organ-specific stem cells with a restricted developmental potency and use these to provide the “spare parts” for replacing damaged cells. In all cases, a substantial reprogramming of the epigenome of these cells accompanies the restructuring process. In vitro strategies have been developed to drive cells back to a pluripotent state, allowing a better understanding of the underlying chromatin adjustments and providing a rich source for cellular therapies.
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Jaspers, M. E. H., and P. Moortgat. "Objective Assessment Tools: Physical Parameters in Scar Assessment." In Textbook on Scar Management, 149–58. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-44766-3_17.
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AbstractObjective assessment tools can be used to evaluate whether (new) scar treatment is effective and to monitor the scar’s response to interventions in clinical practice. It is important to take the clinimetric properties of each tool into account, especially when used for the follow-up of an individual patient. An overview is provided for three important physical scar parameters that can be assessed by noninvasive objective tools: color, elasticity, and perfusion. To assess the color of a scar, an array of tools is available, all using reflectance spectroscopy and determining color by measuring the intensity of reflected light of specific wavelengths. The handheld DSM III ColorMeter offers read-out of erythema and melanin index values as well as CIEL∗a∗b values. The interrater reliability is best for the parameter a∗ of the DSM III ColorMeter. To assess scar elasticity, the Cutometer is the most widely used tool. Scar deformation is measured using negative pressure and reflected in relative and absolute elasticity parameters. On the contrary, firmness or hardness of scar tissue can be quantified by tonometry, a technique that works by exerting pressure on the skin. Lastly, it is of interest to measure scar blood flow (i.e., perfusion) as several treatment regimens work by destructing the microvasculature and/or reducing the blood flow to enhance shrinkage of hypertrophic scar tissue. Laser Doppler imaging and laser speckle imaging can be used to quantify and visualize scar blood flow, but a thorough clinimetric evaluation of these tools in scars is not performed yet.
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Mustafa, El, Sat Parmar, and Prav Praveen. "Premalignant Lesions and Conditions of the Oral Cavity." In Oral and Maxillofacial Surgery for the Clinician, 1845–52. Singapore: Springer Singapore, 2021. http://dx.doi.org/10.1007/978-981-15-1346-6_80.
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AbstractOral cancer develops in precursor lesions referred to as the group of premalignant disorders (PMDs) by the World Health Organization (WHO). Some lesions are relatively common affecting between 1 and 5% of the population (leukoplakia) and may resemble benign and prevalent mucosal disease. These lesions pose a risk for malignancy that is independent of tobacco or alcohol, with a wide range of transformation rates between 13 and 70%. The commonest types are white patches (leukoplakia), red patches (erythroplakia) and submucous fibrosis. Knowledge of the patterns of clinical presentation of PMDs is important in order to screen patients effectively, identifying those who benefit from a close observation, those who require from targeted biopsy and those who may be safely followed up in primary care. We describe clinical features of the most well-documented premalignant disorders discussing lesion-specific risk predictors and treatment options. We also present a brief outline of the less prevalent group or premalignant systemic conditions including those that predispose to the development of mucosal squamous carcinoma and those that associate with the development of cutaneous squamous carcinoma. Genetic pathways involved in the development and progression of PMDs are outlined, and finally, we describe best practices for carrying out a diagnostic tissue biopsy.
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"INTERLEUKIN-7 (IL-7) KNOCK OUT MICE: IMPLICATIONS FOR LYMPHOPOIESIS AND ORGAN-SPECIFIC IMMUNITY." In Cytokines and Cytokine Receptors, 283–304. CRC Press, 2001. http://dx.doi.org/10.1201/9781482283716-16.
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S. Elton, Terry, Md Ismail Hossain, Jessika Carvajal-Moreno, Xinyi Wang, Dalton J. Skaggs, and Jack C. Yalowich. "Maximizing the Efficacy of CRISPR/Cas Homology-Directed Repair Gene Targeting." In CRISPR Technology - Recent Advances [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.109051.
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Clustered regularly interspaced short palindromic repeats/CRISPR-associated system (CRISPR/Cas) is a powerful gene editing tool that can introduce double-strand breaks (DSBs) at precise target sites in genomic DNA. In mammalian cells, the CRISPR/Cas-generated DSBs can be repaired by either template-free error-prone end joining (e.g., non-homologous end joining/microhomology-mediated end joining [NHEJ]/[MMEJ]) or templated error-free homology-directed repair (HDR) pathways. CRISPR/Cas with NHEJ/MMEJ DNA repair results in various length insertions/deletion mutations (indels), which can cause frameshift mutations leading to a stop codon and subsequent gene-specific knockout (i.e., loss of function). In contrast, CRISPR/Cas with HDR DNA repair, utilizing an exogenous repair template harboring specific nucleotide (nt) changes, can be employed to intentionally edit out or introduce mutations or insertions at specific genomic sites (i.e., targeted gene knock-in). This review provides an overview of HDR-based gene-targeting strategies to facilitate the knock-in process, including improving gRNA cleavage efficiency, optimizing HDR efficacy, decreasing off-target effects, suppressing NHEJ/MMEJ activity, and thus expediting the screening of CRISPR/Cas-edited clonal cells.
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Raydugin, Yuri G. "Interactions in a Project System." In Modern Risk Quantification in Complex Projects, 161–74. Oxford University Press, 2020. http://dx.doi.org/10.1093/oso/9780198844334.003.0010.
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This chapter is a bridge from the traditional ‘linear’ definitions of projects and project risks to their ‘non-linear’ versions for complex projects. A project is a dynamic system accompanied by a phased process to deliver a new product, service or facility that is characterized by evolving phase-specific structure (the project structure subsystem (PSS)) matched by a required delivery organization (the project delivery subsystem (PDS)). Risk is a possible aggregated impact on project objectives by an affinity of interacting risks that form dynamic risk patterns supported by a project system. Three types of modified bowtie diagrams are introduced to identify and address instances of risk interactions (internal risk amplification, knock-on interactions, and risk compounding). Risk interactions are described mathematically. Based on the system dynamics experience, even a traditional bowtie diagram for standalone risks in complex projects should be modified to factor in risk-induced work constraints, rework, out-of-sequence work, and lower productivity.
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Bri, Diana, Jaime Lloret, Carlos Turro, and Miguel Garcia. "Measuring Specific Absorption Rate by using Standard Communications Equipment." In Advances in Healthcare Information Systems and Administration, 81–111. IGI Global, 2012. http://dx.doi.org/10.4018/978-1-4666-0888-7.ch004.
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Specific Absorption Rate (SAR) is used to measure the body tissue exposure to electromagnetic fields. This chapter describes how SAR values can be estimated from a deployed Wireless Local Area Network (WLAN). We carried out this work using the Received Signal Strength (RSS) obtained from the access points. This parameter is easily obtained by an ordinary wireless network scanner. RSS variations are measured for a different number of people in the same room and without people. It will allow us to estimate how much energy is absorbed by a group of people and by a single person on average. Moreover, we have included the weight of the people in order to know the RSS lost by kilogram. These measurements were taken at the Higher Polytechnic School of Gandia, Universitat Politècnica de València, Spain, in two placements: the library and inside an anechoic camera.
Conference papers on the topic "Tissue specific knock out"
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Yoshida, Kyoko, Claire Reeves, Jan Kitajewski, Ronald Wapner, Joy Vink, Michael Fernandez, and Kristin Myers. "Anthrax Toxin Receptor 2 Knock-Out and Wild Type Mouse Cervix Exhibit Time-Dependent Mechanical Properties." In ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80732.
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The cervix plays a critical role during pregnancy, acting as a mechanical barrier to keep the fetus inside the uterus until term. In a normal pregnancy, it is hypothesized that the cervix gradually softens until uterine contractions occur. At this point, the cervix dramatically ripens and dilates for delivery. Similar to other collagenous tissues, the extracellular matrix (ECM) is the load-bearing component of cervical tissue. It is composed mainly of a cross-linked network of fibril forming collagen, types I and III, embedded in a viscous proteoglycan ground substance. Studies conducted on animal models suggest that during normal maturation, a shift in ECM components facilitate cervical softening. However, quantitative cervical softness measurements (i.e. material properties) of these previous studies are ill-defined, limiting the comparative ability of the outcome values. Therefore, our goal is to quantify sensitive and specific time-dependent material properties utilizing mouse models of normal and abnormal pregnancy. Our aim is to discern the role of ECM maintenance in cervical softening.
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Bukowski, Michael, Brij Singh, James Roemmich, and Kate Larson. "Lipidomic analysis of TRPC1 Ca2+-permeable channel-knock out mouse demonstrates a vital role in placental tissue sphingolipid and triacylglycerol homeostasis under high-fat diet." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/tjdt4839.
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Placental function including oxygen delivery and nutrient transport are critical determinants of fetal growth, moderating the risks of obesity and metabolic diseases later in life. Previously, we demonstrated in a mouse model that parental diet and exercise play important roles in placental lipid content and inflammation. Transient receptor potential canonical channel 1 (TRPC1) is a Ca2+-permeable integral membrane protein. We have demonstrated that TRPC1 increases total body adiposity in mice by decreasing the efficacy of exercise to limit adipose accumulation under a high fat (HF) diet. Importantly, intracellular calcium may regulate total body adiposity and increased total body adiposity could promote placental lipid accumulation. Similarly, intracellular calcium regulates membrane lipid content via the activation of the protein kinase C. Membrane lipids such as sphingomyelin are key regulators of cell signaling. Maternal HF diets increase placental tissue lipid concentrations resulting in compromised nutrient transport to fetus. However, the specific lipid species that accumulate due to the absence of the placental TRPC1 gene under maternal HF diet feeding is not yet known. We hypothesized that placental tissue response to a maternal HF diet is disrupted in TRPC1 mice fed a maternal HF diet resulting in greater cellular sphingomyelin concentrations. Results showed placentae from TRPC1 KO mice fed high fat diet (45% en, HF) had increased sphingomyelin concentrations compared to control diet (16% en, NF). Placentae from WT mice fed HF diet exhibited diet-dependent increases in ceramide concentration with no concomitant increase in sphingomyelins compared to NF fed WT mice. Additionally, 11 placental triacylglycerol (TAG) species were different based on diet, 16 based on genotype, and 5 were affected by both diet and genotype. These results suggest that during a HF diet, loss of TRPC1 function reduces placental sphingomyelin hydrolysis into ceramide and that placental TAG concentrations respond in diet- and genotype-dependent manner.
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Kloth, B., H. Reichenspurner, T. Eschenhagen, and M. Hirt. "Cardiac Remodeling in Cardiomyocyte-Specific PIEZO2 Knock-Out Mice." In 50th Annual Meeting of the German Society for Thoracic and Cardiovascular Surgery (DGTHG). Georg Thieme Verlag KG, 2021. http://dx.doi.org/10.1055/s-0041-1725677.
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Torday, JS, S. Wei, R. Lee, VK Rehan, and D. Wei. "Characterization of the Lung Mesenchyme-Specific Dermo-1Cre Conditional PTHrP Knock out Phenotype." In American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a3274.
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Ghosh, B., J. Loube, S. Chen, K. Nishida, L. Ying, G. Howard, M. Zaykaner, W. Mitzner, and V. K. Sidhaye. "Characterization of Lung Specific E-Cadherin Knock-Out Model in Obstructive Lung Disease." In American Thoracic Society 2020 International Conference, May 15-20, 2020 - Philadelphia, PA. American Thoracic Society, 2020. http://dx.doi.org/10.1164/ajrccm-conference.2020.201.1_meetingabstracts.a4064.
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Diaz Martinez, Myriam, Masoud Ghamari-Langroudi, Aliya Gifford, Roger Cone, and E. B. Welch. "Automated pipeline to analyze non-contact infrared images of the paraventricular nucleus specific leptin receptor knock-out mouse model." In SPIE Medical Imaging, edited by Barjor Gimi and Robert C. Molthen. SPIE, 2015. http://dx.doi.org/10.1117/12.2082102.
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Rhee, Wonjong, Hanjoong Jo, and Gang Bao. "Live Cell Detection of Specific Messenger RNA for Molecular Analysis of Plaque Formation." In ASME 2007 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2007. http://dx.doi.org/10.1115/sbc2007-176737.
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The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization in response to shear flow can provide unprecedented opportunities for the molecular analysis of atherosclerosis. We carried out an extensive study of the design of molecular beacons to target BMP-4 mRNA, which plays important roles in proatherogenic development in response to unstable flow conditions. Specifically, we selected an optimal molecular beacon design, and found that the fluorescent intensity from targeting BMP-4 mRNA correlated well with the GFP signal after up-regulating BMP-4 and co-expressing GFP using adenovirus. The knock-down of BMP-4 mRNA using siRNA significantly reduced the beacon signal, further demonstrating detection specificity. We found that, due to target accessibility, molecular beacons designed with different target sequences gave very different signal levels, and establishing molecular beacon design rules has significant implications to live cell mRNA detections, especially to the studies of BMP-4 mRNA in endothelial cells under shear flow.
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Sampath, V., R. Mohan, S. Wang, L. Gomez, O. Shoham, and J. Marrelli. "Intelligent Control of Compact Multiphase Separation System (CMSS©)—Part I: Modeling and Simulation." In ASME 2009 Fluids Engineering Division Summer Meeting. ASMEDC, 2009. http://dx.doi.org/10.1115/fedsm2009-78422.
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Performance of compact separators depends on implementation of stable and robust control strategies that are suited for specific applications. In this investigation, an intelligent control system has been developed for Compact Multiphase Separation System (CMSS©) which consists of integrated configurations of three compact separators, namely, Gas-Liquid Cylindrical Cyclone (GLCC©), Liquid-Liquid Cylindrical Cyclone (LLCC©) and Liquid-Liquid Hydrocyclone (LLHC). This is a two-part paper, the first part (current paper) deals with the Modeling and Simulation of the CMSS© and the second part deals with Experimental Investigation. The specific objective of this CMSS© configuration is to knock out free water from the upstream fluids. In mature oil fields, water handling poses a huge problem. Thus water knock out at the earliest stage helps in significant cost savings. A novel fuzzy logic control system has been designed and tested for change in set-point of differential pressure ratio in LLHC. Dynamic models have been developed for each of the above mentioned control systems for design of stable PID parameters. A dynamic simulation platform (DSP) has been developed based on these models in Matlab/Simulink™ for predicting the transient performance of the integrated system. Steady state mechanistic models of individual devices are integrated to the Matlab/Simulink™ platform using look up tables to predict the overall response of the CMSS© for different scenarios.
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Ludu, Andrei, Torsten Baufeld, Harald Philipp, and Carolus Gru¨nig. "Open Chamber Spark Ignited Combustion System Development for High Power Density and Low Emissions." In ASME 2003 Internal Combustion Engine Division Spring Technical Conference. ASMEDC, 2003. http://dx.doi.org/10.1115/ices2003-0677.
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High power density and thermal efficiency combined with very low NOx exhaust emissions are main challenges in today’s gas engine development programmes. Until recently open chamber combustion systems using traditional spark ignition were considered to be limited by knock and misfire in their potential to achieve the above mentioned goals. The main task of the work reported here was to achieve a stable, knock-free combustion using a knock-prone gas fuel of 70 methane number for a specific power density of 25 kW/L at 250 mg/m3N NOx emission according to the TA Luft. The combustion system development was carried out on a single cylinder engine (SCE), thus, a special aspect of the work was to ensure transferability of the single cylinder engine combustion system development results to the targeted V12 multi-cylinder engine. Prior to engine testing, intensive simulation work was undertaken to specify the most promising engine hardware configurations. The main emphasis was on the 3D CFD analysis of the in-cylinder charge motion and its interaction with carefully chosen combustion chamber geometry variants. The ultimate scope was to generate high local turbulence levels during combustion, enabling a short combustion duration with low variation coefficients. The experimental phase included engine tests for several combustion relevant parameters and the detailed combustion analysis with respect to flame propagation and knock centre location using the optical combustion diagnosis system AVL VISIOLUTION. This paper describes the overall work procedure as well as the analytical and experimental methods and tools employed in the combustion system development programme. It also gives information on engine test results, the value and contribution of combustion visualisation, as well as on the engine performance which could be finally reached.
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Corti, Enrico, and Claudio Forte. "A Statistical Approach to Spark Advance Mapping." In ASME 2009 Internal Combustion Engine Division Spring Technical Conference. ASMEDC, 2009. http://dx.doi.org/10.1115/ices2009-76111.
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Engines performance and efficiency are largely influenced by the combustion phasing. Operating conditions and control settings influence the combustion development over the crankshaft angle: the most effective control parameter used by Electronic Control Units (ECU) to optimize the combustion process for Spark Ignition (SI) engines is Spark Advance (SA). SA mapping is a time-consuming process, usually carried out with the engine running in steady state on the test bench, changing SA values while monitoring Brake and Indicated Mean Effective Pressure (BMEP, IMEP) and Brake Specific Fuel Consumption (BSFC). Mean values of IMEP and BSFC for a test carried out with a given SA setting are considered as the parameters to optimize. However, the effect of SA on IMEP and BSFC is not deterministic, due to the cycle-to-cycle variation: the analysis of mean values requires many engine cycles to be significant of the performance obtained with the given control setting. Finally other elements, such as engine or components ageing, and disturbances like Air-to-Fuel Ratio (AFR) or air, water and oil temperature variations, could affect the tests results: this facet can be very significant for racing engines testing. This paper presents a novel approach to SA mapping, with the objective of improving the performance analysis robustness, while reducing the test time. The methodology is based on the observation that, for a given running condition, IMEP can be considered a function of the combustion phasing, represented by the 50% Mass Fraction Burned (MFB50) parameter. Due to cycle-to-cycle variation, many different MFB50 and IMEP values are obtained during a steady state test carried out with constant SA. While MFB50 and IMEP absolute values are influenced by disturbance factors, the relationship between them holds, and it can be synthesized by means of the angular coefficient of the tangent line to the MFB50-IMEP distribution. The angular coefficient variations as a function of SA can be used to feed a SA controller, able to maintain the optimal combustion phasing. Similarly, knock detection is approached by evaluating two indexes: the distribution of a typical knock-sensitive parameter (MAPO, Maximum Amplitude of Pressure Oscillations) is related to that of CHRNET (net Cumulative Heat Release), determining a robust knock index. A knock limiter controller can then be added, in order to restrict the SA range to safe values. The methodology can be implemented in real-time combustion controllers: the algorithms have been applied offline to sampled data, showing the feasibility of fast and robust automatic mapping procedures.
Reports on the topic "Tissue specific knock out"
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Kudaravalli, Rama. Mammary Specific Expression of Cre Recombinase Under the Control of an Endogenous MMTV LTR: A Conditional Knock-Out System. Fort Belvoir, VA: Defense Technical Information Center, October 2001. http://dx.doi.org/10.21236/ada403399.
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Czarneski, Jennifer. Mammary Specific Expression of Cre Recombinase Under the Control of an Endogenous MMTV LTR: A Conditional Knock-Out System. Fort Belvoir, VA: Defense Technical Information Center, April 2000. http://dx.doi.org/10.21236/ada391092.
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Lers, Amnon, and Pamela J. Green. Analysis of Small RNAs Associated with Plant Senescence. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7593393.bard.
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Senescence is an agriculturally significant process due to its negative impact to crop yield and postharvest quality. The genetic regulatory systems controlling senescence induction and progress respond to both developmental and environmental stress signals and involve numerous gene expression changes. Knowledge about the key molecular factors which control senescence is very limited. MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, responses to environmental stresses, and senescence. The long-term goal of this work is to elucidate roles of small RNAs associated with plant senescence. The hypothesis underlying this research is that miRNA-mediated regulation makes important contributions to the senescence process in plants. Specific, original research objectives included: 1) Profiling of small RNAs from senescing plants; 2) Data Analysis and public access via a user-friendly web interface; 3) Validation of senescence-associated miRNAs and target RNAs; 4) Development of transgenic plants for functional analysis of miRNAs in Arabidopsis. Major revisions made in the research compared to the original work plan included 1) Exclusion of the planned work with tomato as recommended by the BARD review panel; 2) Performing miRNA study also in senescing Arabidopsis siliques, in addition to senescing leaves. To identify senescenceregulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel Analysis of RNA Ends (PARE) libraries, which enable the large-scale examination of miRNA-guided cleavage products, were also constructed and sequenced, resulting in over 750 million genome-matched sequences. These massive datasets lead to the identification of new miRNAs, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence. Experiments were initiated for functional analysis of specific senescence-associated miRNAs and respective target genes. Transgenic Arabidopsis plants were generated in which miR408, found in this study to be significantly induced in leaf senescence, was over-expressed either constitutively or under a senescence-specific promoter. These plants are currently being characterized for any altered phenotypes. In addition T-DNA knock out mutants for various target genes identified in this research are being analyzed. This work provides insights about specific miRNAs that contribute to leaf and silique senescence. The knowledge generated may suggest new strategies to monitor and alter the progression of senescence in crops for agricultural improvement.
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Eshed, Yuval, and John Bowman. Harnessing Fine Scale Tuning of Endogenous Plant Regulatory Processes for Manipulation of Organ Growth. United States Department of Agriculture, 2005. http://dx.doi.org/10.32747/2005.7696519.bard.
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Background and objectives: Manipulation of plant organ growth is one of the primary reasons for the success of mankind allowing increasing amounts of food for human and livestock consumption. In contrast with the successful selection for desirable growth characteristics using plant breeding, transgenic manipulations with single genes has met limited success. While breeding is based on accumulation of many small alterations of growth, usually arise from slight changes in expression patterns, transgenic manipulations are primarily based on drastic, non-specific up-regulation or knock down of genes that can exert different effects during different stages of development. To successfully harness transgenic manipulation to attain desirable plant growth traits we require the tools to subtly regulate the temporal and spatial activity of plant growth genes. Polar morphology along the adaxial/abaxial axis characterizes lateral organs of all plants. Juxtaposition of two cell types along this axis is a prerequisite of laminar growth induction. In the study summarized here, we addressed the following questions: Can we identify and harness components of the organ polarity establishment pathway for prolonged growth? Can we identify specific regulatory sequences allowing spatial and temporal manipulation in various stages of organ development? Can we identify genes associated with YABBY-induced growth alterations? Major conclusions and implications: We showed that regulated expression, both spatially and temporally of either organ polarity factors such as the YABBY genes, or the organ maturation program such as the CIN-TCPs can stimulate substantial growth of leaves and floral organs. Promoters for such fine manipulation could be identified by comparison of non-coding sequences of KAN1, where a highly conserved domain was found within the second intron, or by examination of multiple 5” regions of genes showing transient expression along leaf ontogeny. These promoters illustrate the context dependent action of any gene we examined thus far, and facilitate fine tuning of the complex growth process. Implications, both scientific and agricultural. The present study was carried out on the model organism Arabidopsis, and the broad application of its findings were tested in the tomato crop. We learned that all central regulators of organ polarity are functionally conserved, probably in all flowering plants. Thus, with minor modifications, the rules and mechanisms outlined in this work are likely to be general.
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Handa, Avtar K., Yuval Eshdat, Avichai Perl, Bruce A. Watkins, Doron Holland, and David Levy. Enhancing Quality Attributes of Potato and Tomato by Modifying and Controlling their Oxidative Stress Outcome. United States Department of Agriculture, May 2004. http://dx.doi.org/10.32747/2004.7586532.bard.
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General The final goal and overall objective of the current research has been to modify lipid hydroperoxidation in order to create desirable phenotypes in two important crops, potato and tomato, which normally are exposed to abiotic stress associated with such oxidation. The specific original objectives were: (i) the roles of lipoxygenase (LOX) and phospholipids hydroperoxide glutathione peroxidase (PHGPx) in regulating endogenous levels of lipid peroxidation in plant tissues; (ii) the effect of modified lipid peroxidation on fruit ripening, tuber quality, crop productivity and abiotic stress tolerance; (iii) the effect of simultaneous reduction of LOX and increase of PHGPx activities on fruit ripening and tuber quality; and (iv) the role of lipid peroxidation on expression of specific genes. We proposed to accomplish the research goal by genetic engineering of the metabolic activities of LOX and PHGPx using regulatable and tissue specific promoters, and study of the relationships between these two consecutive enzymes in the metabolism and catabolism of phospholipids hydroperoxides. USA Significant progress was made in accomplishing all objectives of proposed research. Due to inability to regenerate tomato plants after transforming with 35S-PHGPx chimeric gene construct, the role of low catalase induced oxidative stress instead of PHGPx was evaluated on agronomical performance of tomato plant and fruit quality attributes. Effects of polyamine, that protects DNA from oxidative stress, were also evaluated. The transgenic plants under expressing lipoxygenase (LOX-sup) were crossed with catalase antisense (CAT-anti) plants or polyamine over producing plants (SAM-over) and the lines homozygous for the two transgenes were selected. Agronomical performance of these line showed that low catalase induced oxidative stress negatively affected growth and development of tomato plants and resulted in a massive change in fruit gene expression. These effects of low catalase activity induced oxidative stress, including the massive shift in gene expression, were greatly overcome by the low lipoxygenase activity. Collectively results show that oxidative stress plays significant role in plant growth including the fruit growth. These results also for the first time indicated that a crosstalk between oxidative stress and lipoxygenase regulated processes determine the outcome during plant growth and development. Israel Regarding PHGPx, most of the study has concentrated on the first and the last specific objectives, since it became evident that plant transformation with this gene is not obvious. Following inability to achieve efficient transformation of potato and tomato using a variety of promoters, model plant systems (tobacco and potato cell cultures, tobacco calli and plantlets, and Arabidopsis) were used to establish the factors and to study the obstacles which prohibited the regeneration of plants carrying the genetic machinery for overproduction of PHGPx. Our results clearly demonstrate that while genetic transformation and over-expression of PHGPx occurs in pre-developmental tissue stage (cell culture, calli clusters) or in completed plant (Arabidopsis), it is likely that over-expression of this enzyme before tissue differentiation is leading to a halt of the regeneration process. To support this assumption, experiments, in which genetic engineering of a point-mutated PHGPx gene enable transformation and over-expression in plants of PhSPY modified in its catalytic site and thus inactive enzymatically, were successfully carried out. These combined results strongly suggest, that if in fact, like in animals and as we established in vitro, the plant PHGPx exhibits PH peroxidase activity, these peroxides are vital for the organisms developmental process.
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Izhar, Shamay, Maureen Hanson, and Nurit Firon. Expression of the Mitochondrial Locus Associated with Cytoplasmic Male Sterility in Petunia. United States Department of Agriculture, February 1996. http://dx.doi.org/10.32747/1996.7604933.bard.
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The main goal of the proposed research was to continue the mutual investigations into the molecular basis of CMS and male fertility restoration [MRF], with the ultimate goal of understanding these phenomena in higher plants. The experiments focused on: (1) dissecting apart the complex CMS - specific mitochondrial S-Pcf locus, in order to distinguish its essential parts which cause sterility from other parts and study its molecular evolution. (2) Studying the expression of the various regions of the S-Pcf locus in fertile and sterile lines and comparing the structure and ultrastructure of sterile and fertile tissues. (3) Determine whether alteration in respiration is genetically associated with CMS. Our mutual investigations further substantiated the association between the S-Pcf locus and CMS by the findings that the fertile phenotype of a population of unstable petunia somatic hybrids which contain the S-Pcf locus, is due to the presence of multiple muclear fertility restoration genes in this group of progenies. The information obtained by our studies indicate that homologous recombination played a major role in the molecular evolution of the S-Pcf locus and the CMS trait and in the generation of mitochondrial mutations in general. Our data suggest that the CMS cytoplasm evolved by introduction of a urs-s containing sublimon into the main mitochondrial genome via homologous recombination. We have also found that the first mutation detected so far in S-Pcf is a consequence of a homologous recombination mechanism involving part of the cox2 coding sequence. In all the cases studied by us, at the molecular level, we found that fusion of two different cells caused mitochondrial DNA recombination followed by sorting out of a specific mtDNA population or sequences. This sequence of events suggested as a mechanism for the generation of novel mitochondrial genomes and the creation of new traits. The present research also provides data concerning the expression of the recombined and complex CMS-specific S-Pcf locus as compared with the expression of additional mitochondrial proteins as well as comparative histological and ultrastructural studies of CMS and fertile Petunia. Evidence is provided for differential localization of mitochondrially encoded proteins in situ at the tissue level. The similar localization patterns of Pcf and atpA may indicate that Pcf product could interfere with the functioning of the mitochondrial ATPase in a tissue undergoing meiosis and microsporogenesis. Studies of respiration in CMS and fertile Petunia lines indicate that they differe in the partitioning of electron transport through the cytochrome oxidase and alternative oxidase pathways. The data indicate that the electron flux through the two oxidase pathways differs between mitochondria from fertile and sterile Petunia lines at certain redox states of the ubiquinone pool. In summary, extensive data concerning the CMS-specific S-Pcf locus of Petunia at the DNA and protein levels as well as information concerning different biochemical activity in CMS as compared to male fertile lines have been accumulated during the three years of this project. In addition, the involvement of the homologous recombination mechanism in the evolution of mt encoded traits is emphasized.
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Meir, Shimon, Michael S. Reid, Cai-Zhong Jiang, Amnon Lers, and Sonia Philosoph-Hadas. Molecular Studies of Postharvest Leaf and Flower Senescence. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7592657.bard.
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Original objectives: To understand the regulation of abscission by exploring the nature of changes of auxin-related gene expression in tomato (Lycopersicon esculatumMill) abscission zones (AZs) following organ removal, and by analyzing the function of these genes. Our specific goals were: 1) To complete the microarray analyses in tomato flower and leaf AZs, for identifying genes whose expression changes early in response to auxin depletion; 2) To examine, using virus-induced gene silencing (VIGS), the effect of silencing target genes on ethylene sensitivity and abscission competence of the leaf and flower AZs; 3) To isolate and characterize promoters from AZ-specific genes to be used in functional analysis; 4) To generate stable transgenic tomato plants with selected genes silenced with RNAi, under the control of an AZ-specific promoter, for further characterization of their abscission phenotypes. Background: Abscission, the separation of organs from the parent plant, results in postharvest quality loss in many ornamentals and other fresh produce. The process is initiated by changes in the auxin gradient across the AZ, and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the initiation of the abscission process, as the AZ becomes sensitized to ethylene. The present project was focused on elucidating these early molecular regulatory events, in order to gain a better control of the abscission process for agricultural manipulations. Major conclusions, solutions, achievements: Microarray analyses, using the Affymetrix Tomato GeneChip®, revealed changes in expression, occurring early in abscission, of many genes with possible regulatory functions. These included a range of auxin- and ethylene-related transcription factors (TFs), other TFs that are transiently induced just after flower removal, and a set of novel AZ-specific genes. We also identified four different defense-related genes, including: Cysteine-type endopeptidase, α- DOX1, WIN2, and SDF2, that are newly-associated with the late stage of the abscission process. This supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses. To facilitate functional studies we implemented an efficient VIGS system in tomato, and isolated two abscission-specific promoters (pTAPG1 and pTAPG4) for gene silencing in stable transformation. Using the VIGS system we could demonstrate the importance of TAPGs in abscission of tomato leaf petioles, and evaluated the importance of more than 45 genes in abscission. Among them we identified few critical genes involved in leaf and flower abscission. These included: PTRP-F1, PRP, TKN4, KNOTTED-like homeobox TF, KD1, and KNOX-like homeodomain protein genes, the silencing of which caused a striking retardation of pedicel abscission, and ERF1, ERF4, Clavata-like3 protein, Sucrose transporter protein, and IAA10 genes, the silencing of which delayed petiole abscission. The importance of PRPand KD1 genes in abscission was confirmed also by antisense–silencing using pTAPG4. Experiments testing the effects of RNAi silencing of few other genes are still in progress, The analysis of the microarray results of flower and leaf AZs allowed us to establish a clear sequence of events occurring during acquisition of tissue sensitivity to ethylene, and to confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes in both AZs. Implication, both scientific and agricultural: Our studies had provided new insights into the regulation of the abscission process, and shaded light on the molecular mechanisms that drive the acquisition of abscission competence in the AZ. We pointed out some critical genes involved in regulation of abscission, and further expanded our knowledge of auxin-ethylene cross talk during the abscission process. This permits the development of novel techniques for manipulating abscission, and thereby improving the postharvest performance of ornamentals and other crops.
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Lers, Amnon, and Gan Susheng. Study of the regulatory mechanism involved in dark-induced Postharvest leaf senescence. United States Department of Agriculture, January 2009. http://dx.doi.org/10.32747/2009.7591734.bard.
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Postharvest leaf senescence contributes to quality losses in flowers and leafy vegetables. The general goal of this research project was to investigate the regulatory mechanisms involved in dark-induced leaf senescence. The regulatory system involved in senescence induction and control is highly complex and possibly involves a network of senescence promoting pathways responsible for activation of the senescence-associated genes. Pathways involving different internal signals and environmental factors may have distinctive importance in different leaf senescence systems. Darkness is known to have a role in enhancement of postharvest leaf senescence and for getting an insight into its regulatory mechanism/s we have applied molecular genetics and functional genomics approaches. The original objectives were: 1. Identification of dark-induced SAGs in Arabidopsis using enhancer/promoter trap lines and microarray approaches; 2. Molecular and functional characterization of the identified genes by analyzing their expression and examining the phenotypes in related knockout mutant plants; 3. Initial studies of promoter sequences for selected early dark-induced SAGs. Since genomic studies of senescence, with emphasis on dark-induced senescence, were early-on published which included information on potential regulatory genes we decided to use this new information. This is instead of using the uncharacterized enhancer/promoter trap lines as originally planned. We have also focused on specific relevant genes identified in the two laboratories. Based on the available genomic analyses of leaf senescence 10 candidate genes hypothesized to have a regulatory role in dark-induced senescence were subjected to both expression as well as functional analyses. For most of these genes senescence-specific regulation was confirmed, however, functional analyses using knock-out mutants indicated no consequence to senescence progression. The transcription factor WARK75 was found to be specifically expressed during natural and dark-induced leaf senescence. Functional analysis demonstrated that in detached leaves senescence under darkness was significantly delayed while no phenotypic consequences could be observed on growth and development, including no effect on natural leaf senescence,. Thus, WARKY75 is suggested to have a role in dark-induced senescence, but not in natural senescence. Another regulatory gene identified to have a role in senescence is MKK9 encoding for a Mitogen-Activated Protein Kinase Kinase 9 which is upregulated during senescence in harvested leaves as well as in naturally senescing leaves. MKK9 can specifically phosphorylate another kinase, MPK6. Both knockouts of MKK9 and MPK6 displayed a significantly senescence delay in harvested leaves and possibly function as a phosphorelay that regulates senescence. To our knowledge, this is the first report that clearly demonstrates the involvement of a MAP kinase pathway in senescence. This research not only revealed a new signal transduction pathway, but more important provided significant insights into the regulatory mechanisms underlying senescence in harvested leaves. In an additional line of research we have employed the promoter of the senescence-induced BFN1 gene as a handle for identifying components of the regulatory mechanism. This gene was shown to be activated during darkinduced senescence of detached leaves, as well as natural senescence. This was shown by following protein accumulation and promoter activity which demonstrated that this promoter is activated during dark-induced senescence. Analysis of the promoter established that, at least some of the regulatory sequences reside in an 80 bps long fragment of the promoter. Overall, progress was made in identification of components with a role in dark-induced senescence in this project. Further studies should be done in order to better understand the function of these components and develop approaches for modulating the progress of senescence in crop plants for the benefit of agriculture.
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Altstein, Miriam, and Ronald Nachman. Rationally designed insect neuropeptide agonists and antagonists: application for the characterization of the pyrokinin/Pban mechanisms of action in insects. United States Department of Agriculture, October 2006. http://dx.doi.org/10.32747/2006.7587235.bard.
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The general objective of this BARD project focused on rationally designed insect neuropeptide (NP) agonists and antagonists, their application for the characterization of the mechanisms of action of the pyrokinin/PBAN (PK-PBAN) family and the development of biostable, bioavailable versions that can provide the basis for development of novel, environmentally-friendly pest insect control agents. The specific objectives of the study, as originally proposed, were to: (i) Test stimulatory potencies of rationally designed backbone cyclic (BBC) peptides on pheromonotropic, melanotropic, myotropic and pupariation activities; (ii) Test the inhibitory potencies of the BBC compounds on the above activities evoked either by synthetic peptides (PBAN, LPK, myotropin and pheromonotropin) or by the natural endogenous mechanism; (iii) Determine the bioavailability of the most potent BBC compounds that will be found in (ii); (iv) Design, synthesize and examine novel PK/PBAN analogs with enhanced bioavailability and receptor binding; (v) Design and synthesize ‘magic bullet’ analogs and examine their ability to selectively kill cells expressing the PK/PBAN receptor. To achieve these goals the agonistic and antagonistic activities/properties of rationally designed linear and BBC neuropeptide (NP) were thoroughly studied and the information obtained was further used for the design and synthesis of improved compounds toward the design of an insecticide prototype. The study revealed important information on the structure activity relationship (SAR) of agonistic/antagonistic peptides, including definitive identification of the orientation of the Pro residue as trans for agonist activity in 4 PK/PBANbioassays (pheromonotropic, pupariation, melanotropic, & hindgut contractile) and a PK-related CAP₂b bioassay (diuretic); indications that led to the identification of a novel scaffold to develop biostbiostable, bioavailable peptidomimetic PK/PBANagonists/antagonists. The work led to the development of an arsenal of PK/PBAN antagonists with a variety of selectivity profiles; whether between different PKbioassays, or within the same bioassay between different natural elicitors. Examples include selective and non-selective BBC and novel amphiphilic PK pheromonotropic and melanotropic antagonists some of which are capable of penetrating the moth cuticle in efficacious quantities. One of the latter analog group demonstrated unprecedented versatility in its ability to antagonize a broad spectrum of pheromonotropic elicitors. A novel, transPro mimetic motif was proposed & used to develop a strong, selective PK agonist of the melanotropic bioassay in moths. The first antagonist (pure) of PK-related CAP₂b diuresis in flies was developed using a cisPro mimetic motif; an indication that while a transPro orientation is associated with receptor agonism, a cisPro orientation is linked with an antagonist interaction. A novel, biostablePK analog, incorporating β-amino acids at key peptidase-susceptible sites, exhibited in vivo pheromonotropic activity that by far exceeded that of PBAN when applied topically. Direct analysis of neural tissue by state-of-the-art MALDI-TOF/TOF mass spectrometry was used to identify specific PK/PK-related peptides native to eight arthropod pest species [house (M. domestica), stable (S. calcitrans), horn (H. irritans) & flesh (N. bullata) flies; Southern cattle fever tick (B. microplus), European tick (I. ricinus), yellow fever mosquito (A. aegypti), & Southern Green Stink Bug (N. viridula)]; including the unprecedented identification of mass-identical Leu/Ile residues and the first identification of NPs from a tick or the CNS of Hemiptera. Evidence was obtained for the selection of Neb-PK-2 as the primary pupariation factor of the flesh fly (N. bullata) among native PK/PK-related candidates. The peptidomic techniques were also used to map the location of PK/PK-related NP in the nervous system of the model fly D. melanogaster. Knowledge of specific PK sequences can aid in the future design of species specific (or non-specific) NP agonists/antagonists. In addition, the study led to the first cloning of a PK/PBAN receptor from insect larvae (S. littoralis), providing the basis for SAR analysis for the future design of 2ⁿᵈgeneration selective and/or nonselective agonists/antagonists. Development of a microplate ligand binding assay using the PK/PBAN pheromone gland receptor was also carried out. The assay will enable screening, including high throughput, of various libraries (chemical, molecular & natural product) for the discovery of receptor specific agonists/antagonists. In summary, the body of work achieves several key milestones and brings us significantly closer to the development of novel, environmentally friendly pest insect management agents based on insect PK/PBANNPs capable of disrupting critical NP-regulated functions.
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Citovsky, Vitaly, and Yedidya Gafni. Viral and Host Cell Determinants of Nuclear Import and Export of the Tomato Yellow Leaf Curl Virus in Tomato Plants. United States Department of Agriculture, August 2002. http://dx.doi.org/10.32747/2002.7585200.bard.
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Tomato yellow leaf curl geminivirus (TYLCV) is a major pathogen of cultivated tomato, causing up to 100% crop loss in many parts of the world. In Israel, where TYLCV epidemics have been recorded since the 1960' s, this viral disease is well known and has been of economic significance ever since. In recent years, TYLCV outbreaks also occurred in the "New World" - Cuba, The Dominican Republic, and in the USA, in Florida, Georgia and Louisiana. Thus, TYLCV substantially hinders tomato growth throughout the world. Surprisingly, however, little is known about the molecular mechanisms of TYLCV interaction with the host tomato cells. The present proposal, a continuation of the project supported by BARD from 1994, expanded our understanding of the molecular mechanisms by which TYLCV enters the host cell nucleus for replication and transcription and exits it for the subsequent cell-to-cell spread. Our project sought two objectives: I. To study the roles of the viral capsid protein (CP) and host cell factors in TYLCV nuclear import. II. To study the roles of CP and host cell factors in TYLCV nuclear export. Our research toward these goals have produced the following major achievements: . Developed a one-hybrid assay for protein nuclear export and import (#3 in the List of Publications). . Identified a functional nuclear export signal (NES) in the capsid protein (CP) of TYLCV (#3 in the List of Publications). . Discovered homotypic interactions between intact TYLCV CP molecules and analyzed these interactions using deletion mutagenesis of TYLCV CP (#5 in the List of Publications). . Showed developmental and tissue-specific expression of the host factor required for nuclear import of TYLCV CP, tomato karyopherin alpha 1, in transgenic tomato plants (#14 in the List of Publications). . By analogy to nuclear import of TYLCV ,identified an Arabidopsis VIPI protein that participates in nuclear import of Agrobacterium T -complexes via the karyopherin alpha pathway (#4,6, and 8 in the List of Publications). These research findings provided significant insights into (i) the molecular pathway of TYLCV entry into the host cell nucleus, and (ii) the mechanism by which TYLCV is exported from the nucleus for the cell-to-cell spread of infection. Furthermore, the obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking viral entry into and/or exit out of the host cell nucleus. Also, as much of our findings is relevant to all geminiviruses, new anti- TYLCV approaches developed based on the results of our research will be useful to combat other members of the Geminivirus family. Finally, in addition to the study of TYLCV nuclear import and export, our research contributed to our understanding of general mechanisms for nucleocytoplasmic shuttling of proteins and nucleic acids in plant cells.