A Comprehensive Analysis of Fibroblast Growth Factor Receptor 2b Signaling on Epithelial Tip Progenitor Cells During Early Mouse Lung Branching Morphogenesis

This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells, via ß-catenin/EP300, controls, through a comprehensive set of developmental genes, morphogenesis, and differentiation. Fibroblast growth factor (FGF) 10 signaling through FGF receptor 2b (FGFR2b) is mandatory during early lung development as the deletion of either the ligand or the receptor leads to lung agenesis. However, this drastic phenotype previously hampered characterization of the primary biological activities, immediate downstream targets and mechanisms of action. Through the use of a dominant negative transgenic mouse model (Rosa26rtTA; tet(o)sFgfr2b), we conditionally inhibited FGF10 signaling in vivo in E12.5 embryonic lungs via doxycycline IP injection to pregnant females, and in vitro by culturing control and experimental lungs with doxycycline. The impact on branching morphogenesis 9 h after doxycycline administration was analyzed by morphometry, fluorescence and electron microscopy. Gene arrays at 6 and 9 h following doxycycline administration were carried out. The relationship between FGF10 and ß-catenin signaling was also analyzed through in vitro experiments using IQ1, a pharmacological inhibitor of ß-catenin/EP300 transcriptional activity. Loss of FGF10 signaling did not impact proliferation or survival, but affected both adherens junctions (up-regulation of E-cadherin), and basement membrane organization (increased laminin). Gene arrays identified multiple direct targets of FGF10, including main transcription factors. Immunofluorescence showed a down-regulation of the distal epithelial marker SOX9 and mis-expression distally of the proximal marker SOX2. Staining for the transcriptionally-active form of ß-catenin showed a reduction in experimental vs. control lungs. In vitro experiments using IQ1 phenocopied the impacts of blocking FGF10. This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells via ß-catenin/EP300 controls, through a comprehensive set of developmental genes, cell adhesion, and differentiation

Use of Wolfram Alpha

This bachelor thesis presents a detailed study of the computational knowledge service Wolfram Alpha. The thesis describes the basic functions of Wolfram Alpha, including its syntax, graphical and mathematical functions, statistics, and data analysis. Moreover, the research outlines practical examples of how Wolfram Alpha can be used in different fields. In addition, a questionnaire survey is conducted in the thesis, which aims to evaluate Wolfram Alpha from the perspective of usability for searching and mathematics. The research provides a useful overview of Wolfram Alpha's functions, its potential uses in various fields and can serve as inspiration for teachers and students utilizing computational technology in education.Tato bakalářská práce se zabývá podrobnou studií výpočetní znalostní služby Wolfram Alpha. V práci jsou popsány základní funkce Wolfram Alpha, včetně jeho syntaxe, grafických a matematických funkcí, statistiky a analýzy dat. Dále jsou popsány konkrétní příklady využití Wolfram Alpha v různých oblastech. V rámci práce je také provedeno dotazníkové šetření, které má za cíl zhodnotit Wolfram Alpha z pohledu využitelnosti pro vyhledávání a matematiku. Práce přináší užitečný přehled o funkci Wolfram Alpha a o možnostech jeho využití v různých oblastech a může sloužit jako inspirace pro učitele a studenty využívající výpočetní technologie v rámci výuky

Generation and Validation of miR-142 Knock Out Mice.

microRNA-142 (miR-142) is an important regulator of many biological processes and associated signaling pathways during embryonic development, homeostasis and disease. The miR-142 hairpin gives rise to the "guide strand" miR-142-3p and the sister "passenger" strand miR-142-5p. miR-142-3p has been shown to play critical, non-redundant functions in the development of the hematopoietic lineage. We have recently reported that miR-142-3p is critical for the control of Wnt signaling in the mesenchyme of the developing lung. miR-142-5p has been proposed to control adaptive growth in cardiomyocytes postnatally and its increase is associated with extensive apoptosis and cardiac dysfunction in a murine heart failure model. Using homologous recombination, we now report the generation and validation of miR-142-null mice. miR-142-null mice show a significant decrease in th expression levels of both the 3p and 5p isoforms. The expression of Bzrap1, a gene immediately flanking miR-142 is not altered while the expression of a long non-coding RNA embedded within the miR-142 gene is decreased. miR-142-null newborn pups appear normal and are normally represented indicating absence of embryonic lethality. At embryonic day 18.5, miR-142-null lungs display increased Wnt signaling associated with the up-regulation of Apc and p300, two previously reported targets of miR-142-3p and -5p, respectively. Adult miR-142-null animals display impaired hematopoietic lineage formation identical to previously reported miR-142 gene trap knockdown mice. We report, for the first time, the homologous recombination-based miR-142-null mice that will be useful for the scientific community working on the diverse biological functions of miR-142

Predicted targets of <i>miR-142-3p</i> and <i>miR-142-5p</i> using Diana-MicroT and Target Scan software prediction tools.

<p><b>(A)</b> Pathway intersection between <i>miR-142-3p</i> and <i>miR-142-5p</i>. <b>(B)</b> Predicted targets of <i>miR-142-3p</i> and <i>miR-142-5p</i> in the Wnt signaling pathway. Orange boxes: common predicted targets for both isoforms; yellow boxes: predicted targets for both isoforms; green boxes: experimentally validated target of <i>miR-142-3p</i>; red box: experimentally validated target of <i>miR-142-5p</i>.</p

<i>miR-142</i> KO mice display hematological abnormalities.

<p><b>(A</b>) Schematic representation of hematopoietic stem cells differentiated into different blood cell types. Peripheral blood cell count in 8 week old <i>miR-142</i>^<i>-/-</i> mice (n = 3) showing a significant decrease in circulating white blood cells (<b>B</b>), lymphocytes <b>(C</b>), platelets (<b>D</b>), eosinophils (<b>E</b>) and monocytes (<b>G</b>) in addition to an increase in mean platelet volume (<b>F</b>).</p

Number of embryos obtained at different stages of embryonic development.

<p># 3 animals died at the age of 2–3 months</p><p>* 1 animal died at the age of 4 months</p><p>Number of embryos obtained at different stages of embryonic development.</p

<i>Increased expression of Apc and p300 is associated with increased Wnt signaling in E18</i>.<i>5 miR-142</i>-null <i>lungs</i>.

<p>(<b>A-L)</b> Immunoflorescence for activated beta-catenin <b>(A-D)</b>, Adenomatous polyposis coli (Apc) <b>(E-H)</b> and p300 <b>(I-L)</b> in wild type (A,B, E,F,I,J) and <i>miR-142</i> ko (C,D,G,H,K,L) lungs showing increased Wnt signaling, Apc and p300 expression. <b>(M-N)</b> qPCR analysis of beta catenin (<i>Ctnnb1</i>), <i>Apc</i> and <i>p300</i> expression in E18.5 wild type and <i>miR-142</i>-null lungs. Scale bar: (A,C,E,G,I,K): 44 μm and (B,D,F,H,J,L): 9 μm.</p

<i>In situ</i> hybridization on E18.5 <i>miR-142</i>^<i>-/-</i> embryonic lung sections.

<p>In situ hybridization for <i>miR-142-3p</i> (<b>A-D</b>) and <i>miR-142-5p</i> (<b>E-H</b>) showing significant reduction of the corresponding expression levels in <i>miR-142</i>^<i>-/-</i> compared to WT E18.5 embryonic lung sections. (<b>I-J</b>). Quantification of the in situ hybridization signals indicating a strong reduction of both isoforms. Scale bar: (A,C,E,G): 20 μm and (B,D,F,H): 4 μm.</p

Human adipose-derived stem cells stimulate neuroregeneration

Traumatic brain injuries and degenerative neurological disorders such as Alzheimer’s dementia, Parkinson’s disease, amyotrophic lateral sclerosis and many others are characterized by loss of brain cells and supporting structures. Restoring microanatomy and function using stem cells is a promising therapeutic approach. Among the many various sources, adipose-derived stem cells (ADSCs) are one of the most easily harvested alternatives, they multiply rapidly, and they demonstrate low immunogenicity with an ability to differentiate into several cell types. The objective of this study was to evaluate the effect of xenotransplanted human ADSCs on post-traumatic regeneration of rat sciatic nerve. Peripheral reconstruction following complete sciatic transection and autonerve grafting was complemented by intra-operative injection of hADSCs into the proximal and distal stumps. The injury caused gliosis and apoptosis of sensory neurons in the lumbar 5 (L5) ganglia in the control rodents; however, animals treated with hADSCs demonstrated a smaller amount of cellular loss. Formation of amputation neuroma, which hinders axonal repair, was less prominent in the experimental group, and immunohistochemical analysis of myelin basic protein showed good myelination 65 days after surgery. At this point, control groups still exhibited high levels of microglia/macrophage-specific marker Iba-1 and proliferating cell nuclear antigen, the mark of an ongoing inflammation and incomplete axonal growth 2 months after the injury. This report demonstrates that hADSCs promote neuronal survival in the spinal ganglion, fuel axonal repair and stimulate the regeneration of peripheral nerves