Identifiying Casc15 as a novel regulator of progenitors’ proliferation and neuronal migration in the developing neocortex

Ein sehr organisiertes vielschichtiges Gewebe im Gehirn von Säugetieren ist der Neokortex, der höhere kognitive Funktionen ausübt wie Erlernen einer Sprache, Denken und räumliches Vorstellungsvermögen. Während der Evolution hat sich der Neokortex vergrößert, um den komplexen kognitiven Bedarf von höher entwickelten Tieren zu bewältigen. Es wird angenommen, dass diese kortikale Expansion primär in der Balance zwischen Proliferation und Differenzierung von neuralen Stammzellen und deren Vorläuferzellen begründet ist. Diese Prozesse sind sehr stark programmiert in Ort und Zeit, was viele Fragen über die involvierten molekularen Netzwerke aufwirft. In den letzten zwei Jahrzehnten haben sich lange nicht-kodierende RNAs (lncRNAs) als attraktive Ziele in der Entwicklungsbiologie dargestellt. Diese Moleküle haben unsere Wahrnehmung von der funktionalen Einheit einer Zelle revolutioniert, da sie bekannt dafür sind, deren Funktion mittels ihrer RNA Struktur auszuüben. Im Gegensatz zu microRNAs, die ihre Funktion über die Regulation von protein-kodierenden Genen auf post-transkriptionaler Ebene ausführen, agieren lncRNAs unterschiedlicher. Vor allem im Gehirn, dem Organ, was die größte Anzahl von lncRNAs exprimiert und das höchste Verhältnis von Gewebe- und Zell-spezifischen lncRNAs besitzt, wurde nachgewiesen, dass sie in fast jedem Prozess während der Entwicklung und bei Erwachsenen involviert sind. Mit dem Ziel eines besseren Verständnisses der molekularen Mechanismen der Gehirnentwicklung, habe ich angestrebt neue Rollen für lncRNAs in der Neurogenese zu identifizieren. Dafür habe ich Gebrauch von einem wirksamen genetischen Instrument gemacht, das zuvor in unserem Labor erzeugt wurde: ein Transkriptom von einer Mauslinie, die es ermöglicht, proliferierende, differenzierende und ausdifferenzierte Zellen zu sortieren. Die Analyse der differentiellen Expression von lncRNAs in den drei Zelltypen enthüllte interessante Kandidaten, die möglicherweise eine Rolle in der Neurogenese spielen. Die Manipulation dieser Kandidaten wurde in vivo getestet durch in utero Elektroporation. In dieser Studie habe ich Casc15 als Regulator von neuraler Stammzellproliferation und neuronaler Migration identifiziert. Überexpression von Casc15 im entwickelnden Kortex verursachte eine Deregulierung von Genen, die in der Entwicklung des Nervensystems und Zellteil-Morphogenese involviert sind. Insbesondere herunterregulierte Gene nach Casc15 Überexpression sind physiologisch angereichert in Neuronen. Diese schließen Gene ein, die verantwortlich für neuronale Migration und Reifung verantwortlich sind. Es wurde gezeigt, dass Casc15 Tbr2, einen neurogenen Transkriptionsfaktor, auf Protein- aber nicht mRNA-Level verringert. Außerdem wurde mittels einer Serie von bioinformatischen Programmen herausgefunden, dass Casc15 eine differentielle Gen-Isoform Benutzung im entwickelnden Gehirn verursacht, was eine Interaktion von Casc15 mit Spleißfaktoren suggeriert. Die Effekte von Casc15 auf Gen- oder Transkript-Expression kann nicht völlig erklärt werden durch Casc15’s Rolle in Neurogenese. Besonders sein Effekt auf Proteintranslation und –stabilität muss adressiert werden. Alles in allem zeigen meine Daten, wenn auch mechanistisch nicht sehr eindeutig, dass Casc15 ein wichtiger Regulator in der Gehirnentwicklung ist. Weiterführende Experimente sind nötig, um die molekularen Aspekte der Casc15 Funktionen zu erörtern.:Introduction 1 1.1 Development of the mammalian neocortex 2 1.2 Neurogenesis in the neocortex 4 1.2.1 Neural stem cells 4 1.2.2 Transient amplifying cells 7 1.2.3 Neurogenesis 9 1.3 Molecular control of neurogenesis in neocortex 11 1.3.1 Signaling pathways influencing the onset and progression of neurogenesis 12 1.3.2 Transcriptional control of neurogenesis 13 1.3.3 Epigenetics, Post-­‐translational modifications and more 15 1.4 Long non-­‐coding RNAs 16 1.4.1 General characteristics of lncRNAs 17 1.4.2 Versatile mechanisms of lncRNAs 20 1.4.3 Expression patterns of lncRNAs 21 1.4.4 lncRNAs in neurogenesis 23 1.5 Btg2RFP/Tubb3GFP mouse line to study cortical development 25 1.6 Aim of the study 28 2 Materials and Methods 29 2.1 Materials 30 2.1.1 Chemicals, buffers and culture media 30 2.1.2 Antibodies 31 2.1.3 Primers 32 2.1.4 Mouse strains 34 2.1.5 Bacterial Strains 34 2.1.6 Vectors 34 2.1.7 Kits and enzymes 35 2.2 Methods 35 2.2.1 Generation of plasmid 35 2.2.2 In utero electroporation 35 2.2.3 Mouse sample collection and treatment 36 2.2.4 Immunohistochemistry 36 2.2.5 Image acquisition and processing 37 2.2.6 Reverse transcription 37 2.2.7 Library preparation and supplemental bioinformatic analyses 37 2.2.8 Quantitative-­‐Reverse Transcriptase-­‐PCRs 39 2.2.9 Bioinformatic analysis 39 2.2.10 Statistical analysis 40 3 Results 41 3.1 Selection of potential regulators of neurogenesis 42 3.1.1 Differential expression analysis for RNA seq data 42 3.1.2 LincRNAs for in vivo manipulation 44 3.2 In vivo manipulation of K13, K10 and Casc15 48 3.2.1 K13 overexpression does not alter progenitors/neurons distribution in the cortex 49 3.2.2 K10 might affect migration of neurons in the developing cortex 50 3.2.3 Casc15 disrupted the distribution of cells across the four cortical layers 50 3.3 Characterization of the cellular phenotype of Casc15 52 3.3.1 Casc15 delays neuronal migration 52 3.3.2 Casc15 does not alter progenitors migration 54 3.3.3 Casc15 does not induce direct neurogenesis 54 3.3.4 Casc15 causes subtle changes on cell distribution after 24 hours 56 3.3.5 Effect of Casc16 on progenitors fate 58 3.4 Molecular effects of Casc15 61 3.4.1 Casc15 minimally changes gene expression in the developing cortex 61 3.4.2 Casc15 changes gene exon usage 64 4 Discussion 68 4.1 Casc15 is a potential regulator of neurogenesis 69 4.1.1 Casc15 induces proliferation of progenitors in the developing cortex 70 4.1.2 Casc15 delays neuronal migration in the developing cortex 71 4.2 Molecular aspects of Casc15 in neurogenesis 72 4.2.1 Casc15 roles in neurogenesis cannot be explained in light of changes in gene expression 73 4.2.2 Casc15 changes the transcriptome at an isoform level 76 4.3 Concluding remarks 78 5 Appendix 79 6 Bibliography 8

Variation of surface properties of ceramic and composite nanostructures and their measurements by a novel approach using perichromic dyes

TiO2 as a model photocatalyst is gaining great interest due to good activity, stability, low toxicity and cost-effectiveness. The rapid recombination between the photogenerated electrons and the holes on the TiO2 surface can be diminished by using carbon-TiO2 composites, especially Graphene (G) and Graphene Oxide (GO), which improve the photocatalytic activity of TiO2 and stability under UV-visible light illumination. The determination of surface properties including surface acidity, polarity and surface area of photocatalysts allows the control and enhancement in the photocatalytic efficiency. This study aims at investigating the surface properties of TiO2-G and TiO2-GO nanocomposites using independent techniques, correlating these surface properties and photocatalytic activity and studying the effect of particle size and the amount of G and GO on these surface properties. TiO2 nanoparticles of different sizes were prepared using sol-gel methodology and varying the rate of hydrolysis of the TiO2 precursor. The as-prepared TiO2 nanoparticles were used to prepare TiO2-G and TiO2-GO nanocomposites by mixing followed by sonication and stirring. The variables in the prepared nanocomposites were the percentage of the added G or GO together with the different TiO2 particle size. Characterization of the formed nanocomposites and the blank samples was performed by using different techniques. The surface acidity was measured using perichromic dyes and NH3-TPD; both measurements are independent. Surface polarity and polarizability were investigated using perichromic dyes to study their impact on the photocatalytic activity. Textural properties were investigated by measuring the surface area and average pore diameter. The structural characteristics of the samples were evaluated using FTIR, Raman and XRD. DRS-UV was employed to measure the band gap energy change with the addition of G or GO. The photocatalytic degradation of methylene blue (MB) dye, as a model water pollutant, was used to evaluate the photocatalytic activity of the samples. The characterization results indicated that two methods of preparation gave two average sizes of 436 ±59 nm and 251 ±32 nm for the samples denoted TL and TS which corresponds to large particle size TiO2 and small particle size TiO2, respectively. Addition of G and GO affected the structure, surface properties and photocatalytic activity of the sample. The results for the photocatalytic activity of the prepared samples, exhibited superior activity over the unmodified TiO2 and the rate enhancement ranged from 9.2 to 69.2% for TS, and 18.8 to 237.5% for TL, showing the clear advantage of using nanocomposites as photocatalysts for pollutants. Photocatalytic activity of the samples is dependent on many factors such as surface acidity, surface area and, to a much lesser extent, the change in band gap energy and the overall effect is a complex combination of all these factors. The formation of Ti-O-C chemical bonds in the samples is evidenced by the Raman results and the addition of G or GO has no effect on the crystal structure of the sample as shown in XRD results. The increase in overall surface acidity in most samples is believed to be primarily due to Brønsted acidity as Lewis acidity is generally lower than the blank in most samples. However, possible stacking of G and GO at higher concentrations resulted in the decrease in overall acidity with the increase in G or GO contents in some of the samples. Surface polarizability, which measures primarily the van der Waals surface interactions, increased with the increase in G content as would be expected due to the hydrophobic nature of G. Further investigations are needed, however, to extend the use of perichromic probes for determination of surface polarizability of solid surfaces. The overall surface acidity of the TL containing samples is generally higher than the TS samples due to higher degree of cross-linking. In conclusion, TiO2-G and TiO2-GO nanocomposites prepared by mixing and sonication were studied for the effects of changing TiO2 particle size, and of the presence and amount of G and GO. It was found that smaller particle size and the presence of G or GO improved the photocatalytic activity of the samples through the change in sample surface properties. Increasing the amount of G or GO in the samples led to limitations in the enhancement of catalytic activity. This is believed to be primarily due to possible blockage of the photocatalytic active sites on the surface of TiO2

GENERALIZED THERMOELASTIC FUNCTIONALLY GRADED HALF SPACE UNDER SURFACE ABSORPTION OF LASER RADIATION

The subject of this paper is to study the thermoelastic behavior of a functionally graded semi-infinite medium heated uniformly by a laser beam having temporally Gaussian distribution. The surface of the medium is taken as traction free. The general solution is obtained in the Laplace transform domain. The inverse of the Laplace transform is computed numerically using the Riemann-sum approximation method. The numerical results for temperature, displacement and stress are obtained and presented graphically for the generalized theory of thermo-elasticity with one relaxation time

Acute toxicity of four heavy metals to Sphaeroma walkeri and Ciroiana bovina (Crustacea:Isopoda)

The median lethal concentrations (LC50) of two Isopoda species exposed to each tested metal (Cu. Co, Cd and Zn) in static tests for different exposure periods are quite variable depending on the tested metal The LC50 values for Sphaeroma walkeri after 24 hours exposure to Cu and Co were estimated graphically to be 11.20 and 7.00 mg/1 respectively. The correspoding values for Cirolana bovina exposed to Cu, Co, Cd and Zn were 3.60, 11.0, 3.80 and 4.80 mg/1 respectively. For 2 days the LC50 of S. walkeri exposed to Cd was 5.60 mg/l, but it was 10.10 mg/l for 3 days exposure to Zn. After prolonged exposure the LC50 values decreased proportionally with the exposure duration of the test the percentages of surviving animals demonstrated a progressive decrease with increasing concentratins as a main factor from the analysis of variance (ANOV A). The sensitivity of adult S. walkeri exposed to the four heavy metals for different exposure times ranked: Cd>Co>Zn>Cu. Cirolana bovina appeared to be more sensitive to Cu. Cd and Zn than to Co. Species in order of increasing sensitivity is C. bovina more than S. walker

Comparative Study of The Effects of Wind and Earthquake Loads on High-rise Buildings

It is very essential to consider the effects of lateral loads induced from wind and earthquakes in the design of reinforced concrete structures, especially for high-rise buildings. The Egyptian Code of Practice for Calculating Loads and Forces in Structural and Building Works, 1993 and 2003 gives simplified methods for calculating such loads. In some cases effects of earthquakes are found to be dominant and more critical than wind effects. This depends on some factors defined by codes. In this research the both effects will be studied and compared according to the Egyptian Code 1993 and 2003. The codes are reviewed for wind and earthquake analysis and discussed to show all factors affecting the design. A computer program is developed to analyze the structural buildings behavior under wind pressure defined as well as equivalent static loads for earthquakes considering all factors in the codes. Application examples for buildings with different heights, floor weights and boundary conditions for both winds and earthquakes such as the intensity of the wind pressure, the seismic zone coefficient, the importance factor, structural system factor and the soil coefficient are analyzed and discussed for the purpose of comparison. Some recommendations are suggested to improve the resistance of the structural and environmental systems of the buildings with respect to lateral loads. Finally Egyptian seismic map, tables of different shape effect and structural systems are provided to help structural designers and researches during design process

Ethoxyquin Inhibits the Progression of Murine Ehrlich Ascites Carcinoma through the Inhibition of Autophagy and LDH

Cancer cells exhibit an increased glycolysis rate for ATP generation (the Warburg effect) to sustain an increased proliferation rate. In tumor cells, the oxidation of pyruvate in the Krebs cycle is substituted by lactate production, catalyzed by LDH. In this study, we use ethoxyquin (EQ) as a novel inhibitor to target LDH in murine Ehrlich ascites carcinoma (EAC) and as a combination therapy to improve the therapeutic efficacy of the conventional chemotherapy drug, cisplatin (CIS). We investigated the anti-tumor effect of EQ on EAC-bearing mice and checked whether EQ can sustain the anti-tumor potential of CIS and whether it influences LDH activity. Treatment with EQ had evident anti-tumor effects on EAC as revealed by the remarkable decrease in the expression of the anti-apoptotic gene Bcl-2 and by a significant increase in the expression of apoptotic genes (BAX and caspase-3). EQ also caused a significant decrease in the autophagic activity of EAC cells, as shown by a reduction in the fluorescence intensity of the autophagosome marker. Additionally, EQ restored the altered hematological and biochemical parameters and improved the disrupted hepatic tissues of EAC-bearing mice. Co-administration of EQ and CIS showed the highest anti-tumor effect against EAC. Collectively, our findings propose EQ as a novel inhibitor of LDH in cancer cells and as a combinatory drug to increase the efficacy of cisplatin. Further studies are required to validate this therapeutic strategy in different cancer models and preclinical trials

A New Design Methodology For Enhancing The Transient Loading Of Low Drop-out Regulators (LDRs)

A new simple design methodology which makes LDR output nearly insensitive to jumps of the load current for long times is proposed. This methodology is tested for more than 104 seconds. Our procedure leans on cross coupling of the time second derivative of the LDR power transistor gate and drain voltages along with their currents. This technique keeps low values of these currents in order of nano or hundreds of micro amperes for undershot or overshot cases, respectively. The introduced methodology has been applied to a standard CMOS of 0.18μm technology for NMOS transistors and validated using MATLAB R2014a

Developing a Jordanian Image of Frost Multidimensional Perfectionism Scale (FMPS) among a Sample of University Gifted and Ordinary Students: A Field Study on the Students of the Faculty of Educational Sciences and Arts (UNRWA)

The aim of this study is to define the psychometric characteristics of Frost Multidimensional Perfectionism Scale FMPS (1990) for Frost and his colleagues to develop a Jordanian image of the scale. The study sample composed of (260) university students, 64 males and 196 females, they were chosen randomly. To achieve the objectives of the study, the original scale was translated into Arabic and applied to an exploratory sample to verify the validity and reliability of the primary image. The results indicated that both the content validity and discriminate were verified. The results of the factor analysis confirmed the validity of its factors. The combined factors (81,069) explained the variation on the scale. The results indicated that the Split-half reliability coefficient of the scale (Spearman - Brown equation) ranged between (0.840-0.776) and using Kronbach Alpha ranged between (0.8344 - 0.7599) which indicates a high degree of reliability. The results also showed statistically significant differences at the level of significance (α≤0.05) in the level of perfectionism among the students in favor of the gifted students. The results did not show statistically significant differences at the level of significance (α≤0.05) in the level of perfectionism due to the difference of gender variable. Keywords: Multidimensional Perfectionism, Neurotic Perfectionism, Psychometric properties, Gifted and Talented student