Analysis of candidate genes for behavioral differences in mice

Organisms have evolved different behavioral strategies for better survival and reproduction. However, the genetic basis for such traits remains still as a longstanding fascinating question in evolutionary biology. Mate choice strategy is one of the behavioral traits which can play an important role in the life history of organisms. A previous study had shown that mate choice preference between two populations of the Western house mouse (M. m. domesticus) is influenced by the genetic background of the fathers. Transcriptome analysis in a follow up study revealed an imprinted cluster on Chromosome 7, known as Prader-Willi Syndrome (PWS) region, and also Peg13 on Chromosome 15 as loci that are highly differentiated between mouse populations and therefore have been suggested as potential regions which may regulate this paternal preference in the house mouse. The present thesis was aimed to investigate the functional role of these two imprinted regions in Western house mice behavior. In the first chapter, I investigated the role of the PWS region on Chromosome 7 through a variety of techniques, including copy number variation analysis, behavioral correlations and transcriptomics. I found that two paternally imprinted tandemly repeated regulatory RNA coding genes (SNORD115 and SNORD116) are of special interest. Their copy number evolves very fast and correlates highly with personality traits between individuals. Further I found that the copy number variation influences the expression of more than 130 genes including genes involved in serotonin regulation, vocalization and bone development. The findings suggest a molecular mechanism for the generation and variability of personality traits in mammals. The second chapter focuses on the analysis of paternally imprinted Peg13 gene, which has so far not been functionally studied. This gene has been known as non-coding gene, but data from ribosomal profiling analysis revealed that this gene could indeed produce a small peptide and it is not a simple non-coding gene. By using knock out mice, I showed even a partial deletion at 3’ part of Peg13 could significantly change expression of hundreds genes in the brain and could thus influence various mouse behaviors. The phenotypic analysis showed a significant effect on mouse sexual and parental behavior. Since RNAseq analysis from mouse brain development timeline showed that this gene has highest expression in day 12.5, I propose that Peg13 may play important role in preoptic area development with possible direct role in sexual and mate choice behavior. 10 Overall the work presented in this thesis describes the first major molecular mechanism underlying mouse personality traits. It presents also the first functional study on the Peg13 gene in mice, which highlights its possible role in mouse brain development and sexual behavior.Contents Summary ................................................................................................................................................. 9 Zusammenfassung ................................................................................................................................. 11 General introduction ............................................................................................................................. 14 Behavior and evolution ..................................................................................................................... 14 Mate choice ....................................................................................................................................... 14 Assortative mating ............................................................................................................................ 15 House mouse and paternal mate choice preference .......................................................................... 16 1-Competitive Ability .................................................................................................................... 17 2-Personality ................................................................................................................................. 18 3-Ultrasonic Vocalization ................................................................................................................. 18 Genomic imprinting .......................................................................................................................... 19 Genomic imprinting and brain development .................................................................................... 19 Imprinted gene and mate choice preference in house mouse ............................................................ 20 Aim of the project ............................................................................................................................. 22 Chapter1 ................................................................................................................................................ 24 Involvement of the Prader-Willi Syndrome (PWS) region genes in mouse behavior .......................... 24 Introduction ....................................................................................................................................... 25 Analysis of SNORD Copy Number Variation .................................................................................. 28 SNORD115 and 116 target genes ..................................................................................................... 30 Link between SNORD copy number and personality ....................................................................... 34 Correlation between SNORD115 and 116 copy numbers ................................................................ 42 Inheritance of SNORD copy numbers .............................................................................................. 45 SNORD copy number differences across tissues .............................................................................. 50 Variation of personality in inbred mice ............................................................................................ 51 SNORD copy number influence on the brain transcriptome ............................................................ 54 SNORD116 copy number and craniofacial features ......................................................................... 60 Differential gene expression through SNORD CNV ........................................................................ 64 Discussion ......................................................................................................................................... 66 SNROD115/ 116 CNV and personality ............................................................................................ 66 Correlation of personality and cognitive ability ............................................................................... 67 Correlation of personality and metabolism ....................................................................................... 68 Correlation of personality traits and vocalization ............................................................................. 68 Personality and craniofacial features ................................................................................................ 69 SNOD115/ 116 and the Prader-Willi syndrome ............................................................................... 69 6 Inheritance of SNROD115 and 116 .................................................................................................. 70 Mice sample ...................................................................................................................................... 71 Mouse keeping .................................................................................................................................. 71 Mouse dissection ............................................................................................................................... 71 DNA extraction ................................................................................................................................. 71 mRNA extraction and cDNA synthesis ............................................................................................ 72 RNAseq analysis ............................................................................................................................... 73 Small RNA extraction, cDNA synthesis, library preparation and sequencing ................................. 73 Read mapping ................................................................................................................................... 74 snoRNA analysis ............................................................................................................................... 75 Droplet digital PCR ........................................................................................................................... 75 1- Finding a suitable reference gene ............................................................................................ 76 2-Primer designing ....................................................................................................................... 76 3-DNA digestion and ddPCR ........................................................................................................ 77 4-Copy Number Calculation ......................................................................................................... 78 Mouse Personality ............................................................................................................................. 81 Behavioral Tests................................................................................................................................ 81 Elevated Plus Maze ........................................................................................................................... 81 Open Field ......................................................................................................................................... 81 Dark/Light Box ................................................................................................................................. 81 Statistical Analysis ............................................................................................................................ 83 Shape phenotyping ............................................................................................................................ 85 Chapter 2 ............................................................................................................................................... 90 Functional analysis of the paternally expressed gene Peg13 ................................................................ 90 Introduction ....................................................................................................................................... 91 Results ............................................................................................................................................... 93 Peg13 and Trappc9 Expression ........................................................................................................ 93 Peg13 expression in the brain ........................................................................................................... 96 Peg13 expression during developmental stages ................................................................................ 97 A possible PEG13 protein ................................................................................................................. 98 Generation of Knock-out mice .......................................................................................................... 99 Peg13 3’-knockout mouse .............................................................................................................. 101 Behavioral tests on Peg13 3’-region knockout mice ...................................................................... 102 RNAseq analysis on Peg13 3’- region knockout mice ................................................................... 108 GO analysis ..................................................................................................................................... 109 7 Discussion ....................................................................................................................................... 109 Expression of Peg13, Kcnk9 and Trappc9 are co-regulated ........................................................... 109 Peg13 is not a simple non-coding RNA .......................................................................................... 110 Behavior of Peg13 3’-region knockout mice .................................................................................. 110 Anxiety behavior of Peg13 3’-KO mice ......................................................................................... 111 Conclusion ...................................................................................................................................... 112 Material and Methods ..................................................................................................................... 113 RNAseq analysis across different tissues ........................................................................................ 113 Ribosomal profiling data analysis ................................................................................................... 113 Generation of knock-out mice by genOway ................................................................................... 113 Novel Object Test ........................................................................................................................... 114 Other procedures ............................................................................................................................. 114 General Discussion ............................................................................................................................. 116 The general background underlying this PhD thesis....................................................................... 116 The PWS region may be associated to the paternal mate choice preference in Western house mouse by regulating mouse personality ..................................................................................................... 117 Evolution of personality .................................................................................................................. 117 1-Selective Neutrality .................................................................................................................. 117 2-Mutation-selection balance ..................................................................................................... 118 3-Balancing Selection ................................................................................................................. 122 Personality and imprinting .............................................................................................................. 123 1-Different recombination rate between male and female .......................................................... 123 2-Co-adapted gene expression .................................................................................................... 123 Peg13 could be involved in sexual behavior in mouse ................................................................... 124 Conclusion .......................................................................................................................................... 126 Reference ............................................................................................................................................ 128 Acknowledgements ............................................................................................................................. 151 Contributions to the thesis .................................................................................................................. 153 Declaration .......................................................................................................................................... 154 Curriculum Vitae ................................................................................................................................ 15

Marriage and Social Identity in The Return of the Native

The Return of the Native presents a world in which “doing means marrying". Thomas Hardy shows how the dominant discourse of the Victorian society defines an individual’s whole life through the conformity to the social code of marriage. This paper clarifies how Hardy’s satirical tone implicitly reflects the voice of the minority, which is not able or eager to follow this conformity code of the majority. Through a detailed analysis of the significance of marriage in defining one’s social identity, family relations, economic ambitions, and individual ideals, the paper focuses on a hermaphrodite character who cannot adapt to the majority’s code because of his physical condition. Such an individual, as the paper presents, is marginalized by the majority and suffers from problems that might lead to psychological disorders. It is Hardy’s implicit satirical tone, which encourages the readers to change their mental set about the role of marriage in defining one’s identity

Caveolin-3 differentially orchestrates cholinergic and serotonergic constriction of murine airways

The mechanisms of controlling airway smooth muscle (ASM) tone are of utmost clinical importance as inappropriate constriction is a hallmark in asthma and chronic obstructive pulmonary disease. Receptors for acetylcholine and serotonin, two relevant mediators in this context, appear to be incorporated in specialized, cholesterol-rich domains of the plasma membrane, termed caveolae due to their invaginated shape. The structural protein caveolin-1 partly accounts for anchoring of these receptors. We here determined the role of the other major caveolar protein, caveolin-3 (cav-3), in orchestrating cholinergic and serotonergic ASM responses, utilizing newly generated cav-3 deficient mice. Cav-3 deficiency fully abrogated serotonin-induced constriction of extrapulmonary airways in organ baths while leaving intrapulmonary airways unaffected, as assessed in precision cut lung slices. The selective expression of cav-3 in tracheal, but not intrapulmonary bronchial epithelial cells, revealed by immunohistochemistry, might explain the differential effects of cav-3 deficiency on serotonergic ASM constriction. The cholinergic response of extrapulmonary airways was not altered, whereas a considerable increase was observed in cav-3â -/- intrapulmonary bronchi. Thus, cav-3 differentially organizes serotonergic and cholinergic signaling in ASM through mechanisms that are specific for airways of certain caliber and anatomical position. This may allow for selective and site-specific intervention in hyperreactive states

Auslander's Formula for contravariantly finite subcategories

A relative version of Auslander's formula with respect to a contravariantly finite subcategory will be given. Dual version will be treated. Several examples and applications will be provided. In particular, we show that under certain circumstances, if relative Auslander algebras of artin algebras $\Lambda$ and $\Lambda'$ are Morita equivalent, then $\Lambda$ and $\Lambda'$ are also Morita equivalent

Modification of bacterial cell membrane to accelerate decolorization of textile wastewater effluent using microbial fuel cells: role of gamma radiation

The aim of the present work was to increase bacterial adhesion on anode via inducing membrane modifications to enhance textile wastewater treatment in Microbial Fuel Cell (MFC). Real textile wastewater was used in mediator-less MFCs for bacterial enrichment. The enriched bacteria were pre-treated by exposure to 1 KGy gamma radiation and were tested in MFC setup. Bacterial cell membrane permeability and cell membrane charges were measured using noninvasive dielectric spectroscopy measurements. The results show that pre-treatment using gamma radiation resulted in biofilm formation and increased cell permeability and exopolysaccharide production; this was reflected in both MFC performance (average voltage 554.67 mV) and decolorization (96.42%) as compared to 392.77 mV and 60.76% decolorization for non-treated cells. At the end of MFC operation, cytotoxicity test was performed for treated wastewater using a dermal cell line, the results obtained show a decrease in toxicity from 24.8 to 0 (v/v%) when cells were exposed to gamma radiation. Fourier-transform infrared (FTIR) spectroscopy showed an increase in exopolysaccharides in bacterial consortium exposed to increasing doses of gamma radiation suggesting that gamma radiation increased exopolysaccharide production, providing transient media for electron transfer and contributing to accelerating MFC performance. Modification of bacterial membrane prior to MFC operation can be considered highly effective as a pre-treatment tool that accelerates MFC performance

Numerical Modeling of Sediment Transport in a Typical Large Reservoir with Respect to Meander Effects on Sediment Flow Path

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchiv

An Efficient Green Synthesis of 3-Amino-1H-chromenes Catalyzed by ZnO Nanoparticles Thin-film

A very simple and environmentally benign approach for the synthesis of 3-amino-1H-chromenes is described using ZnO nanoparticles thin-film as an efficient heterogeneous catalyst in green media, namely water. The mild reaction conditions, reusability of the catalyst, easy work-up and high yields of products make the present protocol sustainable and advantageous compared to conventional methods.KEYWORDS:  3-Amino-1H-chromene, aqueousmedium,hydrothermalsolution method, reusability of the catalyst,ZnOnanoparticles thin-film