Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models.

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.

Au(I) Catalyzed Manipulation of Propargylic Alcohols: A New Route Towards the Synthesis of Indole Alkaloids

In this work we presented several aspects regarding the possibility to use readily available propargylic alcohols as acyclic precursors to develop new stereoselective [Au(I)]-catalyzed cascade reactions for the synthesis of highly complex indole architectures. The use of indole-based propargylic alcohols of type 1 in a stereoselective [Au(I)]-catalyzed hydroindolynation/immiun trapping reactive sequence opened access to a new class of tetracyclic indolines, dihydropyranylindolines A and furoindolines B. An enantioselective protocol was futher explored in order to synthesize this molecules with high yields and ee. The suitability of propargylic alcohols in [Au(I)]-catalyzed cascade reactions was deeply investigated by developing cascade reactions in which was possible not only to synthesize the indole core but also to achieve a second functionalization. Aniline based propargylic alcohols 2 were found to be modular acyclic precursors for the synthesis of [1,2-a] azepinoindoles C. In describing this reactivity we additionally reported experimental evidences for an unprecedented NHCAu(I)-vinyl specie which in a chemoselective fashion, led to the annulation step, synthesizing the N1-C2-connected seven membered ring. The chemical flexibility of propargylic alcohols was further explored by changing the nature of the chemical surrounding with different preinstalled N-alkyl moiety in propargylic alcohols of type 3. Particularly, in the case of a primary alcohol, [Au(I)] catalysis was found to be prominent in the synthesis of a new class of [4,3-a]-oxazinoindoles D while the use of an allylic alcohol led to the first example of [Au(I)] catalyzed synthesis and enantioselective functionalization of this class of molecules (D*). With this work we established propargylic alcohols as excellent acyclic precursor to developed new [Au(I)]-catalyzed cascade reaction and providing new catalytic synthetic tools for the stereoselective synthesis of complex indole/indoline architectures.

The market manipulation regime in the European Union

Market manipulation is an illegal practice that enables a person can profit from practices that artificially raise or lower the prices of an instrument in the financial markets. Its prohibition is based on the 2003 Market Abuse Directive in the EU. The current market manipulation regime was broadly considered as a big success except for enforcement and supervisory inconsistencies in the Member States at the initial. A review of the market manipulation regime began at the end of 2007, which became quickly incorporated into the wider EU crisis-era reform program. A number of weaknesses of current regime have been identified, which include regulatory gaps caused by the development of trading venues and financial products, regulatory gaps concerning cross-border and cross-markets manipulation (particular commodity markets), legal uncertainty as a result of various implementation, and inefficient supervision and enforcement. On 12 June 2014, a new regulatory package of market abuse, Market Abuse Regulation and Directive on criminal sanctions for market abuse, has been adopted. And several changes will be made concerning the EU market manipulation regime. A wider scope of the regime and a new prohibition of attempted market manipulation will ensure the prevention of market manipulation at large. The AMPs will be subject to strict scrutiny of ESMA to reduce divergences in implementation. In order to enhance efficiency of supervision and enforcement, powers of national competent authorities will be strengthened, ESMA is imposed more power to settle disagreement between national regulators, and the administrative and criminal sanctioning regimes are both further harmonized. In addition, the protection of fundamental rights is stressed by the new market manipulation regime, and some measures are provided to guarantee its realization. Further, the success EU market manipulation regime could be of significant reference to China, helping China to refine its immature regime.