Rechtsschutz im österreichischen und slowakischen Vergaberecht

Die Dissertationsarbeit setzt sich mit dem Vergaberecht auseinander, das die Vergabe öffentlicher Aufträge regelt. Im Zentrum der Dissertationsarbeit steht der Vergaberechtsschutz in der Slowakei und in Österreich. Seine genaue Ausgestaltung bestimmt wesentlich die Rechte beschwerter Bieter und Bewerber, die das Vergabeverfahren eines Auftraggebers anfechten wollen.The doctoral thesis analyses the law of public procurement, which regulates the award of public contracts. It focuses mainly on the regulation of the legal protection of bidders and candidates pursuant to the Austrian and the Slovak public procurement law. The respective regulations determine the review procedure before the competent review authorities, which the aggrieved private bidders and candidates shall follow when challenging the conduct of the tender procedure by the public authority

MAIA, Fc receptor–like 3, supersedes JUNO as IZUMO1 receptor during human fertilization

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor–like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes

αV Integrin Expression and Localization in Male Germ Cells

Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science

Biogenesis and Function of the Mitochondrial Ribosome

In this thesis, I present results of my PhD research into the biogenesis and function of the human mitochondrial ribosome. The mammalian mitochondrial ribosome (mitoribosome) is one of the largest ribonucleoprotein complexes in the cell, with the overall molecular mass of the fully assembled 55S monosome being ~2.7 MDa. It is indispensable for cell survival, as it translates 13 polypeptides encoded in mitochondrial DNA, which are essential for oxidative phosphorylation and therefore for supplying the cell with energy in the form of ATP. The mammalian mitoribosome consists of small 28S and large 39S subunits. It is of dual genetic origin, with all protein components encoded in the nucleus and all RNA components encoded in the mitochondrial DNA. A total of 82 proteins, 2 rRNAs and a structural tRNA comprise one mitoribosomal particle. Proper function and assembly of this molecular machine is reliant on numerous associated factors, such as RNA modifying enzymes and assembly factors. Although recent significant progress has been made in our understanding of how the mitoribosome assembles and its functions in translation, we still lack the knowledge about many factors that are necessary for these processes. This work aims to provide insight into the function of selected proteins that were predicted to be involved in biogenesis and/or function of mitochondrial ribosome, namely mitochondrial rRNA methyltransferase 1 (MRM1) and GTP binding protein 8 (GTPBP8). The work also presents genomics and proteomics methods for the study of mitochondrial gene expression machinery and of mitoribosome integrity and assembly, respectively. The first two chapters provide background information for the research performed in the remaining part of the thesis. In the first chapter, I summarise current knowledge of mitochondrial gene expression, with a focus on post-transcriptional RNA modifications and mitochondrial translation. The second chapter describes the materials and methods used. In the third chapter, I present a computational method for analysis of complexome profiling data from experiments that employ stable isotope labelling by amino acids in cell culture (SILAC). This method is implemented in R and is freely available as the Bioconductor package ComPrAn. It provides tools for analysis of peptide-level data as well as normalisation and clustering tools for protein-level data, dedicated visualisation functions and is accompanied by a graphical user interface. Throughout this thesis ComPrAn has been used for quantitative and qualitative analysis of mitoribosomes in studied cell lines. In the fourth chapter, I introduce a CRISPR/Cas9-based screening approach designed to target genes with known or predicted function in mitochondrial gene maintenance and expression. I apply this method to identify genes that show genetic interaction with MRM1. The screen identifies MRM2 as a top candidate for genetic interaction with MRM1. This finding is followed up by the generation of a double knockout cell line which shows severe mitochondrial deficiency, with uridine auxotrophy and disruption of assembly of small mitoribosomal subunit being the most striking effects observed. These findings provide further insight into the role of MRM1 in mitochondria and highlights the complexity of regulation of mitochondrial translation. The fifth chapter focuses on establishing the role of uncharacterised GTPBP8 protein in the cell. I localised GTPBP8 to mitochondria and studied its function by production of a knockout cell line. GTPBP8 knockout presents a strong oxidative phosphorylation defect due to impaired mitochondrial translation. Quantitative analysis of mitoribosome reveals accumulation of both small and large subunits in the knockout, suggesting that GTPBP8 might play a role in very late assembly of either of the subunits, subunit joining or translation initiation. Overall, this work improves our understanding of the regulation of mitochondrial translation by characterising two mitochondrial proteins and their role in mitoribosome biogenesis and function. The CRIPSR/Cas9 screening methodology and ComPrAn R package presented here have potential to be used in the study of other proteins, extending the portfolio of methods available for research of mitochondrial function

Duplexing complexome profiling with SILAC to study human respiratory chain assembly defects

Complexome Profiling (CP) combines size separation, by electrophoresis or other means, of native multimeric complexes with protein identification by mass spectrometry (MS). Peptide MS analysis of the multiple fractions in which the sample is separated, results in the creation of protein abundance profiles in function of molecular size, providing a visual output of the assembly status of a group of proteins of interest. Stable isotope labeling by amino acids in cell culture (SILAC) is an established quantitative proteomics technique that allows duplexing in the MS analysis as well as the comparison of relative protein abundances between the samples, which are processed and analyzed together. Combining SILAC and CP permitted the direct comparison of migration and abundance of the proteins present in the mitochondrial respiratory chain complexes in two different samples. This analysis, however, introduced a level of complexity in data processing for which bioinformatic tools had to be developed in order to generate the normalized protein abundance profiles. The advantages and challenges of using of this type of analysis for the characterization of two cell lines carrying pathological variants in MT-CO3 and MT-CYB is reviewed. An additional unpublished example of SILAC-CP of a cell line with an in-frame 18-bp deletion in MT-CYB is presented. In these cells, in contrast to other MT-CYB deficient models, a small proportion of complex III2 is formed and it is found associated with fully assembled complex I. This analysis also revealed a profuse accumulation of assembly intermediates containing complex III subunits UQCR10 and CYC1, as well as a profound early-stage complex IV assembly defect

Addressing the Compartmentalization of Specific Integrin Heterodimers in Mouse Sperm

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction