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The SARS-CoV-2 virus (Severe acute respiratory syndrome coronavirus 2), the causative agent of the disease COVID-19 (coronavirus disease 2019), is an enveloped virus with a positive RNA genome. After the binding of the virus to the cell receptor and the release of the genome into the cytoplasm, the viral genome is immediately translated. It is translated into a polyprotein consisting of non-structural proteins necessary for viral replication. A part of this polyprotein is also protease 3-CL, which autocatalytically cleaves itself out from this polyprotein and further releases (and thus activates) individual proteins. Due to this key function in virus replication, 3-CL protease has become an important target for the design of specific antiviral drugs. As part of this thesis, we focused on the study of the precursor form of this protease, i.e. the protease that is still embedded in the polyprotein. The precursor form of the 3-CL protease was mimicked by attaching a 100 amino acid extramembrane C-terminal fragment of the nsp4 protein to the N-terminus of the mature form of the protease. The aim was to find out, how the sequence added to the N-terminus of the matured protease affects its enzymatic characteristics. As a part of this work, mutations of the N-terminal autoprocessing site were identified,...

Intrinsic disorder is one of the many traits that can affect the functionality of multiple naturally occurring proteins in biological systems. This thesis reports on the latest findings on mechanisms that intrinsically disordered proteins or intrinsically disordered regions utilize in specific recognition at the molecular level. Here, the general characteristics of intrinsically disordered proteins are summarized, along with the extent of their abundance throughout different lifeforms and the variety of their molecular recognition mechanisms depicted on specific examples. Furthermore, this thesis focuses on protein transitions between ordered and disordered states induced by interaction with its' binding partner. In the last two chapters, characteristic features of intrinsically disordered proteins are described, and attention is paid to the way these features influence cellular signaling pathways such as interactional promiscuity, the role of signaling hubs, alternative splicing, and post- translational modification.

Amplicon high-throughput sequencing is currently the most widely used technique to investigate complex microbial communities. In order to analyze amplicon sequencing data, specialized software and algorithms are required to turn raw sequencing data into biologically meaningful information. This thesis serves as an overview of the current pos- sibilities and an introduction to microbial community analysis. It provides a comparison of the most widely used bioinformatics pipelines as well as a newer pipeline SEED2 and a foundation for users to make a decision based on their preferences. 1

We developed a software framework that allows the analysis of ligand-free (apo) and ligand-bound (holo) forms of proteins that are accessible in PDB. The software downloads the current version of the PDB, divides the structures into groups of the same molecules, and these into apo and holo forms. Finally, it is possible to analyze pairs of apo and holo structures with respect to their different structural characteristics. In addition to the software work itself, we present the results of selected analyses of the current version of the data in the PDB. We also verify the results against previous work.

The thesis deals with presence of runs of homozygosity, their origin and ways of their detection. Based on the analysis of two samples provided by the Institute of Neurology, University College London, we study differences in detection of runs of homozygosity between four different programs. The next part of the thesis is devoted to creating an original visualization for one of the used programs, namely BCFTools/RoH. This visua- lization is created in HTML and Javascript programming languages, thanks to which it can be opened in a web browser. The visualization enables display of the data in a non- traditional form and at the same time it preserves the biological context of the displayed data. 1

For three decades, the ongoing HIV pandemic has taken the lives of tens of millions of people. Still, more tens of millions are fighting this incurable disease today. Current failures in combating this global problem are caused mainly by the virus's extreme ability of mutation, its very effective molecular shield which repels the immune system's attacks, and its immense variability. A breakthrough, achieved relatively recently, is the discovery of the so-called broadly neutralizing antibodies against HIV-1, which carry a very efficient and broad neutralizing response. So far, it's not known how to elucidate the production of these antibodies in the infected hosts to quell or altogether eliminate the virus. This work deals with experimental results, which led to both in vivo and in vitro proof-of-concept of the so-called protein mimetics, the ability to imitate viral surface epitopes, and therefore stimulate an efficient immune response carried by targeted broadly neutralizing antibodies. This effect is mediated by recombinant binding proteins, based on the Myomedin scaffold. This work describes the selection and characterization of these binding proteins mimicking the epitopes of one of the most effective broadly neutralizing antibodies, 10E8. It shows that the binding affinities of selected...

1 Abstract Even though mitochondria possess their own genome and ribosomes, majority of mitochondrial proteins is encoded in the nucleus and translated by cytosolic ribosomes. Hence it was necessary to establish transport complexes allowing the import of proteins from the cytosol. These complexes are best described in yeast. However, we are encountering organisms lacking many of the subunits of these complexes with increasing frequency. Therefore, we are presenting the overview of the distribution of the subunits within eukaryotic organisms. We specifically take a closer look at parasitic protist Giardia intestinalis that is well known for its extreme reductions of the import complexes. There have been only few subunits identified so far. Porin Tom40, that is responsible for translocating all the incoming proteins across the outer mitochondrial membrane, has been identified despite the high divergence, while homolog of Sam50 hasn't been successfully identified yet. Sam50 is however believed to be necessary for insertion of Tom40 into the membrane. Vast part of this thesis is dedicated to this phenomenon that is highly uncommon and maybe unique among the eukaryotic organisms.

Kinases are among the most studied proteins, as they are important in many cellular processes. Today's research shows that their activity is not only dependent on the composition of the domains, but on the non-domain regions as well. This thesis tried to comprehend the influence of the link- ers' composition on the function of multi-domain protein kinases in general. This was done by clustering human two-domain protein kinases with one protein kinase domain by the averaged physicochemical attributes of their inter-domain regions. The uniqueness of Gene Ontology terms and Enzyme Commission numbers within these clusters of proteins with different archi- tectures was then investigated. However, due to multiple missteps, no such influence has been witnessed. 1

Protein phosphorylation is a common post-translational protein modification used in almost all cellular processes. When a phosphate group is added to an amino acid side chain, it may alter the protein conformation and protein-protein interactions due to its size and its negative charge. It may also change the protein function, activity and even localization within the cell. Experimental detection of phosphorylation is still extremely labor demanding and very expensive, even when deploying protein mass spectrometry. For this very reason many bioinformatics scientific groups focus on the prediction of protein phosphorylation sites. Recent analyses of phosphorylation sites studied mainly non-phosphorylated phosphorylation sites and the distribution and representation of amino acids sequentially neighboring them. Since sequentially more distant, but structurally close amino acids can contribute to the recognition of protein substrate by protein kinase, structural environment of phosphorylation sites was studied in this thesis. Furthermore, 3D structures of phosphorylation sites were comprehensively studied for the first time in a phosphorylated state and the results were compared with the results obtained from the analysis of non- phosphorylated sites. Phosphorylation sites were found mostly within...

Zika and Dengue viruses codes their own enzymes which helps them in different stages of the replication cycle. NS3 a NS5 proteins and their cofactors play an essential role in flaviviral life cycle. Although their structure was already solved, many aspects of their function remain unclear. The main subject of this bachelor thesis is the role of these proteins in flaviviral life cycle, polyprotein cleavage, replication and protein-protein interaction. These enzymes keep many particular enzymatic activities such as protease, helicase, methyltrasferase and polymerase. They are both structurally and functionally separated, which is interesting regarding autoactivation and protein-protein interaction. Since Zika and Dengue infections remain a serious health care issue, it is necessary to understand the molecular mechanisms behind their replication. Keywords: protease, polymerase, Zika, Dengue, polyprotein processing, antiviral therapy

Interleukin 10 family (FIL-10) is an important family of cytokines triggering immune response of different outcome, from antiinflammatory factor interleukin (IL) 10 through epithelia related subfamily of IL-19, IL-20 and IL-24, to IL-22 and IL-26 with role in infection immunity. The family is closely related to interferons (IFNs), several of which (IFN s, and in this study alsoλ IFN ) are commonly placed into FIL-10 for its functional and structural similarities with FIL-10γ proteins. FIL-10 interleukins share several receptor subunits, which in different combinations of receptors and interleukin bound result in different immune response. As the family proteins are expressed in as evolutionary old taxa as cartilaginous fish, we presumed a coevolution in the protein family and the corresponding receptors would be detectable in the sequences of genes and subsequently proteins of FIL-10. Using statistical and structure biological methods, evolutionary relations within the group of FIL-10 and group of their receptors were resolved, with notable division of IFN into independent groups of fish and the later vertebrates. Coevolutionγ of the ligand receptor combination in FIL-10 was detected in most cases, with exception of some interactions of IL10RB, the most widely used receptor subunit in the family,...

Plasma membrane is not a static, but rather a dynamic structure that constantly changes its form and local properties. Interactions between building blocks of plasma membrane and external factors are responsible for those changes. In this thesis, I summarize the literature which describes interactions between transmembrane proteins and lipid membranes as well as its consequences. I discuss membrane thickness, respectively thinning, protein sorting and clustering shown to be dependent on the properties of transmembrane domains. Furthermore, the role of proteins in various model of the plasma membrane organization are indicated. Finally, I report on currently discovered impact of the surface roughness of TMDs on local mobility and organization of lipids. All these data indicate importance of detailed understanding of TMDs, their properties and relation to surrounding lipid membranes. Key words: membrane, protein, transmembrane domain, hydrophobic mismatch, protein sorting, models of the plasma membrane

Human carbonic anhydrases are metalloenzymes that are involved in many physiological processes in the body, but also play an important role in the pathogenesis of numerous diseases. Under regular conditions, expression of carbonic anhydrase IX (CAIX) is very limited, unlike that of other 14 human carbonic anhydrase isozymes. But in hypoxic tumors this enzyme is highly overexpressed on the cell surface. For this reason, this enzyme represents a good target for therapy and diagnosis of tumors and thus various anti-CAIX monoclonal antibodies and specific inhibitors are being developed. In this work we investigated the possibility to use fluorescent polymer conjugate carrying a CAIX specific inhibitor for florescent labeling of tumor cells. Specific binding of polymer conjugate to different cell lines was investigated by flow cytometry and confocal microscopy. Binding properties of the polymer conjugate was compared to CAIX specific monoclonal antibody M75 and its single-chain fragment scFv M75. Ability of the polymer conjugate to inhibit CAIX enzyme activity was also investigated. For these experiments, recombinant protein CAII was prepared and purified, which was also used for protein crystallization. Tests of inhibitory activities allowed to identify novel inhibitors CAIX with better inhibitory...

Phosphorylation is one of the most ubiquitous posttranslational modification types. Understanding of itsfunction and regulation has significant impact on diagnosis and treatment of many diseases. This thesis presents and summarizes the results of several publications that analyze phosphorylation on large datasets using bioinformatics tools. In this thesis the general principles how the phosphorylation influences physico-chemical properties of proteins are described. In the first and the second chapter the evolution principles, function and regulation of protein kinases and phosphatases are provided. In the third chapter thesis concentrates on the distributions of phosphorylated sites among organisms (plants and animals). In the last chapter current knowledge of orthosteric and allosteric effects of phosphorylation as well as its effects on 2D and 3D structure of phosphorylated proteins is summarized.

Long non-coding RNAs (lncRNAs) represent a group of transcripts with length greater than 200 nucleotides having low coding potential. It is a group of regulatory ribonucleic acid (RNA) still not fully understood but with significant potential in many biological processes across different species. For animals, many important lncRNA regulators and their roles in a range of events including their involvement in carcinogenic diseases have been reported. However, particular mechanisms of functions are often yet to be discovered. Considering plants, economically important species such as rice, maize or soybean are of particular interest. There are still only several fully annotated transcripts. However, with the constant improvement of sequencing and bioinformatic methods, the importance of lncRNA (for example in pathogen resistance or plant reproduction) becomes clear. This bachelor thesis reviews up-to-date knowledge about lncRNAs and their roles in plants. It also describes the difficulties of lncRNA research and discusses their future potential.

ABCG2 (ABCP/MXR/BCRP) transporters create homodimers through the plasma membrane. They play an important role in transmembrane transport of a wide spectrum of biological substrates. They are essential for renal, intestinal, placental and haematoencephalic barrier function. In particular they perform an excretory function, protect cells against toxic compounds and xenobiotic cumulation. They are also involved in metabolic regulation of stem cells. This bachelor thesis summarizes information about ABCG2 protein function, their physiological role in humans and other mammals. Keywords: ABCG2, BCRP, membrane transporters, multidrug resistention (MDR), ATP binding cassette family (ABC)

Noncovalent interactions are vital for functioning of biological systems. For instance, they facilitate DNA base pairing or protein folding. Recently, in addition to classical noncovalent interactions such as hydrogen bond, nonclassical noncovalent interactions have been discovered. An example of these interactions is halogen bond belonging to the class of σ-hole interactions, the knowledge of which is already being useful for medical compound design. The aim of this work is to find out if the chalcogen bond, also a σ-hole interaction, plays a role in the binding of existing viral inhibitors, too. Following that, we are also interested whether or to what extent can these existing chalcogen bonds be optimized for a greater affinity of the inhibitor binding. Several protein-ligand crystal structures exhibiting geometrical properties favoring a chalcogen bond have been found in the PDB database. We examined the interaction energies and the interaction energy geometrical dependencies of model systems derived from these crystal structures by means of quantum chemical calculations. Further we have optimized their strength by a series of substitutions. We thus propose that chalcogen bond can become a player in rational design of inhibitors of viral enzymes and their protein target. Keywords: Noncovalent...

Prp45 is a SNW protein that is part of a spliceosome and therefore participates in splicing of pre-mRNA into mRNA. In spliceosome Prp45 joins as part of NTC complex before splicing reactions. Prp45 has several known interactional partners (such as small subunit of U2AF, Cyp2, Prp5, Prp22, Clf1 and Cwc3). These proteins serve many functions in the spliceosome from stability to regulation of splicing reactions. Spliceosome consists of several dozens of proteins and several RNA and undergoes massive structural changes during executing its function. Until recently very little was known about the spliceosome structure; however, recent studies allow us to look at interaction network of this machine. In this work we focused on identification of both previously predicted and new interactional partners of Prp45. Using available spliceosome structures we aimed to describe these proteins structurally and functionally. Key words: splicing, Prp45, 3D struktura, spliceosome, binding partners

Dual specificity phosphatases (DUSP) are enzymes that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on one substrate. Most of them specifically dephosphorylate family mitogen-activated protein kinases (MAPK). Number of DUSPs increases with complexity of organisms and in human genome there are 25 DUSPs described. Some DUSPs can dephosphorylate only one protein while other interact with wider spectrum of substrates. Except for substrate specificity DUSPs differ in expression, subcellular localization etc. Although first DUSPs were described about 20 years ago, a clear factor responsible for their substrate specificity is not known. This works uses in silico methods to discover and describe similarities and differences between DUSPs which may be important in determining DUSP specificity. Key words: phosphatase, kinase, DUSP, MAPK, substrate specificity, conservation of residues, phylogenetic tree, in silico methods

Mercaptopurine (6-mercaptopurine) together with azathioprine and 6-thioguanine belong to a group of widely used chemotherapeutics and immunosuppressants. However, insufficient therapy outcome or severe adverse effects such as myelosuppression are still being reported. Technological progress in DNA and RNA sequencing facilitates effective identification of causative genes responsible for the therapy failure, i.e., description of genetic variants for enzymes involved in metabolism of physiological purines as well as thiopurine drugs. Variants of these enzymes may substantially alter concentrations of cytotoxic forms of thiopurines, which affect therapy success rates. Currently, a number of mutations in genes that play role in thiopurine metabolism have been annotated. Nevertheless, molecular mechanisms underlying the effect of these mutations are not fully elucidated. Knowledge of 3D structure for these enzymes may shed light on the effect of the genetic variants to protein function and mechanisms modulating therapeutic efficacy of thiopurines. This thesis focuses mainly on biochemical and structural characterization of thiopurine-S-methyltransferase, fosfatase NUDT15 and cytosolic 5′-nucleotidase II. It summarizes current state of knowledge and emphasizes the importance of structural biology methods for...

A group of small GTPases consist of over twenty protein families in the super class P-loop. It has a very diverse cell functions. Small GTPases regulate the formation of vesicular follicles, cytoskeleton and nuclear transport. They participate also on cell proliferation and signaling. The aim of my work is to find important amino acids that define family and distinguish each other. I focus on families Arf, Rab, Ran, Ras and Rho. At the Rho family I am also devoted to classes Rho, Rac and Cdc42. Amino acids are identified using bioinformatic programs selected Consurf and Sca5. The objective is also to test P2RANK specialized tool developed at the Charles University in Prague that predict ligand binding sites from protein structure in different families. Founding amino acids can have a big role in the functional divergence of individual families and classes of small GTPases and can be the basis for future study example for the proliferation of cancerous cells. 1.1 Keywords Powered by TCPDF (www.tcpdf.org)