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Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
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Generation and analysis of mutant mouse models to study pathophysiological roles of KLK5 and KLK7 in epidermis
Kašpárek, Petr ; Sedláček, Radislav (advisor) ; Stopka, Pavel (referee) ; Machoň, Ondřej (referee)
Kallikrein-related peptidases (KLKs) constitute a family of closely related serine proteases encoded by genes clustered in one chromosomal locus. KLKs are widely expressed in a variety of tissues and numerous in vitro experiments suggest their important roles in many physiological and pathological processes. However, the biological roles of KLKs in vivo are often obscured mainly due to unavailability of suitable animal models. Although gene deficient mouse models were generated for several KLK genes, they had limited use for understanding the roles of individual proteases in the complex environment in vivo. One of the main obstacles which hampers in vivo analysis is partial functional overlap between some KLKs. This makes traditional single-gene deficient animal models an inadequate tool to address the biological impact of the gene deficiency as compensatory mechanisms often result in a lack of phenotype. In this work, we used the transcription activator-like effector nuclease (TALEN) technology to generate several novel mutant mouse models to study the complex KLK proteolytic pathways and their roles in healthy organism and in disease. We prepared a novel mouse model for Netherton syndrome (NS), an autosomal recessive skin disorder caused by mutation in the gene SPINK5, which encodes the KLK-inhibitor...
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Roles of acrosomal proteins in fertilization
Macíčková, Eliška ; Stopka, Pavel (advisor) ; Čermák, Lukáš (referee)
The acrosome is a crucial organelle for sperm cells. Defects in the biogenesis of the acrosome often lead to the impaired development of sperm cells and consequently, to the inability to fertilize the oocyte. Although it is evident that the acrosome is an essential structure, its main function has not been solved yet. The initial hypothesis was that the acrosome helps sperm to pass through the extracellular layers of the egg. This statement is based on evidence that acrosome is full of proteolytic enzymes, and inhibition of these enzymes reduces the ability to fertilize the oocyte. However, this hypothesis began to be questioned when it was found that (i) acrosome-reacted sperm cells are still able to pass through extracellular layers and fertilize the oocyte (ii) most of the sperm cells undergo premature acrosomal reaction before they reach the oocyte. Therefore, we aimed to characterize proteomes of the acrosome and acrosome- reacted sperm cells, identify important proteins using advanced bioinformatics approaches and discuss the function of the acrosome as a vesicle that serves to store and process metabolites after the previous maturation process. In this work, we revealed new findings that contribute to the hypothesis that acrosome is a lysosome-related organelle. Key words: sperm, acrosome,...
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Roles of sperm morphology variation on proteomic content in muroid rodents
Drozdová, Tereza ; Stopka, Pavel (advisor) ; Postlerová, Pavla (referee)
We can find a whole range of morphological differences between the spermatozoa of different animal species, whether at the level of shape, size or intracellular arrangement. It has been proven that these differences are likely to be driven by selective pressure caused by sperm competition. This can cause morphological adaptations that give sperm a selective advantage and increase the individual's success in fertilization. The main goal of my diploma thesis was to determine whether individual morphological variability of spermatozoa is detectable at the proteome level and possibly to determine what changes at the proteome level occur in spermatozoa with changing morphology. Three species of rodents, Mus musculus, Apodemus flavicollis and Microtus arvalis, were selected for this study, and a thorough morphometric measurement of their sperm was performed. Because the greatest variation was detected in the dimensions of the apical hook, only acrosomal proteins were analyzed by mass spectrometry, and then the proteins that distinguished the most sperm with long and short apical hooks were determined by discriminant analysis and machine learning methods. A significant trend among sperm-separating proteins with short and long apical hooks, regardless of species, has been identified for proteins involved...
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Structure and function of selected lipocalins and their role in chamical communication in mammals
Stopková, Romana ; Stopka, Pavel (advisor) ; Sedláček, František (referee) ; Žídek, Lukáš (referee)
Sunrulnv or Pu.D.rHnsrs Chemical communicationis facilitated by various substancesand occurs in all organismal forms from cellular to multicellular organisms. This system of communicationspecializedduring evolutionand incorporatedspecificmeansof transferanddetectionof signalsincludingolfactoryreceptorsanddiversificationof specifictransportersof thesemolecules.Amongtransporters,lipocalinsareproteins thatdueto theirspecificbetabanel formingtertiarysffucutrehavehighafinityto bind various volatile ligands for which they have importantrole in chemical communication. MajorUrinaryProteinsarea well knownexamplein miceaswell asOdorantbinding proteins.However,thereare otherlipocalinsin this proteinfamily of which the function is as yet understudied.In my Ph.D. disertation,I aimed at detailed understandingofthe variabilityin lipocalinfamilyandto suggestotherpotentialroles of theseleastknownproteins.I am presentinga reviewpublicationstemmingout of my bio-informaticanalysis,one paperon Major Urinary Proteinsin two speciesof mice and a paperthatis now submittedand which dealswith novel lipocalin (Obp) genes in Myodes glareolus similar to afrodisin - a protein that was previously describedonly in hamsters.This paperis basedon the sequencesthatwereanalyzed and submittedto GenBank.This last resultopenednew streamof...
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The role of Fbxo38 ubiquitin ligase in mouse spermatogenesis
Zobalová, Eliška ; Čermák, Lukáš (advisor) ; Stopka, Pavel (referee)
Cullin-dependent ubiquitin ligases are responsible for the regulation of most cellular processes. Despite their mutated forms being the cause of many human diseases, their physiological roles are not sufficiently described. In the presented results, we focused on the physiological role of ubiquitin ligase SCFFBXO38 (SKP1-CULLIN1-FBXO38), whose mutated forms are responsible for the progression of distal neuropathy. Preparation of mouse model deficient in FBXO38 revealed that homozygous pups were born in a lower than expected ratio. Animals were growth-retarded, both at the level of the whole organism and individual organs, especially the liver and testes. Males with a deletion in the Fbxo38 gene had significantly lower reproductive capacity, which was associated with lower production of mature sperm and pathological changes in the structure of seminiferous tubules. We found that the FBXO38 protein is functionally expressed in Sertoli cells responsible for regulating spermatogenesis and seminiferous tubules integrity. Detailed analysis of spermatogenic populations revealed a defect at the level of spermatocyte differentiation. The dynamics of this differentiation depend on the hematotesticular barrier functional integrity formed by the intercellular junctions of Sertoli cells. We confirmed that the...
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