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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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Applications of Machine Learning for Detecting and Counting Objects in Cell Biology
Brázdilová, Květa ; Stopka, Pavel (advisor) ; Hoksza, David (referee)
Modern biological research generates large amounts of data, which require automation for efficient analysis. Lately, machine learning solutions are being developed for many of the problems in this field. This thesis focuses on applications of machine learning for image analysis, such as detecting cells in microscopy images and classifying them based on their phenotype. After a brief introduction to machine learning concepts, eight published methods are presented, which employ machine learning for either detecting and classifying, or counting objects in biological images. Five open-source software tools for biological image analysis, which employ some of the methods mentioned above, are introduced. A new project is also described, which aims to create a convolutional neural network for counting bacterial colonies in images of agar plates. The results of this project are discussed. Keywords: machine learning, neural network, pattern recognition, cell biology, segmentation
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Social communication in the house mouse (Mus musculus)
Černá, Zuzana ; Stopka, Pavel (advisor) ; Havlíček, Jan (referee)
Chemical communication is the most important way of communication in the house mouse. Traditionally, the urine of the house mouse was the most studied source of signals. Typical way of scent marking, relatively simple way of collection and analysis was the main reason. The analysis of physical interaction of two mouse conspecifics has shown that there are several other very important sources of chemical signals. My ethological analysis has revealed that during the first interaction of two individuals of opposite sex, the most investigated body part is the orofacial region, and that the oral contact with salivary exchange is very frequent. At the same time, the results of this research have demonstrated the significant influence of reproductive status on behavioural patterns in individuals of both sex. One of these important sources of chemical signals is the saliva. During the interaction between two individuals, saliva is actively investigated by sniffing and physically exchanged. Gas chromatography and mass spectrometry (GC-MS) was used for chemical analysis of saliva. It has revealed that saliva is a sexually dimorphic signal, which may represent individual odour profiles. Uniqueness of this odour is not based on the presence of special substances typical just for one sex, unique is the blend,...
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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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Using Sperm Transcriptome in Search for Novel Biomarkers of Male Infertility
Semyakina, Anastasiya ; Liška, František (advisor) ; Stopka, Pavel (referee)
Currently infertility affects 10 to 15 percent of couples. In nearly 50% of all cases male factor contributes to infertility of the couple. Majority of causes of male infertility remains unexplained. For this reason, finding simple and clinically useful tools for improving male infertility diagnostics can be important at present. Based on the analysis of sperm transcriptome, in this diploma thesis we aimed to find genes that show differential expression between normal and pathological sperm, this could provide information about molecular basis of male infertility, moreover, expression profile of such genes in sperm could be employed for noninvasive diagnosis of male infertility. This study was conducted by using 67 sperm samples, including 16 control sperm samples from fertile men. The samples of infertile patients were divided into 3 groups according to morphology and motility using results of standard sperm evaluation according to WHO. First group included 20 patients diagnosed with astenoteratozoospermia (low sperm motility and abnormal morphology), second group was 15 patients diagnosed with asthenozoospermia (normal morphology and low sperm motility), the third group comprised 16 samples from infertile patients with normal spermiogram. 16 control samples were from fertile men (conception of...
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