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Vliv délky ekvilibrace na motilitu spermií býků po rozmrazení
KRÁTILOVÁ, Karolína
The aim of the study was to monitor the change in motility of bull sperm post-thawing depending on the time of equilibration before freezing of insemination doses. The percentage of progressive sperm activity post-thawing was determined from the average progressive sperm activity after collection. The evaluation was performed on 6 bulls, approximately the same age, reared under the same conditions at the insemination station. 15 ejaculates were taken from each bull and subsequently processed into insemination doses, which were divided into four groups according to the length of equilibration: 1, 3, 8 and 24 hours. Equilibration was performed in a refrigerator at 5 ° C. The doses were then frozen and evaluated after one week of storage in liquid nitrogen. The mean values of the input parameters were: progressive motility at sampling was 74.63 ? 9.07, the amount of ejaculate 10.08 g and the density 0.91x 106 ml-1 sperm. The results document higher progressive sperm motility with longer equilibration times (8 and 24 hours) as opposed to shorter times (1 and 3 hours) (p <0.01). The best procentual progressive motility was recorded at the equilibration time of 8 hours, when it was 58.74 ? 13.92 % of the initial value (43,84 % of real progresive aktivity). When evaluating the progressive motility of bull sperm after thawing, statistical differences were observed between bulls (p <0.05). Determination of the progressive motility of bull sperm is important in terms of assessing bull fertility. The equilibration time is only one of many factors influencing the quality of the produced insemination doses.
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Dýchání spermií ryb: druhová specifičnost a vliv teploty prostředí
RAHI, Deepali
The presented studies were designed to investigate the role of mitochondrial respiration, glycolysis, and fatty acid oxidation in the energy supply of spermatozoa in freshwater, externally fertilizing fish species possessing differences in sperm motility duration and spawning temperature. The first study conducted on Siberian sturgeon (Acipenser baerii, Acipenseriformes) revealed that mitochondrial respiration is essential for the energy supply of spermatozoa, while glycolysis and fatty acid oxidation have minor importance at both states - before and after attaining motility. Spermatozoon oxygen consumption rate (OCR) was higher than one detected for coldwater teleosts, such as trout, and lower than in marine species, for example, turbot. The presented study also revealed that sturgeons belong to the group of fishes whose spermatozoa possess the capacity to enhance oxidative metabolism after motility activation and uncoupling of oxidative phosphorylation (OXPHOS). In the second study conducted on spermatozoa of coldwater species burbot (Lota lota, Gadiformes), the functioning of the most important bioenergetic pathways (mitochondrial respiration, glycolysis, and fatty acid oxidation) was investigated at spawning temperature (4 °C) and maximum critical temperature (CTmax, 15 °C). At spawning temperature, similar to the results revealed for Siberian sturgeon spermatozoa, the role of glycolysis and fatty acid oxidation in energy supply was insignificant. Mitochondrial respiration was detectable at both quiescent and motile states, but no inhibition was observed after exposure of sperm to the respiratory inhibitor. Also, the OCR range was the lowest compared to the available data for OCR in teleost spermatozoa at motile or immotile state. Additionally, the OCR was not enhanced after motility activation or after treatment with an uncoupling agent. Contrastingly, at CTmax, the activity of OXPHOS became predominant. The OCR was enhanced, and that could be inhibited by using a respiratory inhibitor. Furthermore, to explore the long-lasting motility of sturgeon spermatozoa, succeeding steps were taken by studying the phosphocreatine-creatine (PCr-Cr) shuttle system apart from the previously studied pathways - OXPHOS, glycolysis, and fatty acid oxidation. The results obtained for sterlet (A. ruthenus, Acipenseriformes) revealed that PCr-Cr shuttle, known for maintaining a homogenous concentration of ATP molecules throughout the flagellar length, plays an active role while spermatozoa are at a quiescent state. No significant contribution of this shuttle in energy supply after sterlet sperm motility activation was detected. In addition to this, presented results also revealed that sturgeon spermatozoa, similar to carp and trout sperm, can be reactivated after the termination of the first motility period, and these secondly activated spermatozoa can successfully fertilize eggs. Moreover, for the first time, it was revealed that OXPHOS and PCr-Cr shuttle systems are the most crucial bioenergetic pathways during the sperm reactivation process. Overall, it was concluded that in the studied fish species, despite taxonomical differences or varying spawning temperature , sperm energy production and utilization strategy remained the same: most of the spermatozoon energy was derived from stored ATP that had been synthesized via OXPHOS during a quiescent but bioenergetically active state. Also, sturgeons are the group of fishes that possess an ability of sperm reactivation, with mitochondrial respiration and PCr-Cr shuttle system being the major contributing pathways for energy supply during reactivation process.
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Molecular identification, typing and adaptation of Achromobacter spp. during the course of chronic infection
Učíková, Barbora ; Dřevínek, Pavel (advisor) ; Nemec, Alexandr (referee)
Achromobacter spp. is an emerging pathogen, especially in chronic respiratory infections in patients with cystic fibrosis. MALDI-TOF mass spectrometry provides reliable identification only at the genus level. The nrdA gene sequence is used for species identification of representatives. Clonality studies using multilocus sequence typing can determine whether a patient is still infected with the same clone or whether reinfection with a new strain occurs over time. Time-collected isolates of Achromobacter spp. from patients with cystic fibrosis were included in our study. Patients were divided into three groups according to the time interval between collections. In the first group, the external interval between collections was approximately 10 years, in the second group 7 to 12 months, and the remaining group consisted of single isolates. In the course of chronic infection, Achromobacter spp. adapt to the exposed antibiotics and to the host. Isolates sampled at an interval of 10 years showed a higher number of mutations than isolates with a sampling interval of up to one year. During chronic infection, loss of motility occurs, which we demonstrated phenotypically at the level of motility, reduction in flagella number and changes in flagellar genes. Increased resistance was observed in some isolates by...
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Study of effect of Bacillus subtilis yxkO gene on motility during stress response to osmotic upshift.
Streitová, Eliška ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
Bacillus subtilis is gram-positive soil bacteria. In its natural environment it is constantly exposed to changes of chemical and physical conditons, including changes of osmolality. It responds to high osmolality by transporting of potassium ions and afterthat transporting and/or synthetising of compatible solutes. In last years the mutant strain Bacillus subtilis L-42 was isolated with non-specific insertional mutagenesis (mini Tn10) in our laboratory. This strain displays limited growth and inability to cope with hyperosmotic shock in a defined medium with potassium concentration of < 1 mmol/l. Insertion of transposon was located in yxkO gene which encodes a protein of unknown biological function. Some other data also indicate a possible role of disruption of yxkO gene in regulation of expression of hag gene, which encodes flagelin - a pivotal protein of bacterial flagellum. The goal of this thesis was to clarify if the disruption of yxkO gene influences motility and whether is affected the transcription of hag gene. With integrative vector pMUTIN4 a mutant strain with specific mutation of yxkO gene was prepared. Vector was pasted into chromosome of Bacillus subtilis strain 1A839 - genotype of this strain allows to extrude the known transcriptional regulation of hag gene. Cell's motility was...
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Uloha kataninu, ATPázy štěpící mikrotubuly, při modulaci buněčné motility a proliferace glioblastomových buněk.
Uhlířová, Jana ; Dráber, Pavel (advisor) ; Libusová, Lenka (referee)
Glioblastomas are the most common and the deadliest types of brain tumours. Due to their highly invasive behaviour, they are incurable by convencial therapeutical strategies. It was shown that some components of microtubules, namely class III β-tubulin, γ-tubulin and microtubule severing protein spastin are overexpressed in glioblastoma cell lines as well as glioblastomas. This diploma thesis is focused on the expression, subcellular distribution and function of katanin, another microtubule-severing enzyme, in gliobastoma cell lines. Katanin is formed by catalytic (p60) and regulatory (p80) subunits. Expression and cellular localization of both katanin subunits was studied in panel of human glioblastoma cell lines isolated form adults (T98G, U87MG, U118MG and U138 MG) and child (KNS42). Data presented in this thesis demonstrated that katanin subunits were overexpresed both on transcript and protein levels in T98G, U87MG and KNS42 cell lines, but not in U138MG and U118MG cell lines when compared to normal non- transformed human astrocytes. Immunofluorescence microscopy revealed that both katanin subunits were diffusively distributed in cytoplasm and concentrated on spindle poles of mitotic cells and on leading edges of migrating cells. Examination of cell motility revealed that velocities in...
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The role of cell polarity signaling in the plasticity of cancer cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Cvrčková, Fatima (referee)
Throughout the last few years cancer research has focused on studying the origin of secondary tumors, i.e. metastases, which are a direct outcome of the ability of cancer cells to disseminate from the primary tumor and invade the adjacent tissue. Generally, cancer cells migrate by two distinct mechanisms- amoeboid or mesenchymal. Whereas the mesenchymal migration mode can be described as "path generating", the amoeboid mode resembles a "path finding" way of migration. Both types of invasion are regulated by divergent signaling pathways that are closely related to cell polarity and cytoskeleton reorganization. Responsible for cell polarization are not only the polarity complexes Par, Scribble and Crumbs, but also phosphoinositides and Rho GTPases Rac, Rho and Cdc42, which, additionally, regulate the dynamics of the cytoskeleton. By a mutual interplay they regulate cell motility. It cannot come as a surprise that their deregulation commonly results in tumorigenesis. A more thorough comprehension of the signaling pathways leading to cancer cell invasiveness is a necessary step towards understanding the complex problem of metastasis. Key words: invasiveness, amoeboid, mesenchymal, cell polarity, motility, Rho GTPases, polarity complexes
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Effect of knock out of yxkO gene on environmental stress adaptation in genus Bacillus
Tkadlec, Jan ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...
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Rozdíly v aktivitě spermií hřebců
NOVOTNÁ, Lucie
One of the requirements for a successful artificial insemination is the quality of stallion´s ejaculate. This thesis deals with influence of sperm velocity fraction on fertility. Semen samples in this thesis were evaluated by using an objective computerized method CASA, system SCA. Sperm motility in monitoring was performed in 75 samples from 12 stallions involved in artificial insemination fresh sperm in ZH Písek. Total and progressive motility, rapid, medium and slow motility were monitored. Stallion´s age was from 3-21 years. The average of fertility was 56,19 %. The average of total motility was 78,07 %. The average of progressive motility was 38,35 %. Furthermore, the spermatozoa were divided into 3 velocity fractions and the average percentage of the individual fractions in the samples was as follows: 27,30 % rapid spermatozoa, 21,27 % medium spermatozoa and 29,50% slow spermatozoa. Then the average percentage of rapid progressive spermatozoa (13,43 %), medium progressive spermatozoa (24,92 %) and non-progressive spermatozoa (39,72 %) were found. There was a positive correlation between fertility and the rate of rapid motile spermatozoa (r=0,34). The difference between individual stallions (p-value < 0,01) in the mean percentage of rapid spermatozoa in the ejaculate was demonstrated. The stallion with the highest rate of rapid motile spermatozoa and the best pregnancy was welsh part-bred stallion. On the other hand, the worst pregnancy results and one of the lowest percentages of rapid motile spermatozoa had noriker stallion. The highest success rate of pregnancy was found after the second insemination.The results of this work have shown that the quality of the ejaculate has a very significant effect on good pregnancy but it is just one of many acting factors.
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Uloha kataninu, ATPázy štěpící mikrotubuly, při modulaci buněčné motility a proliferace glioblastomových buněk.
Uhlířová, Jana ; Dráber, Pavel (advisor) ; Libusová, Lenka (referee)
Glioblastomas are the most common and the deadliest types of brain tumours. Due to their highly invasive behaviour, they are incurable by convencial therapeutical strategies. It was shown that some components of microtubules, namely class III β-tubulin, γ-tubulin and microtubule severing protein spastin are overexpressed in glioblastoma cell lines as well as glioblastomas. This diploma thesis is focused on the expression, subcellular distribution and function of katanin, another microtubule-severing enzyme, in gliobastoma cell lines. Katanin is formed by catalytic (p60) and regulatory (p80) subunits. Expression and cellular localization of both katanin subunits was studied in panel of human glioblastoma cell lines isolated form adults (T98G, U87MG, U118MG and U138 MG) and child (KNS42). Data presented in this thesis demonstrated that katanin subunits were overexpresed both on transcript and protein levels in T98G, U87MG and KNS42 cell lines, but not in U138MG and U118MG cell lines when compared to normal non- transformed human astrocytes. Immunofluorescence microscopy revealed that both katanin subunits were diffusively distributed in cytoplasm and concentrated on spindle poles of mitotic cells and on leading edges of migrating cells. Examination of cell motility revealed that velocities in...
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