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Fundamentals of proteomics
Harant, K. ; Křenková, Alena
The first part of the course will be an introduction to proteomics with the aim to show current methods and tools used for proteomic data acquisition and evaluation. In the first part, we will discuss in abbreviated form all steps of the experimental procedure from 1) designing the experiment to be suitable for subsequent LC/MS analysis, 2) sample preparation using examples of immunoprecipitation and complex lysate, to 3) measurement options using Data Dependent or Data Independent acquisition. The course will also include a critical evaluation of the quality of the measured data. The evaluation will focus on the possibilities of data processing in freeware software such as MaxQuant and DIAnn. We will also briefly introduce you to commercial programs - Proteome Discoverer, PEAKS, Spectronaut. The second part of the course will include an introduction to statistical processing of quantitative proteomic data in Perseus software. Emphasis will be placed on understanding important statistical concepts (data distribution, t-test, ANOVA, p-value, FDR, etc.) and understanding all necessary steps of statistical analysis such as data filtering, data normalization, statistical testing, etc. There will be model data that participants will process during the course.
A method for identification of foreign amylases in honey
Erban, Tomáš ; Shcherbachenko, Elena ; Talacko, Pavel ; Harant, Karel
Honey is a unique natural product. Honey has been used as a sweet and delicious foodstuff since ancient times. However, it is also valued for its multifaceted currative properties. Unfortunately, honey is one of the most adulterated foods. Nothing may be added to or modified from the honey. Honey also needs to be handled with care. Honey quality can negatively be affected by the way of processing such as heating and storage. Despite great progress in analytical methods, it is not possible to prove all adulterated honeys. Some methods of adulteration are quite sophisticated. Therefore, it is necessary find new approaches and methods for identification of honey adulteration. To be sold, honey must comply with internationally valid standards, which are also valid at national levels with possible minor modifications. One of the important parameters for honey is the level of diastase or amylase enzyme activity, which is a recognized indicator of the freshness and quality of honey. Lower diastase activity below the established level may indicate old honey, but it may also be the result of careless handling of honey. Last but not least, diastase activity may be reduced due to adulteration of honey such as its dilution with sugar substitutes. It is possible that amylase activity can be artificially adjusted by the addition of enzymes. Honey adulterated by the artificially added amylase meets the legislative requirements for placing honey on the market, but this violates the rules laid down by law. The methods used so far have not made it possible to prove this way of honey adulteration. Therefore, this methodology focuses on the identification of foreign amylases that may occur in honey. The methodology enables to identify practically any foreign amylase in honey by bottom-up shotgun proteomic approach. Based on the obtained results with specific peptides can be further used for the development of a targeted method for the identification of foreign amylases.
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Regulation of cell cycle in Bacillus subtilis.
Zelenka, Tomáš ; Lichá, Irena (advisor) ; Harant, Karel (referee)
2 Abstract Relations between several events running in bacterial cell during cell cycle were the subject of many studies during last years. More advanced techniques showed, that bacterial cell life has much more variable factors, than we supposed before. Relatively recent researches managed to reveal function and in few events molecular principle of several mechanisms coordinating those events such as progression of replication and its initiation, segregation of newly replicated chromosomes and after all synchronization of complex cell division machinery. Furthermore it showed variability of those events during changing living conditions of the cell. Keywords: Cell cycle, regulation, initiation, replication, segregation of chromosome, cytokinesis, Bacillus subtilis
Implication of eukaryotic DNA repair machinery in viral replication
Hron, Tomáš ; Španielová, Hana (advisor) ; Harant, Karel (referee)
Eukaryotic DNA damage response is an important mechanism which ensures genome stability. Its components are also mobilized during viral infection as a reaction against extraneous nucleic acid. Additionally, DNA repair machinery seems to be activated by some viruses purposely to provide their replication. This activation is mediated mainly by viral proteins which are able to interact with cellular factors. In many cases, key components of DNA damage mechanisms are associated with viral replication centre and likely participate in this process. Furthermore, cellular DNA damage signaling is exploited to provide competent environment for viral reproduction. However, particular mechanisms how these cellular factors participate in viral infection are still largely unclear. In this thesis, the principles of relationship between viral infection and eukaryotic DNA damage response are summarized and main viral families which are known to activate and utilize these mechanisms for its genom replication are described.
Cytomegalovirus infection in transplant patients
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Harant, Karel (referee)
Cytomegalovirus (HCMV) is a ubiquitous human -herpesvirus highly prevalent in the population. HCMV is transmitted by close contact between individuals. In infected person this virus causes mainly asymptomatic primary infection, after which the latency is established. In pregnant women HCMV infection can lead to abortions, defects of the fetus and congenital abnormalities of newborn babies. Even more serious complications are caused by this virus in the immunocompromised patients, especially those infected by HIV and in patients who undergo solid organ transplantation and hematopoietic stem cell transplantation. This work is a complex report about HCMV biology with emphasis on complications which HCMV causes in patients after solid organ transplantation and hematopoietic stem cell transplantation. This article also contains summary of the methods used for diagnostic of HCMV infection and monitoring and prevention of HCMV associated diseases. Keywords: Cytomegalovirus, hematopoietic stem cell transplantation, solid organ transplantation, detection, monitoring, polymerase chain reaction, cellular immunity, humoral immunity
Application of mass spectrometry methods for identification and localization of important molecules from parasitic helminths
Siegel, Tomáš ; Kašný, Martin (advisor) ; Harant, Karel (referee)
The thesis summarises the fundamental technological solutions, processes and principles of mass spectrometry with special focus on their use in parasitology. In the first part, the particular phases of processing of the biological sample by using mass spectrometry are defined - from ionisation to the evaluation of results. In the text, the various methodological approaches are presented mainly on the published examples concerning the identification of molecules from parasitic helminths, especially proteins of fasciolid flukes (e.g. Fasciola hepatica). The second part of the thesis comprises the methods of identification and localization of molecules by using mass spectrometry imaging and the use of this combined technique in experimental biology, parasitology, is discussed. There is also proposed the procedure including the combination of laser microdissection and mass spectrometry techniques which could be applied for identification and localisation of important proteins of the fluke Fascioloides magna.
Stable isotopes and the study of insects ecology
Javůrková, Jaroslava ; Šípek, Petr (advisor) ; Harant, Karel (referee)
Animal foraging ecology is an important part in the puzzle ecosystem relationships. This bachelor thesis presents review in the field of food ecology, especially insects. It focuses on the method of analysis of stable isotopes, which is one of the current and in recent decades a modern approach for studying this issue. Stable isotope analysis is compared with other frequently used methods for animal food ecology. The aim of this work is to introduce this method and its possible use in the study of insect food ecology. The first part provides the necessary theoretical background, practical application in laboratory and then it deals with the problems typically adressed by this method. Key words: stable isotopes, stable isotopes analysis, food ecology, food preferences, insects

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