Tau protein, biomarker Alzheimerovy choroby: in vitro fosforylace a charakterizace tau reaktivních protilátek

Tau protein je s mikrotubuly asociovaný protein, který se nachází v axonech neuronů. Je klíčovou molekulou podílející se významně na patogenezi Alzheimerovy nemoci (AN), která je nejčastější příčinou stařecké demence. Tau patří do skupiny tzv. přirozeně nesbalených proteinů postrádajících globulární strukturu a vysoce náchylných k post-translačním modifikacím. Za patologických podmínek je tau protein abnormálně modifikován a chybně sbalován, což v důsledku vede k jeho oligomerizaci a agregaci do párově helikálních filament, které jsou základní jednotkou neurofibrilárních klubek, histopatologických útvarů typických pro AN. Současné léky používané v léčbě AN neurodegenerativní procesy pouze zpomalují a lék zcela zastavující vlastní rozvoj nemoci stále chybí. V současnosti je velmi slibným a rozvíjejícím se terapeutickým přístupem imunoterapie. Testována je také imunomodulace pomocí intravenózních imunoglobulinových preparátů (IVIG), které obsahují velké množství přirozených protilátek běžně se vyskytujících u zdravé populace. Objevení přirozeně se vyskytujících protilátek reaktivních s tau proteinem (nTau-Ab) v tělních tekutinách u pacientů s AD, zdravých kontrol, i jejich přítomnost v IVIG preparátech odstartovaly snahy lépe pochopit jejich úlohu u AN a jejich potenciální terapeutické využití....Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,...Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

Modes of micromolar host-guest binding of beta-cyclodextrin complexes revealed by NMR spectroscopy in salt water

Multitopic supramolecular guests with finely tuned affinities toward widely explored cucurbit[n]urils (CBs) and cyclodextrins (CDs) have been recently designed and tested as functional components of advanced supramolecular systems. We employed various spacers between the adamantane cage and a cationic moiety as a tool for tuning the binding strength toward CB7 to prepare a set of model guests with K-CB7 and K beta-CD values of (0.6-5.0) x 10(10) M-1 and (0.6-2.6) x 10(6) M-1, respectively. These accessible adamantylphenyl-based binding motifs open a way toward supramolecular components with an outstanding affinity toward beta-cyclodextrin. H-1 NMR experiments performed in 30% CaCl2 /D2O at 273 K along with molecular dynamics simulations allowed us to identify two arrangements of the guest@beta-CD complexes. The approach, joining experimental and theoretical methods, provided a better understanding of the structure of cyclodextrin complexes and related molecular recognition, which is highly important for the rational design of drug delivery systems, molecular sensors and switches.Internal Funding Agency of the Tomas Bata University in Zlin [IGA/FT/2020/001]; Czech Science FoundationGrant Agency of the Czech Republic [18-05421S]; Ministry of Education, Youths and Sports of the Czech Republic (MEYS CR) [LQ1601]; CIISB, Instruct-CZ Center of Instruct-ERIC EU consortium - MEYS CR infrastructure project [LM2018127]; MEYS CR from the Large Infrastructures for Research, Experimental Development and Innovations [CZLM2018140]Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT: CZ-LM2018140, LM2018127; Grantová Agentura České Republiky, GA ČR: 18-05421S, LQ1601; Univerzita Tomáše Bati ve Zlíně: IGA/FT/2020/001; Masarykova Univerzita, M

Effect of Separation Methods to the Functional Properties of Peripheral Blood Granulocytes

Diplomová práce je členěna na dvě hlavní osnovní části: teoretickou část a experimentální část. V teoretické části jsou shrnuta fakta o nejvíce zastoupené populaci fagocytujících granulocytů - neutrofilech. Je blíže popsán proces fagocytózy a základní mechanismy v obraně proti cizorodým agens. Následuje popis průtokového cytometru a jeho hlavních součástí. Jsou ukázány možnosti využití průtokového cytometru v analýze funkčních vlastností neutrofilů jako významného analytického nástroje v diagnóze imunodeficiencí souvisejících s buněčnou přirozenou imunitou. Cílem předkládaného experimentu byla optimalizace metody třídění granulocytů periferní krve na průtokovém třídiči buněk s ohledem na následně prováděnou analýzu fagocytární aktivity měřenou jako míru oxidativního vzplanutí po stimulaci buněčnou suspenzí inaktivovaných bakterií E.coli. Byla také analyzována míra apoptózy. V prvé řadě byla nutná úprava vzorku nesrážlivé periferní krve před vlastním tříděním granulocytů. Na základě výsledků fagotestu byla úprava prováděna sedimentací periferní krve v inzulinových stříkačkách o objemu 1 ml po dobu 30 minut při laboratorní teplotě. Měnícím parametrem průtokového třídiče buněk byl průměr otvoru nozzlu. Analýzy byly prováděny při třech různých průměrech: 70 ?m, 85 ?m a 100 ?m. Bylo zjištěno, že již vlastní separace granulocytů třídičem buněk měla vliv na jejich schopnost fagocytózy a tvorbu ROS. Byly zjištěny významné rozdíly středních hodnot fluorescence (MFI) granulocytární populace v závislosti na použitém nozzlu.The thesis is divided into two main warp parts: a theoretical part and an experimental part. The theoretical section summarizes the facts about the most abundant population of phagocytic granulocytes - neutrophils. It is described in more detail the process of phagocytosis, and the fundamental mechanisms of defense against foreign agents. The following part is a description of the flow cytometer and its main components. There is also indicated the possibility of using flow cytometry in the analysis of functional properties of neutrophils as an important analytical tool in the diagnosis of immunodeficiency associated with cellular innate immunity. The aim of the experiment was to optimize methods for sorting of granulocytes in peripheral blood by cell sorter with regard to the subsequent analysis of phagocytic activity as measured by degree of oxidative burst after stimulation with cell suspension of inactivated E.coli . It was also analyzed the rate of apoptosis. First, it was necessary to optimalize sample preparation of human peripheral blood. Based on the results of phagotest, the peripheral blood had been settling in the insulin syringes of 1 ml for 30 minutes at room temperature. Nozzle diameter in the flow cytometer was the parameter that was changed. Analyses were performed at three different diameters : 70 micron , 85 micron and 100 micron. It was found that already own cell sorter separation of granulocytes had an impact on their ability to phagocytosis and ROS production . There were significant differences of mean values of fluorescence (MFI) of granulocyte population depending on the nozzle.Katedra biologických a biochemických věd1. Prezentace výsledků diplomové práce 2. Diskuze k posudkům vedoucího a oponenta diplomové práce 3. Diplomantka zodpověděla všechny dotazy a připomínky k D

Card players as a theme in visual arts

The bachelor thesis is theoretical and practical form. Subject of interest is art group Osma and theirs art work from years 1907-1909 with theme of card players. First chapter introduces the reader with the group Osma, focusing on the common theme of card players. This part is also based on the socio-historical context of time, in which the group produced their works. Reader is introduced with problematics of communication of the group and its surroundings. Second chapter presents informations of each individual member of the group. Thesis is then focused on analysis of works in terms of visual side and also technological side. The theoretical part includes extensive visual attachment. The practical art part processes knowledge from the theoretical part and reflects the works of the Osma group with the subject of card players. The results are studies and final drawings on a paper pad. The entire process of creation is captured by photographic documentation

Tau protein, a biomarker of Alzheimer's disease: in vitro phosphorylation and tau-reactive antibodies characterization

Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,..

Tau protein, a biomarker of Alzheimer's disease: in vitro phosphorylation and tau-reactive antibodies characterization

Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,..

Tau protein, a biomarker of Alzheimer's disease: in vitro phosphorylation and tau-reactive antibodies characterization

Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,..

Charakterizace tau rekativních protilátek izolovaných z IVIg produktu, plazmy pacientů s Alzheimerovou chorobou a kognitivně zdravých jedinců

The presence of natural tau-reactive antibodies was reported in human blood. In this study, we isolated and characterized natural tau-reactive antibodies occurring in IVIG product Flebogamma, plasma of patients with Alzheimer's disease (AD) and older cognitively normal persons (controls). Using blotting immunoassays and ELISA, we showed reactivity of antibodies obtained from IVIG and controls against a recombinant fragment of tau (155–421 aa) and aggregates present in brains of AD patients. In contrast, antibodies isolated from plasma of AD patients reacted mainly with the recombinant full-length tau form and tau monomeric forms in brain tissue.Přítomnost přirozeně se vyskytujících tau rekativních protilátek v lidské krvi byla již popsána. V této práci byly tyto přirozené tau reaktivní protilátky vyskytující se v IVIg produktu Flebogamma, plazmě pacientů s Alzheimerovou chorobou (ACH) a kognitivně zdravých jedinců (kontrola) izolovány a následně podrobeny charakterizaci. Za použití blotovacích imunoesejí a ELISA metody byla ukázána reaktivita těchto protilátek izolovaných z IVIg a kontrolní plazmy proti rekombinantnímu fragmentu tau (155-421 aminokyselin) a agregátům vyskytujícím se v ACH mozkové tkáni. Oproti tomu, protilátky izolované z plazmy pacientů s ACH reagovaly především s celou formou rekombinantního tau proteinu (1-441 aminokyselin) a monomerními formami tau proteinu v mozkové tkáni

Quality evaluation of monoclonal antibodies suitable for immunomagnetic purification of native tau protein

Tau protein plays a crucial role in the neuronal cytoskeleton stabilization. Under the pathological conditions, it can be abnormally phosphorylated which leads to the aggregation and formation of neurofibrillary tangles representing pathological hallmark of Alzheimer’s disease (AD). For its association with neurodegenerative diseases, tau protein is intensively studied in various diagnostic and therapeutic applications. Since there is no standard of tau protein involving essential post-translational modifications, it is often necessary to purify it directly from cerebrospinal fluid (CSF) or blood of healthy or AD clinical signs exhibiting organism. The immunomagnetic purification based on the isolation of the target protein using a specific antibody bound to a magnetic carrier is the most effective tool for this purpose. High quality antibodies are the main prerequisite of successful purification, but many commercial antibodies do not comply with the challenging requirements for the immunosorbent preparation. In this work, we compared four different anti-tau monoclonal antibodies currently available on the market (clones HT7, BT2, 8F10, 7E5). The evaluation criteria were set along the intended use for the preparation of specific magnetic immunosorbent subsequently applicable for the native tau protein purification. We evaluated the characteristics declared by producers as specificity, purity and homogeneity. We also tested the binding affinity and IgG stability during the covalent immobilization to the surface of magnetic microparticles and during the immunoprecipitation of intact tau protein or tryptic tau fragments. The results are summarized and discussed here