Advanced Separation Techniques and Data Processing in Capillary Electrophoresis

Metody kapilární elektroforézy jsou esenciálními postupy pro kvalitativní i kvantitativní analýzu široké řady látek. Nacházejí uplatnění v mnoha chemických oborech, počínaje farmakologickým průmyslem přes potravinářství až po vysoce specializovaná biotechnologická výzkumná pracoviště. Za svou více než stoletou historii jsou z teoretického hlediska podrobně prostudovány. Současný výzkum je tedy cílen především na vývoj nových, stále specializovanějších aplikací. K nejvýznamnějším pokrokům v oblasti afinitní kapilární elektroforézy došlo v 90. letech 20. století, přičemž mnoho autorů používalo pro popis obdobných jevů rozdílnou terminologii. První část práce se zaměřuje na ucelení problematiky afinitní kapilární elektroforézy a sjednocení používaného názvosloví. Současně jsou zde diskutovány úkazy, ke kterým v průběhu měření může docházet, a jak mohou ovlivnit výsledek. V rámci studia ACE se zabýváme podmnožinou této metody, konkrétně metodou afinitní kapilární elektroforézy s částečným plněním, která dosud nebyla dostatečně teoreticky prozkoumána. Představujeme zde její matematický popis pro využití ke stanovování komplexačních konstant a uvádíme i limitace dané metody. Ve druhé části této práce se věnujeme vývoji a demonstraci využití počítačových programů pro optimalizaci experimentů a...Techniques of capillary electrophoresis are essential for both the quantitative and qualitative analysis of a large variety of compounds. They find application in many areas of chemistry, from pharmacological industry and food processing, up to highly specialized biotechnological laboratories. Over the last hundred years, their mathematical model has been described with precision. Thus, current research mainly aims for the development of more advanced and more specialized applications. During the greatest boom of affinity capillary electrophoresis within the nineties, many authors would describe similar phenomena under different names. The first part of this work focuses on the consolidation and unification of the problematics and terminology of this method. It also discusses the phenomena which can affect electromigration and separation during electrophoretic processes. We will focus on a specific subset of affinity capillary electrophoresis, partial filling capillary electrophoresis, which, to our knowledge, has not been fully theoretically explored yet, and present a mathematical model allowing the determination of complexation constants and state some limitations of this approach. The second part of this thesis focuses on the development and application of computer softwares, which are meant...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemieFaculty of SciencePřírodovědecká fakult

The characterization of the collimated beams of fast neutrons with the clid detection system

A new detection device for the measurements of light ions (p, d, t, α) emitted as the products of the nuclear reactions induced by fast neutrons (5-33 MeV) was recently developed at the Nuclear Physics Institute of the Czech Academy of Sciences. The main objective of the Chamber-for-Light-Ion-Detection (CLID) is to produce new differential nuclear data of high interest for the material applications related to fusion and aerospace technologies and to potentially test and validate models of nuclear reactions. Hereby the experimental set-up for the measurements with the CLID is described in detail. The experimental characterization of the collimated fast neutron beams produced by the cyclotron-driven converter (p(35 MeV)+Be(2.5 mm)) is presented. In particular, the implementation of the Proton-Recoil-Telescope technique used for neutron energy spectra determination with the CLID is described

Kiralna separacija beta-blokatora tekućinskom kromatografijom visoke djelotvornosti i određivanje enantiomera bisoprolola u površinskim vodama

Beta-blockers are chiral compounds with enantiomers that have different bioactivity, which means that while one is active, the other can be inactive or even harmful. Due to their high consumption and incomplete degradation in waste water, they may reach surface waters and affect aquatic organisms. To address this issue we developed a chromatographic method suitable for determining beta-blocker enantiomers in surface waters. It was tested on five beta-blockers (acebutolol, atenolol, bisoprolol, labetalol and metoprolol) and validated on bisoprolol enantiomers. Good enantioseparation of all analysed beta-blockers was achieved on the Chirobiotic V column with the mobile phase composed of methanol/acetic acid/triethylamine (100/0.20/0.15 v/v/v) at a flow rate of 0.5 mL/min and column temperature of 45 °C. Method proved to be linear in the concentration range from 0.075 μg/mL to 5 μg/mL, and showed good recovery. The limits of bisoprolol enantiomer detection were 0.025 μg/mL and 0.026 μg/mL and of quantification 0.075 μg/mL and 0.075 μg/mL. Despite its limitations, it seems to be a promising method for bisoprolol enantiomer analysis in surface water samples. Further research could focus on waste water analysis, where enantiomer concentrations may be high. Furthermore, transferring the method to a more sensitive one such as liquid chromatography coupled with tandem mass spectrometry and using ammonium acetate as the mobile phase additive instead of acetic acid and triethylamine would perhaps yield much lower limits of detection and quantification.Beta-blokatori su kiralni spojevi s enantiomerima različite bioaktivnosti, dakle, dok je jedan enantiomer aktivan, drugi može biti neaktivan ili čak štetan za organizam. Zbog njihove visoke potrošnje i nepotpune razgradnje u pogonima za preradu otpadnih voda, postoji mogućnost da se pojave u prirodnim vodama i negativno utječu na vodene organizme. Stoga je u ovom radu razvijena kromatografska metoda za određivanje enantiomera beta-blokatora u prirodnim vodama. Metoda je testirana na pet beta-blokatora (acebutolol, atenolol, bisoprolol, labetalol i metoprolol) te validirana za enantiomere bisoprolola. Dobra enantioseparacija svih analiziranih beta-blokatora postignuta je na koloni Chirobiotic V sastava mobilne faze metanol/octena kiselina/trietilamin (100:0,2:0,15 v/v/v) pri protoku od 0,5 mL/min i temperaturi od 45 °C. Metodom je postignuta dobra linearnost u području od 0,075 μg/mL do 5 μg/mL s dobrim analitičkim povratom. Granice detekcije pojedinih enantiomera bisoprolola bile su 0,025 μg/mL i 0,026 μg/mL, a granice kvantifikacije 0,075 μg/ mL za oba enantiomera. Unatoč ograničenjima metode, pokazala se kao obećavajuća metoda analize enantiomera bisoprolola u površinskim vodama. Daljnja istraživanja mogla bi se izvoditi na otpadnim vodama, gdje bi koncentracije enantiomera mogle biti više. Također, korištenjem osjetljivije metode, primjerice vezanoga sustava tekućinske kromatografije i tandemne spektrometrije masa, te korištenjem amonijeva acetata kao aditiva mobilnoj fazi umjesto octene kiseline i trietilamina, mogle bi se postići znatno niže granice detekcije i kvantifikacije

The characterization of the collimated beams of fast neutrons with the clid detection system

A new detection device for the measurements of light ions (p, d, t, α) emitted as the products of the nuclear reactions induced by fast neutrons (5-33 MeV) was recently developed at the Nuclear Physics Institute of the Czech Academy of Sciences. The main objective of the Chamber-for-Light-Ion-Detection (CLID) is to produce new differential nuclear data of high interest for the material applications related to fusion and aerospace technologies and to potentially test and validate models of nuclear reactions. Hereby the experimental set-up for the measurements with the CLID is described in detail. The experimental characterization of the collimated fast neutron beams produced by the cyclotron-driven converter (p(35 MeV)+Be(2.5 mm)) is presented. In particular, the implementation of the Proton-Recoil-Telescope technique used for neutron energy spectra determination with the CLID is described

Inhibitors of Dipeptidyl Peptidase IV and Aminopeptidase N Target Major Pathogenetic Steps in Acne Initiation

Acne is a chronic disease hallmarked by sebaceous hyperplasia, follicular hyperkeratosis, and inflammation. Parallel targeting of these factors is required to treat acne effectively. Inhibitors of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) show strong anti-inflammatory effects on immune cells and therapeutic efficacy in autoimmune disorders. Our investigation focused on the expression and functional relevance of these ectopeptidases in three cell types which exhibit an altered phenotype in early acne lesions. We showed for the first time expression of DP IV and APN on human sebocytes. In the SZ95 sebocyte cell line, the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide and the APN inhibitors actinonin and bestatin suppressed proliferation, enhanced terminal differentiation, and slightly decreased total neutral lipid production. The anti-inflammatory and differentiation-restoring cytokine IL-1 receptor antagonist was significantly upregulated in SZ95 sebocytes and the HaCaT keratinocyte cell line in the presence of inhibitors. Furthermore, the inhibitors suppressed proliferation and IL-2 production of Propionibacterium acnes-stimulated T cells ex vivo and enhanced the expression of the immunosuppressive cytokine transforming growth factor-β1. Our data provide first evidence for a functional role of DP IV and APN in the sebaceous gland apparatus and for their inhibitors, used alone or in combination, as completely new substances possibly affecting acne pathogenesis in a therapeutic manner

Oligogenic genetic variation of neurodegenerative disease genes in 980 postmortem human brains.

BACKGROUND: Several studies suggest that multiple rare genetic variants in genes causing monogenic forms of neurodegenerative disorders interact synergistically to increase disease risk or reduce the age of onset, but these studies have not been validated in large sporadic case series. METHODS: We analysed 980 neuropathologically characterised human brains with Alzheimer's disease (AD), Parkinson's disease-dementia with Lewy bodies (PD-DLB), frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) and age-matched controls. Genetic variants were assessed using the American College of Medical Genetics criteria for pathogenicity. Individuals with two or more variants within a relevant disease gene panel were defined as 'oligogenic'. RESULTS: The majority of oligogenic variant combinations consisted of a highly penetrant allele or known risk factor in combination with another rare but likely benign allele. The presence of oligogenic variants did not influence the age of onset or disease severity. After controlling for the single known major risk allele, the frequency of oligogenic variants was no different between cases and controls. CONCLUSIONS: A priori, individuals with AD, PD-DLB and FTD-ALS are more likely to harbour a known genetic risk factor, and it is the burden of these variants in combination with rare benign alleles that is likely to be responsible for some oligogenic associations. Controlling for this bias is essential in studies investigating a potential role for oligogenic variation in neurodegenerative diseases

Frequency and signature of somatic variants in 1461 human brain exomes.

PURPOSE: To systematically study somatic variants arising during development in the human brain across a spectrum of neurodegenerative disorders. METHODS: In this study we developed a pipeline to identify somatic variants from exome sequencing data in 1461 diseased and control human brains. Eighty-eight percent of the DNA samples were extracted from the cerebellum. Identified somatic variants were validated by targeted amplicon sequencing and/or PyroMark® Q24. RESULTS: We observed somatic coding variants present in >10% of sampled cells in at least 1% of brains. The mutational signature of the detected variants showed a predominance of C>T variants most consistent with arising from DNA mismatch repair, occurred frequently in genes that are highly expressed within the central nervous system, and with a minimum somatic mutation rate of 4.25 × 10-10 per base pair per individual. CONCLUSION: These findings provide proof-of-principle that deleterious somatic variants can affect sizeable brain regions in at least 1% of the population, and thus have the potential to contribute to the pathogenesis of common neurodegenerative diseases.Wellcome Trus

Scaling violations: Connections between elastic and inelastic hadron scattering in a geometrical approach

Starting from a short range expansion of the inelastic overlap function, capable of describing quite well the elastic pp and $\bar{p}p$ scattering data, we obtain extensions to the inelastic channel, through unitarity and an impact parameter approach. Based on geometrical arguments we infer some characteristics of the elementary hadronic process and this allows an excellent description of the inclusive multiplicity distributions in $pp$ and $\bar{p}p$ collisions. With this approach we quantitatively correlate the violations of both geometrical and KNO scaling in an analytical way. The physical picture from both channels is that the geometrical evolution of the hadronic constituents is principally reponsible for the energy dependence of the physical quantities rather than the dynamical (elementary) interaction itself.Comment: 16 pages, aps-revtex, 11 figure