This thesis describes studies and research conducted as part of the development, standardization, and validation of a new laboratory protocol for measuring the bioaccumulation of sediment-associated contaminants in freshwater organisms. The test species used in this method are the oligochaete Lumbriculus variegatus, the mayfly nymph Hexagenia spp., and the juvenile fathead minnow Pimephales promelas. Bioaccumulation methods in the literature were critically reviewed to properly guide the development and standardization of methods. This enabled data gaps to be addressed and the conditions and exposure techniques of the new method to be standardized, properly justified, and based on experimental evidence. Method development included the investigation of the effect of the density of organisms on bioaccumulation in the three test species. The importance of standardizing loading density to total organic carbon (TOC) in sediment was demonstrated, as was the appropriateness of using a ratio of TOC to organism dry weight of 27:1 as a standard loading density for the different test species. To validate the new method and assess the relative effectiveness of the three test species for accumulating different contaminants, a variety of field-contaminated sediments were tested, representing a range of contaminants, levels of contamination, and physical properties of sediment. It was observed that differences in bioaccumulation between the three species may, but do not always, exist, and can vary with contaminant and sediment type. It was also demonstrated that estimates of bioaccumulation, such as biota-sediment accumulation factors (BSAFs), can be species- and site-specific, supporting the need and use of standardized bioaccumulation methods and test species to facilitate comparisons across sites or over time. Comparisons of laboratory- and field-based estimates of bioaccumulation further validated the new laboratory method. Good agreement was observed between laboratory and field estimates for fish, while bioaccumulation was higher in laboratory-exposed invertebrates compared to mussels caged in situ. The laboratory method generally overestimated the relative bioavailability of contaminants compared to the field, but provides a conservative estimate of bioaccumulation. A kinetic study investigated the uptake and elimination of PCBs in the three test species and demonstrated that a 28-d test duration was a sufficient standard for both invertebrate species to reach steady-state concentrations. There was conflicting evidence of whether steady-state concentrations were truly reached in the fish and uncertainty remains as to the appropriateness of a 28-d test for these organisms, for which additional testing is necessary.

This thesis describes the development of an instrument for assessing shared understanding in teams. The purpose was to develop an instrument that would be usable, understandable, objective, flexible and self-explanatory. Teams working in naturalistic settings are expected to have a shared understanding concerning common goals and how to achieve these. The problem investigated in this thesis is that current techniques and instruments for assessing shared understanding in teams generally suffer from one or more of the following drawbacks, namely that they are expensive, difficult to use, time-consuming, requiring expertise, and are often based on subjective perceptions. Departing from existing theory in team cognition techniques and theories, the research questions posed in this thesis are: 1) How can shared understanding be measured without the disadvantages of existing methods? 2) How can shared understanding be assessed without the bias of self-ratings and/or assessments by experts/observers? 3) Can team performance be better understood by the outcomes of an instrument that measures shared understanding? These research questions are answered through six studies that are presented in this thesis. Over the six studies an instrument was iterated and subsequently developed, called the “shared priorities instrument”. When using this instrument, team members are instructed to generate items and rank these in order of importance. By comparing these rank orders from different participants, a team measure of shared understanding can be calculated. The advantages of this instrument compared to earlier measures are that it is less expensive, easier to use, less time-consuming, does not require subject matter expertise, and that the instrument is distanced from subjective perceptions. Furthermore, the final study provides results where outcomes from the shared priorities instrument correlate with performance, supporting earlier research connecting shared understanding in teams with team performance. A structural equation model, a result of the final study, shows that the instrument is both valid and reliable. / Denna avhandling beskriver utvecklingen av ett mätinstrument för att värdera delad förståelse hos team. Syftet har varit att utveckla ett mätinstrument som är användbart, förståeligt, objektivt, flexibelt och självförklarande. Team som arbetar i naturalistiska miljöer förväntas ha en delad förståelse för gemensamma mål och hur dessa ska uppnås. Befintliga tekniker och mätinstrument för värdering av delad förståelse hos team är att de ofta lider av ett eller flera av följande problem: de är dyra, svåra att använda, tidskrävande, kräver expertis, och bygger många gånger på subjektiva bedömningar. Genom att utgå från teoribildningen inom teamkognition ställs följande forskningsfrågor: 1) Hur kan delad förståelse i team mätas utan nackdelarna hos befintliga metoder? 2) Hur kan delad förståelse i team mätas utan att riskera att färgas av partiskheten hos egenbedömningar och/eller experters värderingar? 3) Kan teamprestation förstås bättre med hjälp av ett instrument som mäter delad förståelse? Dessa frågeställningar besvaras i de sex delstudier som presenteras i denna avhandling där ett instrument (som kallas shared priorities) utvecklats för att mäta delad förståelse. Tillämpningen innebär att medlemmarna i ett team individuellt får generera och rangordna faktorer som de anser vara viktiga för att teamet ska nå sitt/sina gemensamma mål och därefter rangordna varandras faktorer. Genom att beräkna överensstämmelsen i dessa rangordningar erhålls ett mått på teamets delade förståelse. Fördelen med detta instrument, i jämförelse med tidigare mått, är att det kostar mindre, är lättare att använda, tar mindre tid, inte kräver någon domänexpertis, och att mätmetoden inte bygger på rent subjektiva bedömningar. I den sista delstudien erhålls resultat där instrumentet shared priorities korrelerar med prestation, vilket stöder tidigare forskning om delad förståelse. En statistisk modell (SEM) visar på instrumentets validitet och reliabilitet.

This thesis explored new methods for understanding the fate and transport of halogenated organic contaminants in the environment. A theoretical method of hazard assessment of chemical mixtures containing large numbers of components was developed and its application illustrated using polychlorinated alkanes, toxaphene, and halogenated dibenzo-para-dioxins and furans. Partitioning properties predicted by high-throughput quantitative structure property relationships were used to locate mixture constituents on plots displaying equilibrium phase distribution in various environmental compartments and the potential for bioaccumulation and long range transport. Potentially hazardous components were identified graphically for more detailed assessments. The applicability of XAD-resin based passive air samplers (XAD-PAS) for studying neutral polyfluoroalkyl substances (nPFAS) in the atmosphere was tested empirically. XAD-PASs have sufficiently high uptake capacity to yield temporally averaged nPFAS concentrations over period as long as a year. When applied as part of the Global Atmospheric Passive Sampling network, nPFAS were found to be truly global contaminants.

This thesis explored new methods for understanding the fate and transport of halogenated organic contaminants in the environment. A theoretical method of hazard assessment of chemical mixtures containing large numbers of components was developed and its application illustrated using polychlorinated alkanes, toxaphene, and halogenated dibenzo-para-dioxins and furans. Partitioning properties predicted by high-throughput quantitative structure property relationships were used to locate mixture constituents on plots displaying equilibrium phase distribution in various environmental compartments and the potential for bioaccumulation and long range transport. Potentially hazardous components were identified graphically for more detailed assessments. The applicability of XAD-resin based passive air samplers (XAD-PAS) for studying neutral polyfluoroalkyl substances (nPFAS) in the atmosphere was tested empirically. XAD-PASs have sufficiently high uptake capacity to yield temporally averaged nPFAS concentrations over period as long as a year. When applied as part of the Global Atmospheric Passive Sampling network, nPFAS were found to be truly global contaminants.

The aim of this diploma thesis was to develop a systematic procedure for the development of HPLC methods using the design of experiments. The system was developed based on the development of three HPLC methods for the determination of the purity of active substances using the design of experiments approach. The HPLC method for determining the purity of esomeprazole was selected to develop the systematic process for the development of HPLC methods by statistical approach. Experimental space was explored to find more suitable separation conditions. The second method used to develop the systematic procedure was the method for determining the purity of bisoprolol. This method was converted to a column of smaller size and the composition of the aqueous part of mobile phase was modified compared with the original pharmacopoeial method. Experimental space was explored to find more suitable separation conditions using the design of experiments. Last the method for determining the purity of risperidone was chosen. The composition of the aqueous part of mobile phase was changed in contrast to the original pharmacopoeial method. Experimental space was explored to find more suitable separation conditions using the design of experiments. For all studied HPLC methods, the values of the monitored chromatographic...

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In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform. In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow. Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma. In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.      Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.     A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Monika Dohnalová Supervisor: Ing. Vladimír Kubíček, CSc. Title of Diploma Thesis: HPLC analysis of drugs The diploma thesis deals with the development and optimization of HPLC method for simultaneous determination of selected polyphenolic compounds: apigenin, acteoside and luteolin. During the experiments, the most suitable conditions for separation were sought, various mobile phases and various types of gradient elution were tested. The Zorbax Eclipse XBD C18 column 250 x 4.6 mm; 5 µm was used for analysis. The detection was performed with diode array detector at wavelengths 249 nm and 350 nm,the column was thermostated at 30 řC. The injected volume was 10 µl. The best results were achieved using mobile phase consisting of acetonitrile and aqueous solution of formic acid (0.03 mol/l) at a flow rate 1ml/min.

Synthetic piperazines are chemically-produced compounds that contain a six-member ring with two opposing nitrogen atoms. Several piperazine derivatives, namely 1- benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), and 1-(3- chlorophenyl)-piperazine (mCPP), have fallen into the “designer drugs” category due to their increasing recreational use as a “legal” alternative to ecstasy (3,4-methylenedioxymethamphetamine). These compounds share similar stimulant and physiological effects with amphetamines which make them desirable to young adults in party-type atmospheres. BZP, a Schedule I drug for its high abuse potential and no accepted medical use, is the only recreationally-abused synthetic piperazine currently federally controlled in the United States. The purpose of this research was to develop and validate a reliable method to identify and quantify eight forensically significant synthetic piperazines in blood and urine using ultra-fast liquid chromatography-electrospray ionization-tandem mass spectrometry (UFLC-ESI-MS/MS). The method was validated according to the Scientific Working Group for Forensic Toxicologists (SWGTOX) guidelines for quantitative analysis for both matrices and includes the following analytes: 1-benzylpiperazine (BZP), 1-(4-fluorobenzyl)-piperazine (FBZP), 4-methyl-1-benzylpiperazine (MBZP), 1-(4-methoxyphenyl)-piperazine (MeOPP), 1-(para-fluorophenyl)-piperazine (pFPP), 1-(3-chlorophenyl)-piperazine (mCPP), 2,3-dichlorophenylpiperazine (DCPP), and 1-(3-trifluoromethylphenyl)-piperazine (TFMPP). All samples were prepared by fortifying 100 µL of certified drug-free whole blood and urine (UTAK Laboratories, Inc., Valencia, CA, U.S.A.) with certified reference standards (Cayman Chemical, Ann Arbor, MI, U.S.A.) of each analyte at desired concentrations and standard additions of 1-benzylpiperazine-d7, 1-(3-chlorophenyl)-piperazine-d8, and 1(-3-trifluoromethylphenyl)-piperazine-d4 internal standards (Cerilliant, Round Rock, TX, U.S.A). After pretreatment with 1 mL phosphate buffer, samples underwent solid phase extraction (SPE) on mixed-mode copolymeric columns (Clean Screen®, UCT Inc., Levittown, PA, U.S.A.). Eluents were evaporated to dryness with low heat (65°C) and nitrogen gas. Samples were reconstituted with a 50:50 mixture of methanol and 2mM ammonium formate buffer with 0.2% formic acid before being analyzed by a UFLC (Shimadzu Corporation, Kyoto, Japan) and 4000 QTRAP ESIMS/MS (SCIEX, Framingham, MA, U.S.A.) system. Analyses were performed with multiple reaction monitoring scans in positive ionization mode using ions and voltages obtained from a manual compound optimization. Analytes were separated on a reversed phase column (Kinetex® F5, Phenomenex®, Torrance, CA, U.S.A.) with a binary gradient consisting of a 2mM ammonium formate buffer with 0.2% formic acid and methanol with 0.1% formic acid. The flow rate was 0.400 mL/min. Analyst™ (SCIEX) software was used for data collection and MultiQuant™ (SCIEX) software was used for quantitation. The total run time was 11.5 minutes with equilibrations. All calibration curves in both matrices exhibited R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 20-2000 ng/mL was used for all analytes in both matrices, except for BZP in urine which ranged from 50-2000 ng/mL. In blood, the limit of quantitation was 10 ng/mL for mCPP and TFMPP and 20 ng/mL for BZP, FBZP, MBZP, MeOPP, pFPP and DCPP. In urine, the limit of quantitation was 10 ng/mL for MeOPP, mCPP, TFMPP and DCPP, 20 ng/mL for FBZP, MBZP and pFPP and 50 ng/mL for BZP. When a 200 ng/mL concentration was evaluated, the SPE procedure showed percent recoveries ranging from 80-95% for blood; except for BZP, FBZP, and MeOPP which had recoveries of 60%, 60%, and 105%, respectively. Percent recoveries ranged from 82-94% for urine; except for BZP and FBZP which had recoveries of 66% and 68%, respectively. Bias and precision were assessed at concentrations of 50, 200, and 700 ng/mL. All samples were calculated within ±20% bias and within ±20% coefficient of variation. The highest concentration evaluated that did not produce carryover in subsequent matrix blanks was 5000 ng/mL. Ionization was suppressed for all analytes in both matrices by 45-95%. Matrix effects were present but were determined to be insignificant. Of the drugs evaluated, caffeine, dibenzylpiperazine, and 1-(4-chlorophenyl)-piperazine (pCPP) produced chromatographic peaks in the method; however, pCPP was the only substance that affected quantitation of an analyte. It increased the peak area of mCPP by almost 50% when present at the same concentration which suggests this method is unable to differentiate between isomeric pairs. This is a sensitive, reliable, and robust method with a wide linear dynamic range to account for the presence of these analytes in both blood and urine. This research will provide for the identification and quantitation of these substances in forensic casework.

<p>After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. </p><p>Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.</p>