A comprehensive investigation of sensitive ecosystems in South Florida with the main goal of determining the identity, spatial distribution, and sources of both organic biocides and trace elements in different environmental compartments is reported. This study presents the development and validation of a fractionation and isolation method of twelve polar acidic herbicides commonly applied in the vicinity of the study areas, including e.g. 2,4-D, MCPA, dichlorprop, mecroprop, picloram in surface water. Solid phase extraction (SPE) was used to isolate the analytes from abiotic matrices containing large amounts of dissolved organic material. Atmospheric-pressure ionization (API) with electrospray ionization in negative mode (ESP-) in a Quadrupole Ion Trap mass spectrometer was used to perform the characterization of the herbicides of interest. The application of Laser Ablation-ICP-MS methodology in the analysis of soils and sediments is reported in this study. The analytical performance of the method was evaluated on certified standards and real soil and sediment samples. Residential soils were analyzed to evaluate feasibility of using the powerful technique as a routine and rapid method to monitor potential contaminated sites. Forty eight sediments were also collected from semi pristine areas in South Florida to conduct screening of baseline levels of bioavailable elements in support of risk evaluation. The LA-ICP-MS data were used to perform a statistical evaluation of the elemental composition as a tool for environmental forensics. A LA-ICP-MS protocol was also developed and optimized for the elemental analysis of a wide range of elements in polymeric filters containing atmospheric dust. A quantitative strategy based on internal and external standards allowed for a rapid determination of airborne trace elements in filters containing both contemporary African dust and local dust emissions. These distributions were used to qualitative and quantitative assess differences of composition and to establish provenance and fluxes to protected regional ecosystems such as coral reefs and national parks.

Since their 'discovery' at the turn of the 21st century, giant viruses of the amoeba have captured the fascination of virologists. They have raised a plethora of questions regarding their evolution and ecological significance and have greatly defied a century's old definition of viruses. By now, it is understood that a handful of giant viruses enter the amoeba via the phagosomal pathway. This thesis chooses to focus on the giant Melbournevirus (MelV) regarding its entry and uncoating pathway. We now conclude that the initial attachment between MelV and amoeba cells is built upon glycan interactions based on evidence that mannose competitively inhibits MelV binding. This attachment likely entails an approximately 70 kDa mannose containing glycoprotein on the MelV. Mannose and other glycans induce secretion of proteins including phagosomal enzymes from amoeba. Based on these findings, it is hypothesised that the mannose-induced phagosomal enzymes could play a role in the uncoating of the MelV. The results further reveal isolated phagosomes, also to some extent the glycan-induced protein secretions, to have high levels of proteins involved in cell redox homeostasis. This implies that the highly oxidative environment of the phagosome may be an overlooked physiological factor when regarding the uncoating of the MelV. A deeper understanding of the physiological uncoating conditions can be used for studying internal structures of giant viruses, such as the enigmatic Large and Dense Body (LDB) of the MelV particle.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22015号 / 工博第4627号 / 新制||工||1721(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 梅田 眞郷, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

This diploma thesis focuses on polar pesticides and their analysis in agricultural commodities. One of the aims was establishing of the method for quantitative determination of diquat and paraquat using liquid chromatography with tandem mass spectrometry. Optimization parameters based on the European reference laboratories recommendations, availability of laboratory equipment and method suitability for routine analyses were taken into account. Extraction of analytes was based on shaking with acidified methanol with formic or hydrochloric acid. Considering the matrix complexity, purification with sorbents and/or acetonitrile precipitation were applied. Method validation was carried out on several levels for selected representative agricultural commodities. Part of the thesis is a field experiment where potatoes plants were sprayed with the diquat active substance. Samples of treated plants and tubers were analyzed for diquat residues. The validated method was also used for screening of diquat and paraquat residues in feed samples collected within official controls of the Central Institute for Supervising and Testing in Agriculture in 2020. The final method enables to analyze diquat and paraquat with sensitivity suitable for the maximal residue limit controls.

Background: Paediatric poisoning is a common presentation to emergency departments worldwide. There is a paucity of data on the role of liquid chromatography-tandem mass spectrometry (LC-MS/MS), in the management of paediatric poisoning in low-and middle-income countries (LMICs). In high-income countries, most studies are retrospective, and few include children. Objective: The study describes the prevalence of liquid chromatography-tandem mass spectrometry confirmed paediatric poisoning at Red Cross War Memorial Children's Hospital, Cape Town, South Africa. Methods Children admitted with suspected poisoning between 1 January 2017 and 31 December 2017, were recruited. All patients had a urine and/or blood sample sent for LC-MS/MS toxicology. Data collected included demographic data, clinical features, investigations, management, outcome and social interventions. Results 152 children, with median age of 39 (IQR 25 -61) months were enrolled of which 128 (84%) were poisoning cases. Of the 128 poisoning cases, 88 (69%) presented with a history of ingesting a known substance, 16(12%) an unknown substance and 24(19%) were cases of occult poisoning. LC-MS/MS was able to identify a substance in 92% of the cases of occult poisoning. In those who had presented with a seemingly known substance, LC-MS/MS found a different substance in 15 cases. LC-MS/MS was also able to detect multiple drugs in 40 patients. Of the poisoning cases, six (5%) cases were attempted homicide cases and 5 (4%) cases were attempted suicide cases. No children died. Individualized social interventions were instituted in poisoning cases. Emergency placement safety reasons was required in 6 children. Conclusion: When the limitations are known, LC-MS/MS is useful in identifying cases of occult poisoning; identifying patients who have ingested multiple substances and/or an unknown substance and when targeted towards child protection. As LC-MS/MS is an expensive test, it should be used judiciously in LMICs.

The presented thesis deals with the issue of mycotoxins in brewing materials and beer. Attention was devoted mainly to the selected fusarium mycotoxins (deoxynivalenol, zearalenol, T-2 toxin, and HT-2 toxin) ochratoxin A and aflatoxins B1, B2, G1, and G2. The aim of the thesis was to optimize and validate analytical methods for the determination of the above mentioned mycotoxins in the brewing materials and beer. Analytes were separated using high-performance liquid chromatography with mass – spectrometric detection (HPLC-MS/MS) and ultra-performance liquid chromatography with fluorescence detection (UPLC/FLR). These analytical methods were then applied for mapping the occurrence of fusarium mycotoxins in malting barley crops in the Czech Republic and monitoring the level of contamination with mycotoxins in malting and brewing industries. In addition, experiments studying over-foaming of beer were conducted as primary gushing – over-foaming of beer – is connected, similarly as mycotoxins, with the presence of microscopic filamentous fungi in the raw materials for beer production. Studies describing in detail these methods are part of this thesis (Annex I – V). From all published results, it is evident that the occurrence of mycotoxins in cereals including barley is natural and cannot be completely prevented, not even if all conditions of correct agricultural practice are observed. It is known that some mycotoxins present in contaminated malting barley pass to the final product – beer due to their chemical and physical properties. However, the mycotoxin concentrations found do not mean any significant health risk for consumers.

Malaria is a disease caused by the protozoan parasite Plasmodium where the species that causes the most severe form of malaria in humans is known as Plasmodium falciparum. At least 40% of the global population is at risk of contracting malaria with 627 000 people dying as a result of this disease in 2012. Approximately 90% of all malaria deaths occur in sub-Saharan Africa, where approximately every 30 seconds a young child dies, making malaria the leading cause of death in children under the age of five years old. The malaria parasite has a complex life cycle utilising both invertebrate and vertebrate hosts across sexual and asexual stages. The erythrocyte invasion stage of the life cycle in the human whereby the invasive merozoite form of the parasite enters the erythrocyte is a central and essential step, and it is during this stage that the clinical symptoms of malaria manifest themselves. Merozoites invade erythrocytes utilising multiple, highly specific receptor-ligand interactions in a series of co-ordinated events. The aim of this study was to better understand the interactions occurring between the merozoite and erythrocyte during invasion by using modern, cutting-edge proteomic techniques. This was done in the hope of laying the foundation for the discovery of new key therapeutic targets for antimalarial drug and vaccine-based strategies, as there is currently no commercially available antimalarial vaccine and no drug to which the parasite has not at least started showing resistance. In this study healthy human erythrocytes were treated separately with different protein-altering enzymes and chemicals being trypsin, the potent oxidant sodium periodate (NaIO4), the amine cross-linker tris(2-chloroethyl)amine hydrochloride (TCEA) and the thiol cross-linker 1,11-bis(maleimido)triethylene glycol (BM(PEG)3). The resulting erythrocyte protein alterations were visualised as protein band differences on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), where treated and untreated control erythrocyte ghost protein fingerprints were visually compared to one another. The protein bands showing differences between treated and control samples were in-gel digested using trypsin then sequenced by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified using proteomics-based software. In this way, the erythrocyte proteins altered by each enzyme/chemical treatment were identified. Malaria invasion assays were performed where each treatment group of erythrocytes as well as the control erythrocytes were incubated separately with schizont stage malaria parasites for the duration of one complete life cycle. Using fluorescent staining and flow cytometry, the invasion inhibition efficiency for each treatment group was evaluated. By utilising these methods, the identification and the relative importance of the erythrocyte proteins involved in the invasion process were determined. Protein fingerprints of control and treated erythrocyte ghosts were visualised and optimised on SDS PAGE where induced protein band differences were successfully identified by LC-MS/MS. It was found that each treatment altered erythrocyte proteins with changes found in Band 3, actin, phosphoglycerate kinase 1, spectrin alpha, spectrin beta, ankyrin, haemoglobin, Bands 4.1 and 4.2, glycophorin A and stomatin. The invasion assays revealed that TCEA inhibited invasion to the greatest extent as compared to the other treatments, followed by BM(PEG)3 and trypsin. Sodium periodate-treated erythrocytes could not be assessed using the invasion assay due to auto-haemolysis. Band 3, glycophorin A, Band 4.1 and stomatin appear to be of higher relative importance in the invasion process as compared to the other altered erythrocyte proteins. These results confirmed the known roles of spectrin alpha, spectrin beta, glycophorin A, Band 3 and Band 4.1 in invasion, and suggested that ankyrin, Band 4.2 and stomatin may also be involved. This study highlighted the potential that modern, cutting-edge proteomic techniques provide when applied to previous comparative studies found in older literature, as previously unidentified proteins that can be involved in invasion were revealed. These results can be used as a foundation in future studies in order to identify new key targets for the development of new antimalarial drug- and vaccine-based strategies, with the hope of preventing the suffering of the millions of malaria-inflicted people worldwide, and ultimately eradicating this deadly disease. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Pharmacology / MSc / Unrestricted

Störungen des Lipidstoffwechsels sind entscheidend an der Entstehung atherosklerotisch bedingter kardiovaskulärer Erkrankungen (CVD) beteiligt. Insbesondere erhöhte Plasmakonzentrationen von Low-density-lipoprotein Cholesterin (LDL-C) tragen zur Entstehung von Atherosklerose bei. Apolipoproteine sind strukturelle und funktionelle Bestandteile von Lipoproteinen und damit als mögliche Biomarker und therapeutische Angriffspunkte der CVD Gegenstand aktueller Forschung. Zur experimentellen Untersuchung von kardiovaskulären Erkrankungen ist die Maus das bevorzugte Tiermodell. Im Mausmodell basiert quantitative Proteinanalytik bislang auf Antikörper-abhängigen Methoden (z.B. Western Blot), welche teils ungenau und materialaufwendig sind. Ein Verfahren zur effizienten und quantitativen Analyse von Apoliproteinen (Apos) in Mäusen, welches Flüssigchromatographie gekoppelt mit Massenspektrometrie (LC-MS/MS) nutzt, wurde bislang noch nicht beschrieben. In der vorliegenden Arbeit wurde eine LC-MS/MS Methode zur simultanen Quantifizierung von apoA-I, apoA-II, apoA-IV, apoB total, apoB-100, apoC-I, apoE und apoJ aus murinen Plasmaproben (3 µL) etabliert und validiert. Ausgehend von einer für humane Proben bestehenden Methode wurden die analytischen Bedingungen (z.B. Prüfung der Peptide, Dauer des tryptischen Verdaus, Linearität, Reproduzierbarkeit, Vergleich mit immunologischen Methoden) im Mausmodel ermittelt und optimiert. Anschließend wurde das Verfahren angewendet, um Apo-Plasmakonzentrationen in normocholesterinämischen C57BL/6, sowie in hypercholesterinämischen LDL-Rezeptor-defizienten (LDLR0) und apoE-defizienten Mäusen (ApoE0) zu charakterisieren. Dabei ermittelten wir moderate Unterschiede der Apo-Plasmakonzentrationen zwischen gefastetem und postprandialem Zustand, wobei apoA-IV und apoC-I postprandial erhöht waren und apoJ erniedrigt war. ApoE war in LDLR0 Mäusen 6-fach höher konzentriert als in C57BL/6 Mäusen und wurde erwartungsgemäß in ApoE0 Tieren nicht detektiert. Apo-B48 zeigte die höchste relative Konzentration in Apo-E0 Mäusen (93% des apoB-total), verglichen mit 61% in LDLR0-Mäusen. Zudem wurden Apo-Konzentrationen auf isolierten HDL-Partikeln bestimmt und zwischen den Mauslinien verglichen. Das entwickelte Verfahren kann zur weiteren Charakterisierung von Apos in verschiedenen Mausmodellen eingesetzt werden und damit als Grundlage für weitere Arbeiten zum Verständnis der Pathophysiologie und Funktionsweise von Apos dienen.

The purpose of this report is to compare seven different methods for biomarker detection and quantification based on previously published papers. The methods investigated are ELISA, LC-MS/MS, UPLC-MS/MS, LC-IM/MS, IA-LC-MS/MS, MSIA-HR/AM, HTRF and AlphaLISA ® . The focus lies on biomarkers relevant for diabetes, obesity and cardiovascular diseases.Namely insulin, proinsulin, glucagon and C-peptide. Particular significance is assigned to the comparison of the currently widest used method, ELISA, with various types of LC-MS/MS. The report concludes ELISA being superior to LC-MS/MS methods in terms of recovery and precision, while LC-MS/MS is superior in accuracy, multiplexing, specificity, throughput and sample cost. This suggests that different types of LC-MS/MS has the potential to gain momentum in the field of biomarker quantification if they become more available.

This research study successfully completed three objectives: 1) validate liquid-liquid, supported-liquid, and solid-phase extractions for the quantitation of three barbiturates (amobarbital, butalbital, and phenobarbital) in blood and urine using liquid chromatography-tandem mass spectrometry; 2) to compare the efficiency and effectiveness among methods in accomplishing extraction of barbiturates under the laboratory setting at Boston University School of Medicine; and 3) to report all the analytical data to RTI International for interlaboratory comparison. For the validation study, a six-point linear calibration model (20-2000 ng/mL) with inversely weighted concentration (1/x) was reproducible in all three sample preparation methods for both blood and urine with r2 greater than or equal to 0.994. Bias and precision evaluated from three controls throughout the range of the curve were within ±20% and ±20%CV, respectively. Neither carryover nor interference was observed. Detection limits were evaluated down to 5 ng/mL depending on the extraction procedure. Samples were able to be diluted up to 50 times prior to instrumental analysis. Samples were stable on autosampler at room temperature up to 72 hours after their initial analysis. Recovery of barbiturates from blood and urine all ranged from 45% to 86%. The effect of ionization suppression or enhancement was found to have minimal impact on the validation. For choosing the most suitable method quantifying barbiturates, efficiency and effectiveness were studied. Efficiency evaluates the time and ease of sample preparation required to prepare a sample for analysis. Supported-liquid extraction was found to be the most efficient method for extracting barbiturates as it required the least amount of time to perform and could be easily automated with minimal training. Effectiveness is an assessment of one’s ability to selectively recover target analyte at a reasonably low concentration. By considering a method’s recovery, extract cleanliness, detection limits, and reproducibility, liquid-liquid extraction was the best at quantifying barbiturates in blood and supported-liquid extraction was the most suitable method for extracting barbiturates from urine. For interlaboratory comparison, all the data collected has been reported to RTI International. These findings can be used for examining the overall reliability and reproducibility of the validated methods. Results obtained can also be used to explore the possibility for streamlining sample preparation in the forensic laboratory, and hence reducing the case backlog.