Stress conditions such as heat shock, UV, alkylating agents, and H2O2 have been shown to result in the modification of a variety of protein targets via the production of reactive electrophiles. These modifications can directly impact protein function or can alter posttranslational modifications, thus leading to a disruption of cellular regulatory processes. Recent studies have shown that stress-induced protein modifications can modulate posttranslational modification by the small ubiquitin related modifier (SUMO) family of proteins. Unlike ubiquitination, which primarily targets proteins for proteasomal degradation, sumoylation exerts a variety of effects including protein stabilization, subcellular localization, and the alteration of protein-protein interactions and transcriptional activity. To investigate the effects of alkylation and oxidative stress on sumoylation, HEK293 cells were treated with iodoacetamide, hydroquinone, benzoquinone, Texas Red C5 bromoacetamide, hydrogen peroxide, and 4-hydroxynonenal (HNE), a highly reactive product of lipid peroxidation associated with oxidative stress. Western blot analysis revealed that the agents tested resulted in concentration-dependent changes in the patterns of SUMO-1 and SUMO-2/3 protein conjugation. Localization studies using western blot analysis and confocal immunofluorescence microscopy demonstrated that SUMO-1 protein conjugates were located primarily in the nucleus, whereas SUMO-2/3 protein conjugates were more equally distributed between the nucleus and the cytoplasm. SUMO-associated proteins were harvested from vehicle- and HNE-treated non-transfected HEK293 cells using agarose conjugated anti-SUMO-1 antibodies or from HA-SUMO-1- and HA-SUMO-3-expressing HEK293 cells using immunoaffinity chromatography. Multidimensional liquid chromatography-tandem mass spectrometry analyses resulted in the identification of 54 HA-SUMO-1-associated proteins and 37 HA-SUMO-3-associated proteins in vehicle-treated cells and 21 HA-SUMO-1- and HA-SUMO-3-associated proteins in HNE treated cells. Additionally, 27 SUMO-1-associated proteins were identified in the HNE-treated non-transfected cells. The functional classes of proteins targeted included RNA binding and processing proteins, metabolic enzymes, cytoskeletal regulators, and chaperone proteins. HNE treatment resulted in a near complete redistribution of both SUMO-1 and SUMO-3 to different targets. There was a 15% overlap in SUMO-1 and SUMO-3 associated proteins in vehicle-treated cells and a 10% overlap in HNE-treated cells indicating that SUMO proteins target distinct protein groups. These results indicate that protein modifying reactive electrophiles can regulate protein functions through the indirect alteration of endogenous posttranslational modifications.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The use of pesticides always has been associated with the effective control of pests or invasive weeds to ensure an increase in the food production. However, the indiscriminate use of these substances has caused the degradation of water resources. In Brazil, the Ministry of Health through Ordinance 2914 defines several parameters of potability, among them, the maximum limits allowed for same pesticides. In this work it was developed and validated a method for the determination of residues of 70 pesticides in drinking water using (SPE) for sample preparation and determination by Gas and Liquid Chromatography coupled to tandem mass spectrometry, triple quadrupole analyzer (GC-(TQ)MS/MS and LC- (TQ)MS/MS). It was evaluated different sample volume, sorbents and solvent of elution. The best results were obtained using 100 mL sample acidified at pH 2.5, Oasis® SPE cartridge HLB 60 mg/3 mL and dichloromethane/methanol as eluent. Analytical curves were linear between 10 and 250 Sg L-1, with r2 values greater than 0.99 for all compounds. The values of method LOQ were 0.02 Sg L-1 for aldrin, dieldrin and chlordane and 0.5 Sg L-1 for the other compounds. To evaluate accuracy the blank samples ware fortified at 0.5, 1.5 and 4.0 Sg L- 1 and an extra level at 0.02 Sg L-1 for aldrin, dieldrin and chlordane. The method showed good precision, with RSD values below to 20% and good accuracy, with recoveries between 70 and 120%. Only the compounds methamidophos, aldicarb, benfuracarb, terbufos, benomyl and thiophanate methyl were not recovered adequately. The matrix effect was evaluated, showing upper 10% for the most compounds. In order to compensate this effect, analytical curves were obtained with standarts prepared in blank extracts of the matrix. The validated method was applied to 12 samples of drinking water of different characteristics (river, shed, well and treated water), and just one of the river samples presented residues of lambda-cyhalothrin. The results indicate that the proposed method is suitable for analysis of pesticides residues in drinking water, since all the validation parameters met the suggested limits and parameters for validation of chromatographic methods. / O uso de agrotóxicos sempre esteve associado à efetividade no controle de pragas ou plantas invasoras para aumentar a produção de alimentos. No entanto, o uso indiscriminado dessas substâncias vem causando a degradação dos recursos hídricos. No Brasil, o Ministério da Saúde através da Portaria 2914 define diversos parâmetros de potabilidade, entre eles, os limites máximos permitidos para alguns agrotóxicos. No presente trabalho foi desenvolvido e validado um método para a determinação de resíduos de 70 agrotóxicos em água potável, utilizando Extração em Fase Sólida (SPE) para o preparo de amostra e determinação por Cromatografia Gasosa e Líquida, acopladas à Espectrometria de Massas sequencial, com analisador triplo quadrupolo (GC-(TQ)MS/MS e LC- (TQ)MS/MS). Foram avaliados diferentes volumes de amostra, sorventes e solventes de eluição. Os melhores resultados foram obtidos com 100 mL de amostra acidificada em pH 2,5, cartucho SPE Oasis® HLB 60 mg/3 mL e diclorometano/metanol como eluente. As curvas analíticas apresentaram linearidade entre 10 e 250 Sg L-1, com valores de r2 maiores que 0,99 para todos os compostos. Os valores de LOQ do método foram 0,02 Sg L-1 para aldrin, dieldrin e clordano e de 0,5 Sg L-1 para os demais compostos. Para avaliação da exatidão as amostras branco foram fortificadas em 0,5, 1,5 e 4,0 Sg L-1 e um nível extra em 0,02 Sg L-1 para os compostos aldrin, dieldrin e clordano. O método apresentou boa precisão, com valores de RSD inferiores a 20% e boa exatidão, com recuperações entre 70 e 120%. Apenas os compostos metamidofós, aldicarbe, benfuracarbe, terbufós, benomil e tiofanato metílico não foram recuperados de forma adequada. O efeito matriz foi avaliado, mostrando-se superior a 10% para a maioria dos compostos. A fim de compensar este efeito, utilizou-se curvas analíticas preparadas no extrato branco da matriz. O método validado foi aplicado em 12 amostras de água potável de diferentes características (rio, vertente, poço e água tratada), sendo que apenas uma das amostras de água de rio apresentou resíduos de lambda-cialotrina. Os resultados indicam que o método proposto é adequado à análise de resíduos de agrotóxicos em água potável, visto que todos os parâmetros de validação atenderam os limites e parâmetros sugeridos para validação de métodos cromatográficos.

Insects represent very useful experimental model in various branches of biological research. The investigation is driven by economic importance of many insect species, and also by biological features of insects as model organisms such as short period of reproduction, easy breeding and manipulation and, in particular, the minimal regulatory requirements which are associated to the management of vertebrates. Here we report robust and efficient LC/MS/MS methodology for the determination of the physiologically important lipid molecular species in insects. The target metabolites represent polar glycerophos-phopholipids (GPL) and nonpolar lipids diacylglycerols (DG) and triacylglycerols (TG). Combination of the LC/MS data with the subsequent GC fatty acid analysis enables complete structural elucidation of particular lipid species including their fatty acid compositions. The developed methodology was applied to studies of the chill tolerance of the firebug Pyrhocorris appterus. Fields and laboratory experiments were conducted to separate the triggering effects of low temperature, desiccation and diapause progression on the physiological characteristics related to chill tolerance with emphasis on the restructuring of GPL composition. The same effect on the GPL composition was observed during acclimatization in the field and cold acclimation in laboratory. By contrast, the GPL changes related to desiccation and diapause progression were relatively small (Tomčala et al, 2006). In adults of Drosophila melanogaster it has been found that acclimation at 15, 20 and 25°C during preimaginal development affects thermal tolerance and composition of membrane GPLs. Low temperature acclimation was associated with increase in proportion of ethanolamine at the expense of choline in GPLS. Relatively small, but statistically significant changes in lipid molecular compositon were observed with decreasing acclimation temperature (Overgard et al, 2008). Hormonal treatment studies on insect model Locusta migratoria showed a heterogeneous distribution of individual DGs in haemolymph after the hormone application and revealed that mobilization of the DGs is molecular species-specific with the highest proportion of DG 16:0/18:1 and forming in summary about 20% of the total mobilized DG content. Additional analysis of fat body triacylglycerols revealed that the AKH mobilizes the DGs specifically with the preference of those possessing the unsaturated C18 fatty acids (FAs). The fat body FAs with more than 18 carbons did not participate on the mobilization (Tomcala et al, 2009). The LC/MS methodology was further applied to lipid composition studies of several samples with very diverse biological origin (fish, human blood etc.) and was proved to be universally applicable to the wide scope of biological samples.

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. The procedure involves a simple liquid-liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r2 ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.

Widespread sedentary lifestyle and unhealthy eating habits in the last few decades have resulted in a dramatic increase of the number of people affected by obesity, type 2 diabetes, and cardiovascular diseases. The study of these pathological conditions revealed that impaired metabolism often causes these disorders. Lipid metabolism research has contributed significantly to determining mechanisms underlying metabolic disorders. Omega-3 fatty acids are an interesting target for lipidomics studies because they were shown to lower risk of cardiovascular diseases and are hypothesized to regulate lipid metabolism. In this work, I optimized lipid extraction and chemical modification methods for analysis of fatty acids profile of tissue samples and biofluids using comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GCxGC-MS). At first, I evaluated the relative amount of omega-3 fatty acids in red blood cells (Omega-3 index) of people living in Czech Republic in either the capital city (n=476) or the rural region (n=388). For this large-scale project, I extracted phospholipids from red blood cell (RBC) membranes, transesterified them into fatty acid methyl esters (FAMEs), and measured their profile by GCxGC-MS. The mean Omega-3 index was 3.56 mol % and I detected no significant...

The detection and quantification of hormones are important to assess the reproductive and stress status of experimental models and for the diagnosis of diseases in human and veterinary clinics. The peptide secretoneurin (SN) has been proposed as a new sex hormone, but effective quantification methods are challenging. Traditional methods require the use of antibodies with either radioactive or non-radioactive tracers. There are difficulties with these methods in terms of sensitivity, specificity, and inter-laboratory repeatability. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can circumvent many of these challenges. Another source of variation is the extraction of lipophilic steroidal compounds, which is incompatible with the extraction of hydrophilic peptide hormones. I have developed efficient extraction and sensitive detection methods of SN with numerous other peptide and steroid hormones in the same tissue sample in mice and zebrafish. The extraction efficiency for both peptide and steroid analytes is over 85%. The standard deviation for extraction and LC-MS/MS analysis for each compound varies between 5-10%. The steroid hormones can be quantified in the low to medium fmol/µL range. We quantified peptide hormones in the high fmol/µL to low pmol/µL range. Mouse SN levels were measured and compared against the levels of GnRH 1, oxytocin, vasopressin, E2, and P4 in multiple tissues at 3 important periods through the estrous cycle. In addition, SN levels were found to be moderately related to GnRH 1 levels in the hypothalamus in the estrous cycle. This is important because it is GnRH 1 that stimulates the luteinizing hormone surge in the pituitary that regulates ovulation in all vertebrate species. I also determined that SNa and SNb were both within the 2-8 pmol/µL range in the brain or pituitary harvested from a single female zebrafish. This makes it feasible for the first time to study the correlation between the SNs and other peptides and steroid hormones by quantifying them simultaneously in very small tissue samples. Untargeted peptidomics determined that the SN peptides in zebrafish can be further processed into smaller discrete fragments. This implies not only active synthesis and selective peptide processing but suggests that the are unknown functions of the SN peptide fragments that await discovery. This cost-effective package was used for the detailed assessment of hypothalamic-pituitary-gonadal function in mice and zebrafish and may be adaptable to many other hormones across species.

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

In the early 19th century a ground-breaking discovery was made that linked a dietary deficiency of a fat-soluble vitamin with the childhood disease known as rickets. The vitamin was named vitamin D and extensive research regarding the physiological importance of this vitamin followed ever since. It is currently known that vitamin D plays an important role in maintaining the calcium and phosphate homeostasis in the human body. Less clear evidence states the medical importance of vitamin D in the prevention and cancer, autoimmune disease and diabetes. Current literature shows that vitamin D has five distinct forms, vitamin D1 to D5, of which vitamin D2 and D3 are the most studied forms. The term “vitamin D” is often wrongfully used to include the vitamin D mother molecule, the vitamin D status indicator (25(OH)D), the biologically active form (1,25(OH)2D) and biologically inactive form (24,25(OH)2D). The interest for measurement of these vitamin D analogues is a continuously growing field both on individual and epidemiological level. For decades laboratories have struggled to produce a robust method capable of quantifying these different vitamin D analogues and uncovered a new form of complexity regarding the analysis of these analogues. The identification of the C3-epimeric forms of vitamin D metabolites has forced laboratories to rethink their analytical methods and several concerns were raised regarding the overestimation of the true vitamin D status by current analytical methods. The quantification of the biologically active and inactive forms of vitamin D is reported to be difficult and to date very few LC-MS/MS methods reported in the literature are able to quantify various vitamin D analogues. However, to our knowledge none of these methods are able to include the precursor vitamin D, the 25-hydroxylated metabolites, the biologically active and inactive metabolites, C3-epimers and isobaric compounds in a single run. Therefore the aim of this study was to develop, optimise and validate a LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run. This was done by optimising the underlying LC-MS/MS parameters to ensure optimal analytical sensitivity in positive ESI mode and sufficient chromatographic separation between analytes with similar chemical properties. Furthermore, the optimised method was validated to ensure the accuracy and precision of the method before implementation into a clinical environment. The vitamin D analogues included in this study were vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi- 25(OH)D3, 7(OH)4C3 and 1α(OH)D3. A double liquid-liquid extraction with hexane and ethyl acetate were found to be the most efficient at extracting the vitamin D analogues from a serum matrix after matrix modification with sodium hydroxide. Recoveries of > 95 % (CV <10 %) were achieved for all the analytes. It was noted that a precursor adduct other than the molecular mass ion for a specific vitamin D analogue can produce a more abundant MS1 signal and that the ESI source parameters vary between analytes with different chemical properties and should therefore be optimised individually for each analyte. Various columns were assessed and sufficient chromatographic separation between the relevant analytes was achieved with an Agilent Technologies Pentafluorophenyl column. Baseline separation was achieved between 25(OH)D3 and 3-epi-25(OH)D3 as well as 25(OH)D2 and 3-epi-25(OH)D2, which is a requirement for this method to be viable. The method was subjected to a series of validation steps to ensure the accuracy and precision of the method. These included the assessment of the analytical range, LOD, LOQ, inaccuracy, imprecision, stability, interference and recovery. It was found that the optimised method had good linearity (r > 0.995), acceptable repeatability (CV < 10 %) and within-lab precision (CV < 15%) and excellent method accuracy (systematic error < 6.60 %). Furthermore, all the analytes proved to be stable for 48 hours after sample preparation with no interferences found for co-eluting analytes. Finally, based on the sigma metric scale specifications, it was calculated that this method proved to be “world class” and very little QC is needed to ensure the quality of the data derived from this method. Based on the findings in this study, it was concluded that a novel LC-MS/MS method for the quantification of twelve vitamin D analogues in a single run was successfully developed. All the LC-MS/MS parameters were optimised to ensure optimal analytical sensitivity for each analyte and the method was validated based on a series of method validation steps required for implementation into a clinical laboratory. This validation proved this method to be ready for implementation into a clinical environment. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

<p>Under en individs liv erfars i regel många livsomvälvande händelser som fungerar förändrande och utvecklande. I vissa fall leder en företeelse till att en individs identitet genomgår en förändring. Denna studies fokus riktas mot förhållandet mellan ett reviderat självkoncept och den personliga identiteten. En kvalitativ metod genomfördes där åtta individer med sjukdomen MS intervjuades. Studien visade att intervjupersonernas upplevelse av den egna kroppen, sig själva och de värderingar de attribuerade till sig själva, det vill säga självkonceptet, förändrades till följd av de hinder och begränsningar sjukdomen bidragit till. Detta fick till följd att den personliga identiteten omskapades i fråga om karaktärsdrag och målsättningar. Studien bidrar med fördjupade kunskaper om förändringar i självkonceptet och den personliga identiteten vid sjukdom.</p> / <p>During an individual’s life many events develop and change the person and some may even change the individuals’ identity. Focusing on the relationship between an altered self-concept and the personal identity, a qualitative attempt was made. Eight interviews were made with individuals who suffered from MS. The result showed that the way the participants experienced themselves, their bodies and the way they attributed themselves changed dependent on the obstacles and limitations the disease contributed to. This was followed by changes in the personal identity in terms of characteristics and goals. The study contributes with advanced knowledge about alterations of the self-concept and the personal identity concerning illness.</p>

No description available.