Antibiotic resistant bacteria are threatening human health, and the resistance is progressingfaster than the development of new antimicrobial compounds. Antibiotic resistant infections cost enormous sums of money and resources, but most importantly human lives. Therefore, early prediction and detection of antibiotic resistance in bacteria are research areas of high priority. The use of analytical instruments such as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) is a powerful tool for research in antibiotic resistant bacteria, both as quick microbe identification and for other areas in the field.Due to its relatively easy interpretable spectra provided by the soft ionization technique, and ability to ionize macromolecules without compromising sample integrity, it has also been used for biomarker screening for detection of antimicrobial resistance. Although these studies have shown promising results, the area is still progressing and needs further method development and standardized protocols. This study aimed to use MALDI-TOF MS for carbapenemaseassociated screening in Klebsiella oxytoca. The presumed key spectral peaks would derive from presence of the enzyme Verona integron-encoded metallo-β-lactamase, type 1 (VIM-1), and would not be found if VIM-1 were absent. The isolates, carrying the enzyme, used in the study were isolated from wastewater and river water in Örebro, Sweden. Bacteria genus and species was determined by MALDI-TOF identification, whereupon the microbes were tested for antibiotic susceptibility using the disk diffusion method. Carbapenem hydrolysis assay was used to confirm the presence/absence of functional carbapenemase. A genotypic confirmation was performed by polymerase chain reaction and gel electrophoresis. Mass spectra from MALDI-TOF were compared for identification of possible biomarker peaks that could indicate carbapenem resistance. Nine key mass spectral peaks were found that could potentially be used as biomarkers in future studies. The peaks differentiated two groups of Klebsiella oxytocaisolates, one group producing functional carbapenemase and one group that did not, consistent with the aim of this study.

Die vorliegende Arbeit behandelt die elektrochemische Synthese (elektrochemische Polymerisation und Copolymerisation) und die Charakterisierung der Redox- und sensorischen Eigenschaften neuer funktionalisierter Polymere für die Ionensensorik. Die Funktionalisierung wird sowohl in der Polymer-Hauptkette (Polysalene) als auch in der Polymer-Seitenkette (ein Thiophen-Copolymer: 3-Methylthiophen/6-Hydroxy-2-(2-(3-thienyl)-ethoxy)-acetophenon) dargestellt. Die Redox-Prozesse der funktionalisierten Polymere wurden mit spektroelektrochemischen Methoden: ESR-, UV-Vis-NIR- und FTIR-Spektroelektrochemie charakterisiert. Durch diese Methoden konnten während der elektrochemischen Oxidation von funktionalisierten leitfähigen Polymeren verschiedene Polymer- bzw. Copolymer-Ladungsträger nachgewiesen werden: Polaronen, Bipolaronen beim Thiophen-Copolymer, zwei Polaronen auf einer Polymerkette im Singulettezustand beim Poly(3-methylthiophen) und eine diamagnetische Spin-Spin-Wechselwirkung zwischen ungepaarten Elektronen der Cu(II)-Ionen und der ungepaarten Elektronen von bisphenolischen Ligand-Kationradikalen beim Poly[Cu(II)-salen]. Sensorische Eigenschaften gegenüber Ni(II)-Ionen wurden durch Potentiometrie an einem Poly[Ni(II)-salen]-Derivat getestet. Es zeigt eine gute potentiometrische Ni(II)-Ionenselektivität (der Logarithmus des potentiometrischen Selektivitätskoeffizienten liegt im Bereich von -0.5 bis -1.5) in Anwesenheit von Cd(II), Mn(II), Zn(II) und Na(I).

Die vorliegende Arbeit behandelt die elektrochemische Synthese (elektrochemische Polymerisation und Copolymerisation) und die Charakterisierung der Redox- und sensorischen Eigenschaften neuer funktionalisierter Polymere für die Ionensensorik. Die Funktionalisierung wird sowohl in der Polymer-Hauptkette (Polysalene) als auch in der Polymer-Seitenkette (ein Thiophen-Copolymer: 3-Methylthiophen/6-Hydroxy-2-(2-(3-thienyl)-ethoxy)-acetophenon) dargestellt. Die Redox-Prozesse der funktionalisierten Polymere wurden mit spektroelektrochemischen Methoden: ESR-, UV-Vis-NIR- und FTIR-Spektroelektrochemie charakterisiert. Durch diese Methoden konnten während der elektrochemischen Oxidation von funktionalisierten leitfähigen Polymeren verschiedene Polymer- bzw. Copolymer-Ladungsträger nachgewiesen werden: Polaronen, Bipolaronen beim Thiophen-Copolymer, zwei Polaronen auf einer Polymerkette im Singulettezustand beim Poly(3-methylthiophen) und eine diamagnetische Spin-Spin-Wechselwirkung zwischen ungepaarten Elektronen der Cu(II)-Ionen und der ungepaarten Elektronen von bisphenolischen Ligand-Kationradikalen beim Poly[Cu(II)-salen]. Sensorische Eigenschaften gegenüber Ni(II)-Ionen wurden durch Potentiometrie an einem Poly[Ni(II)-salen]-Derivat getestet. Es zeigt eine gute potentiometrische Ni(II)-Ionenselektivität (der Logarithmus des potentiometrischen Selektivitätskoeffizienten liegt im Bereich von -0.5 bis -1.5) in Anwesenheit von Cd(II), Mn(II), Zn(II) und Na(I).

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle's, less in Huxley's/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as it is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in tR between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) for the pituitary and recombinant preparations. The linearity of the dose-response curve (r = 0.9965, n = 15) for this RP-HPLC methodology, as well as an inter-assay precision with relative standard deviation less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behavior and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the accurate molecular mass of the individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for characterization and quality control of this hormone that is not yet described in the main pharmacopoeias.

Philosophiae Doctor - PhD / Obesity is a medical condition in which an energy imbalance leads to excessive accumulation of body fat. Obesity leads to a reduction in life expectancy through its association with chronic diseases of lifestyle. The prevalence of obesity is rapidly increasing throughout the world. It is now accepted that most cases of obesity result from an interaction between genetic and environmental factors. This rapid increase in obesity generally leads to an increase in morbidity and mortality from chronic diseases such as cardiovascular disease, type 2 diabetes, osteoarthritis and cancer of which obesity is a risk factor. There is a lack of information in molecular research to explain how obesity predisposes individuals to these diseases. Proteomics is a molecular tool and a set of techniques used to identify changes at protein level from a diseased state. This study aims to identify differentially expressed proteins in serum of obese rats fed different isocaloric diets using proteomics.

Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as it is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in tR between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) for the pituitary and recombinant preparations. The linearity of the dose-response curve (r = 0.9965, n = 15) for this RP-HPLC methodology, as well as an inter-assay precision with relative standard deviation less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behavior and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the accurate molecular mass of the individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for characterization and quality control of this hormone that is not yet described in the main pharmacopoeias.

Blütenstaub ist eine der Hauptursachen für allergische Erkrankungen des Menschen. Insbesondere die Matrix-unterstützte Laser-Desorption/Ionisation-Flugzeit Massenspektrometrie stellt eine geeignete Technik dar, um solch komplexe, biologische Proben zu charakterisieren. Bei MALDI-TOF MS-Analysen werden die Bestandteile detektiert, die zuvor durch die Matrixauftragung und/oder während der Laser-Desorption von der Oberfläche des Pollenkorns abgelöst werden. Hier zeigte sich, dass die Verwendung eines leitfähigen Klebebandes zur Probenpräparation sowohl die Fixierung der Pollenkörner als auch die Extraktion der Analyten aus diesen verbessert. Die gezeigten Ergebnisse belegen, dass sich Pollen verschiedener Pflanzenarten über den Vergleich von massenspektrometrischen Peakmustern differenzieren lassen. Zur Beurteilung der spektralen Muster wurden in dieser Arbeit multivariate, statistische Verfahren, speziell die Hauptkomponentenanalyse verwendet. Diese hebt Unterschiede in den Datensätzen hervor und ermöglicht eine varianzgewichtete Darstellung und verbesserte Klassifizierung. Zur effizienten multivarianten Analyse wurde dieses Verfahren in eine graphische Benutzeroberfläche eingebunden. Neben der individuellen Charakterisierung einzelner Pollenproben liegt eine weitere Herausforderung in der massenspektrometrischen Analyse von Pollenkornmischungen, da es durch Vermischungs- und Suppressionseffekte zur Diskriminierung einzelner, möglicherweise charakteristischer MS-Peaks kommen kann. Deshalb wurde in dieser Arbeit die bildgebende MALDI Massenspektrometrie angewendet. So konnten in einem Modellsystem einzelne Pollenkörner unterschiedlicher Arten nachgewiesen und ihren ursprünglichen Positionen in der Probe zugeordnet werden. Der im Rahmen der Arbeit vorgestellte Ansatz stellt eine neue, zuverlässige Methode zur Pollenbestimmung dar, die nicht auf der individuellen, visuellen Beurteilung, sondern auf einer spektrometrisch-analytischen Basis beruht. / Pollen represent one of the major causes for allergies in humans. Matrix-assisted laser desorption/ ionization time-of-flight is a suitable technique to characterize such complex biological samples. In pollen analyses, MALDI-TOF MS detects components that are extracted from the grain surface by the matrix solution and during laser desorption. Like any biological structure, pollen grains contain specific proteins, peptides, lipids and carbohydrates that produce characteristic signal patterns in the mass spectrum. The sample preparation applied in this work showed that the use of a conductive adhesive tape improves both the fixation of the pollen grains and the extraction of the analytes from them. The shown results prove that pollen of different plant species can be differentiated by comparing their respective mass spectral patterns. For the evaluation of such spectral patterns multivariate statistical methods, and in this work especially principal component analysis, are needed. PCA highlights differences in data sets and enables variance-weighting and improved classification. For efficient multivariate analysis, this method was integrated into a graphical user interface. In addition to the individual characterization of pollen samples, the mass spectrometric analysis of pollen grain mixtures posed a further challenge, since mixing and suppression effects can lead to discrimination of individual, possibly characteristic MS-peaks. For this purpose, the MALDI MS Imaging technique was applied. For the evaluation of such imaging data sets, further multivariate methods were applied, investigated, and evaluated. Thereby, in a model system individual pollen grains of different species could be identified and assigned their original position in the sample. The presented approach represents a new, reliable method for pollen determination, based on spectrometric-analytical basis rather than an individual visual assessment.

In this thesis, the vital cell functions performed by integral membrane proteins (IMPs) are briefly discussed. Such proteins are under-represented in most protein studies due to the hydrophobic nature of IMPs, which seriously complicate their solubilization, sample handling, preparation, separation and analysis. Conventional analytical techniques include for example matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS), capillary electrophoresis (CE) and reversed phase high-performance liquid chromatography (RP-HPLC). Presented here are methods and protocols, which have been developed especially for IMP and hydrophobic peptide analysis, using the abovementioned techniques. The fluorinated organic solvent hexafluoroisopropanol (HFIP) has previously been shown beneficial as an additive for solubilization of hydrophobic analytes, which are poorly soluble in commonly used organic solvents or water. In Papers I-IV, HFIP is successfully exploited as solvent for the investigated IMPs and peptides. The simple fabrication and the focusing effect of a new structured MALDI target plate are presented in Paper I. This target plate contains concentrating sample spots, specifically designed to provide increased sensitivity for hydrophobic protein and peptide MALDI-MS analysis. When replacing a regular steel target with this new structured MALDI plate, more than a five-fold increase in average sensitivity is achieved for HFIP solubilized hydrophobic peptides. The full-length IMP bacteriorhodopsin (BR) and a cyanogen bromide digest thereof are used as model samples for the development of sample handling procedures in Paper II, and the peptides were used for evaluation of the MALDI-target plate in Paper I. Furthermore, the CE separation of the peptides, fractionation onto the structured MALDI plate and following MS analysis is presented in Paper III. Nine of the ten theoretical BR peptides were detected using this method. A protocol for the purification and analysis of chloroplast membrane proteins from the green macroalga Ulva lactuca has been described in Paper IV. The highest protein yield was achieved when proteins were extracted in HFIP, directly from the chloroplasts. The MALDI-MS analysis of samples with and without previous RP-HPLC fractionation revealed proteins with molecular weights ranging between 1 and 376 kDa. In Paper V, a closed-open-closed CE system is presented, containing an open microchannel for off-line MALDI detection. The electroosmotic flow and band broadening of this system has been evaluated. / QC 20100916

In Chapter I is a general description of novel metal complexes which hydrolytically cleave peptides, proteins, DNA, and other biological molecules. These reagents are becoming the more important as potential therapeutic agents. A panel of ligands was investigated for coordination to ZrIV and other metals in groups 4, 5, and 6 to effect the greatest degree of hydrolysis. Chapter II describes a ZrIV complex which is capable of hydrolyzing a 30 amino acid peptide, insulin chain B, with amino acid specificity. Oxidized insulin chain B peptide was hydrolyzed after only 4 h of treatment at pH 7.0 and 60 °C using ZrCl4 in the presence of 4,13-diaza-18-crown-6. MALDI-TOF and ESI LC-MS mass spectra indicated that insulin chain B was hydrolyzed by ZrIV at the Gly8-Ser9, Ser9-His10, and Gly20-Glu21 amide bonds within the oligopeptide. To our surprise, the cysteine sulfonic acid sequences Cys(SO3H)7-Gly8 and Cys(SO3H)19-Gly20 were also cleaved. To the best of our knowledge, this constitutes the first example of metal-assisted hydrolysis of a Cys(SO3H)-Xaa amide bond. This is significant in light of the fact that cysteine sulfonic acid formation in proteins is triggered by oxidative stress and has been associated with amyloid fibril formation, Parkinson’s disease, and other deleterious, physiological processes. Chapter III describes the metal-assisted hydrolysis of sphingomyelin which is a principle phospholipid component of animal cell membranes. The sphingomyelin assays showed evidence of metal-assisted hydrolysis after 20 h of treatment at lysosomal pH 4.8 and cytosolic pH 7.0 at both physiological temperature 37 °C and 60 °C. The metal ion CeIV was the most reactive, followed by ZrIV, and then HfIV. The goal of this work is to develop metal-based reagents to reverse the lethal build-up of sphingomyelin that occurs in lysosomes of patients suffering from Niemann-Pick disease.