Objectives: Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass-Spectrometry holds promise as a non invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may improve the early diagnosis of oral cancer tremendously. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS. Material and Methods: In the first step, a data base consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as a basis for further classification of the blind study composed of additional 26 samples including HNSCC, oral lesions and healthy mucosa. Results: By analyzing spectral patterns of the blind study, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%. Conclusion: ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to a more impartial early diagnosis of HNSCC.

>Magister Scientiae - MSc / Salvia hispanica L. is an annual pseudocereal food crop, locally known as chia that has the ability to grow in water stress environments. The importance of chia dates back to the pre-columbian era where it was consumed as staple food by the indigenous South Americans due to its high nutritional and medicinal benefits. A single chia plant produces two seed variants: white seed genotype (denoted as WSG) and black seed genotype (denoted as BSG). Chia seeds have been proven to have a huge potential as a healthy food source and contained various medicinal properties. However, these plants are still prone to environmental stress conditions such as salinity that is one of the major abiotic stresses that influence crop production and yield worldwide. Despite the nutritional impact of the chia seeds, limited information regarding their molecular responses to abiotic stress conditions are known. This study was divided into two distinct parts. Firstly, the study comparatively analysed the leaf proteomes of two chia genotypes using gelbased proteomic analysis coupled with mass spectrometry. Total soluble proteins were extracted from chia leaves and subjected to 2-D PAGE analysis. Proteins were visualized by CBB and identified by MALDI-TOF MS/MS. A total of 284 and 209 spots were detected in WSG and BSG, respectively. Using mass spectrometry, 36 differentially expressed protein spots were successfully identified based on their protein abundance using homology database searches. Interestingly, two defensive-related proteins (osmotin-like protein and the chalcone isomerase) were only present in WSG and absent in BSG. In light of previous information regarding the nutritional profiles (no significant difference) of these two genotypes, this study has shown that there are distinct molecular differences between these genotypes. Therefore, WSG will be used in further downstream analysis. The second part of this study focused on the influence of salt stress (imposed by 100 mM NaCl) on the leaf proteome of WSG. Using gel-based proteomic analysis, 61 differentially expressed proteins were identified and classified into nine functional categories. Most of the proteins identified in this study were upregulated by salt stress. Interesting to note, 12 proteins identified in this study were only present in response to salt stress but were absent in the control. These proteins include ATP-dependent zinc metalloprotease FTSH 2 (spot 48), HSP70 proteins (spots 46 and 47), superoxide dismutases (spots 10, 41 and 42) and an ascorbate peroxidase (spot 56). All these proteins are important antioxidants that play a significant role in scavenging reactive oxygen species (ROS). Previous studies have shown that these antioxidants play vital roles in stress tolerance. These proteins could serve as potential biomarkers that could be used to enhance salt stress tolerance in pseudocereals and cereal food crops. / National Research Foundation (NRF) and Agricultural Research Council

Microarrays on gold have been used to develop new methodologies for biocatalysis and proteomic applications. The technology applies the logic of solid phase supported chemistry using self-assembled monolayers (SAMs) on a gold chip. The advantages of this technology are: i) an easy to handle platform, ii) parallel screening (64 reactions at once), iii) microliter scale reactions (1µL per sample), iv the use of mild conditions (buffers and t=37 °C), v) the absence of purification steps (only chip washing is required), vi) quick and accurate analysis by MALDI ToF MS.The metallopeptidase thermolysin was studied in peptide-coupling reactions to profile reactivity and specificity (Chapter 2). Reactivity was further investigated in transpeptidation reactions. Comparing the serine peptidase chymotrypsin with the zinc dependent thermolysin, it was found that transpeptidations proceed via N-transacylation reactions independent of a specific enzymatic catalytic mechanism (Chapter 3). These transacylations may be exploited for modifications of biocompatible and selective surfaces in ‘bottom-up’ bionanofabrication technologies. Selected peptidases with different catalytic mechanism were also arrayed to investigate polymerisation ability of dipeptides (Chapter 4). It was shown that oligomerisation can be obtained under mild conditions and a set of peptides was synthesised. Chapter 5 describes a new chemical methodology by which crude tryptic peptide digests can be trapped on chip and analysed by MALDI ToF MS without further purification steps. This dramatically improves time and cost efficiency.Finally, a new stepwise native chemical ligation methodology is proposed for amino acids, and peptides containing N-terminal cysteine residues (Chapter 6).

Abstrakt: Sepsis (blodförgiftning) är ett livshotande tillstånd som innebär att bakterier eller svampar tar sig in i blodbanan. Det sker ofta genom njurarna, lungorna eller sår i skadad hud. Symptom innefattar feber och allmän sjukdomskänsla. Positiva blododlingar prepareras och analyseras med Matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF MS) där blandas preparerade prov med matrix och bestrålas sedan med en laser. Bestrålningen medför att proteinerna i provet joniseras och rör sig mot en detektor. Proteinerna detekteras som ett spektrum som i sin tur jämförs med ett referensspektrum av en känd bakterie/svamp som finns lagrat i en databas. Dessutom erhålls ett score-värde över hur väl provet liknar ett referensprov. Arbetets syfte var att se vilken provberedningsmetod som gav ett tillförlitligt resultat i MALDI-TOF och ett bra score-värde för identifiering av bakterier till art-nivå på kortast tid. Preparationsmetoderna innefattar rening av blod från röda blodkroppar och andra partiklar som stör MALDI TOF-instrument. Metoderna jämfördes med nuvarande Saponinmetod. Två av metoderna (Ferroni och Huang) gav dåliga resultat och jämförelsen avbröts efter två försök. Ett kommersiellt kit Sepsityper extraktion gav bra resultat där 79 % av analyserna gav identifiering till art-nivå. Motsvarande resultat med saponin-metoden var 33 %. Slutsatsen är att Sepsityper-extraktion-metoden gav betydligt bättre resultat än nuvarande Saponin-metod. Metoden var robust, användarvänlig, snabb och gav bra resultat både för gramnegativa och grampositiva bakterier. / Abstract: Sepsis (blood poisoning) is a life-threatening condition caused by bacteria or fungi entering the bloodstream. Pathogens often again access through the kidneys, lungs or wounds in damaged skin. Symptoms include fever, chills and general feeling of illness. Positive blood cultures are prepared and analyzed with Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF MS) prepared samples are mixed with matrix and then irradiated with laser beam. The laser beam will be directed to each position in the MALDI plate that causes the proteins in the sample to ionize and move towards a detector. The protein pattern is presented in a spectrum that is compared with a reference spectrum of known bacteria / fungi, stored in a database. In addition, a score value was obtained on how well the sample is aligned to the reference sample. The aim of this study was to compare three methods in order to achieve the best score value for identification to species level. The time factor was also important. The preparation methods include purification of blood removing red blood cells and other particles that interfere with MALDI analysis. The methods were compared with the current Saponinmethod. The results of two methods (Ferroni and Huang) were not satisfactory and further comparison was interrupted after two experiments. A commercial kit Sepsityper with extraction gave good results with 79 % identification to species level. Corresponding results for the current Saponin-method was 33 %. In conclusion, the Sepsityper-extraction-method was superior to the current Saponin-method. The method was robust, user-friendly, rapid and gave good results from both gramnegative and grampositive bacteria.

The thesis is focused on electrical discharge in liquid solutions, in particular in ethanol solutions. In the theoretical section, the plasma is defined along with its important parameters, types of plasma generation and usage of plasma technologies. Main part is focused on the discharges in water solutions. Electric discharges in liquid solutions enable physical processes (shock waves, UV radiaton, electric current) as well as chemical ones (generation of radicals and ions). Both types of the processes are used in many fields – medicine (tool sterilization, destruction of pathogens), consumer technologies (surface adjustemen, processing of the textiles, surface clearing) of environmental protection (clearing water, decomposition of organic compounds). Goal of the thesis was to analyze discharge products on ethanol solution. Multiple samples were measured and concentration od selected products was monitored during the measurement – ethanol, acetone and acetaldehyde. PTR-TOF-MS method was used to collect and analyze the data. This method is well used thanks to quick response and immediate measurement. It produce the reset accurate enough for our purpose. It would be more efficient to use this method in combination with another analytical metohod in the future research, e.g. gas chromatography.

This thesis deals with the passive sampling of polar compounds from water using passive samplers POCIS (Polar Organic Chemical Integrative Sampler). These devices were exposed for 4 weeks in the waste water at the inflow of municipal waste water treatment plant in Brno – Modřice, and also at the outflow of the treated water. After exposition sequestered compounds were released by extraction with a mixture of methanol, toluene and dichlormethane. Extracts were analysed by liquid chromatography/mass spectrometry with electrospray ionization. Further, methylated and trimethylsilylated extracts were analysed by orthogonal comprehensive two-dimensional gas chromatography with mass spectrometric detection. Separated compounds were identified on the base of their retention and mass spectra.

The diploma thesis will be focuses on the isolation of fragrances of natural origin by steam vapor distillation, ultrasonic extraction, microwave extraction and pressure solvent extraction. The extracts were characterized by GCxGC/TOF-MS. To study the surface structure of the leaf by SEM, a sample of the medulla medical was selected. Photos were taken before and after extraction.

Differenzierung aviärer Brachyspiren mit PCR-basierten Methoden und MALDI-TOF-MS

ANA-buljongen är utvecklad för att kunna anrika både aeroba och anaeroba bakterier vid inkubering i aerob miljö. Odlingsbuljongen innehåller antioxidanter som skyddar anaeroberna mot reaktiva syreradikaler, vilket främjar anaerob anrikning. Syftet med arbetet var att utvärdera om ANA-buljongen kan ersätta de två befintliga odlingsbuljongerna som idag används vid anrikning av bakterier på avdelningen för klinisk mikrobiologi, Kronoberg Blekingesjukhuset. Elva bakteriearter och fyra blandfloror användes i utvärderingen av ANA-buljongen. En mikroliter (µl) inokulat som innehöll ca 100 colony forming units tillsattes till ANA-buljong samt aeroba buljongen Brain Heart Infusion (BHI) eller den anaerobbuljongen Fastidious Anaerobe Broth (FAB). Prov från buljongerna odlades ut efter två, fyra och sju dygns inkubering. Tillväxtsgrad vid olika inkubationstider jämfördes mellan ANA-buljongen och BHI-buljongen/FAB. ANA-buljongen utvärderades även för odling av kliniska prov. De kliniska proven odlades ut på agarplattor samt inokulerades till ANA-, BHI-buljong och FAB. Vid utodling från buljongerna jämfördes fynden från BHI-buljongen och FAB med fynden från ANA-buljongen. Tillväxten verkade vara snabbare i ANA-buljongen jämfört med BHI-buljong och FAB. Vissa arter, exempelvis Cutibacterium acnes, uppvisade en betydligt snabbare tillväxt i ANA-buljongen. Vissa bakteriearter tillväxte men överlevde inte längre inkubationstider och kunde efter dessa inte detekteras. Anrikningen av blandfloror i ANA-buljongen resulterade i överväxt av en art vilken konkurrerade ut de övriga arterna. Detta fenomen uppstod inte i FAB. Att ersätta de två kliniska buljongerna med ANA-buljongen hade varit fördelaktigt både praktiskt, ekonomiskt och ur ett hållbarhetsperspektiv. Eftersom ANA-buljongen enbart visade sig användbar vid odling av renkulturer men inte vid odling av blandfloror är den inte optimal för klinisk diagnostik. Men den skulle eventuellt kunna nyttjas vid ortopediska Kamme-Lindberg-odlingar då det oftast är fråga om renkulturer. / ANA-broth is developed to enrich both aerobic and anaerobic bacteria by incubation in an aerobic environment. The broth contains antioxidants that protect anaerobic bacteria from reactive oxygen species and make it possible for anaerobic bacteria to grow in an oxygenated environment. The purpose of the study was to evaluate the ANA-broth and see if the broth could replace the two broths that are being used clinically today.  Eleven bacterial species and four mixed floras were used to evaluate the ANA-broth. One microliter (µl) inoculum with about 100 colony forming units was used to inoculate ANA-broth, Brain Heart Infusion (BHI) broth and Fastidious Anaerobe Broth (FAB), respectively. Samples from the broths were then used to inoculate agar plates after two, four and seven days of incubation, to evaluate the difference in enrichment effectiveness at different incubation times in relation to the clinically used broths. The ANA-broth was also evaluated by using clinical samples from patients. The findings from the ANA-broth were compared with the findings from the BHI-broth and FAB. The growth in the ANA-broth was quicker compared to the BHI-broth and FAB. Some bacterial species grew but became non-viable in the ANA-broth after a longer period of incubation. Bacterial species within mixed floras were not able to grow in coexistence in the ANA-broth. One bacterial species took over and competed out the residual bacteria. This phenomenon did not occur in FAB. If the ANA-broth could replace the broths now used clinically then that would be advantageous both in terms of practicality, economy and from a sustainability perspective. Though, the ANA-broth is not optimal for clinical use considering that only one bacterial species within a mixed flora was able to grow. As the broth has greater potential for cultures containing only one bacterial species it could possibly be suited for orthopaedic Kamme-Lindberg cultivation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20240号 / 医博第4199号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 安達 泰治, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM