Dissertations / Theses on the topic 'Volatile Biomarkers'

Bladder cancer is an important global disease. The gold standard for diagnosis is cystoscopy and biopsy; both are invasive and require highly trained personnel. In a majority of cases, treated patients are followed up by frequent cystoscopies lasting several years. The discovery of biomarkers indicating which individuals should proceed to cystoscopy would be an important addition to bladder cancer management. The odour of urine is produced by volatile organic compounds, (VOCs), detectable by gas chromatography and mass spectrometry (GC-MS). An analysis of the VOCs in urine from various groups of individuals including patients with bladder cancer is undertaken in search of possible discriminating compounds, which could be harnessed in future as a potential screening tool or adjunct in bladder cancer management. Methods First void urine was obtained from 64 patients with new non-muscle invasive bladder cancer, 71 cancer free patients with haematuria and 51 asymptomatic volunteers. After equilibration, the headspace above these pH adjusted urine samples was extracted for 20minutes, using a carboxen / polydimethylsiloxane solid phase micro-extraction fibre (SPME). This was followed by desorption and VOC identification by GC-MS. Results Urine headspace VOCs under acidic conditions, (pH of modified urine 2), were found to be discriminating. Identified compounds were analyzed using forward stepwise discriminant analysis: 9 VOCs when used together, gave 84.7% correct classification of samples (Haematuria control v Bladder cancer) with no change on cross validation of results. The calculated sensitivity and specificity of this model is 76.6% and 92.9% respectively, with Positive predictive value of 90.7% and Negative predictive value of 92.9%. These results are comparable, and in some cases better than those obtained using commercially available urinary bladder cancer biomarkers. Conclusion Volatile organic compounds in urinary head space change with the development of bladder cancer. Urinary VOCs are exciting novel potential biomarkers in the detection of bladder cancer.

Early identification of malignancy has the ability to improve long term morbidity and mortality. This is certainly the case for colorectal cancer and hepatocellular carcinoma (HCC), two of the commonest gastrointestinal malignancies. Both diseases are subject to screening programmes. The UK national Bowel Cancer Screening Programme (BCSP) has been shown to significantly improve 5 year cancer survival by identifying colorectal cancer at an earlier stage, but also by identifying advanced, pre-malignant adenomatous disease and removing it. Screening of cirrhotic patients at risk of HCC is a bit more controversial but is recommended by national and international bodies, with some evidence to suggest an improved survival from cancer associated death. Biomarkers are currently employed in the diagnosis and patients selection process for these screening programmes, in particular alpha fetaprotein (AFP) for HCC. The sensitivity and specificity, and thus the valid application of these biomarkers has been brought in question, in the case of alpha feta protein, leading to its removal from screening protocols. Volatile organic compounds have been proposed as biomarkers for various disease processes, including gastrointestinal malignancies. They may therefore have an application in disease screening and/or monitoring. The work presented here explores volatile organic compounds (VOCs) emitted from stool and urine, in order to detect disease specific differences that may be utilised as biomarkers for colorectal cancer and hepatocellular carcinoma. It also explores the driver-passenger model of colorectal cancer and biological plausibility via the detection of volatile organic compounds emitted from cultures of Fusobacterium nucleatum and Campylobacter showae. Finally, it assesses the utility of the stool based tM2-PK assay as a marker of colorectal neoplasia in a novel secondary care cohort. Solid phase micro-extraction of headspace gas followed by gas chromatography mass spectrometry was used to isolate and identify candidate volatile organic compounds. Statistical analysis, including logicistic regression modelling and 10 fold cross validation, were applied to assess biomarker utility. Analysis of VOCs emitted from stool was able to differentiate those with higher risk neoplastic disease with the greatest confidence, including established colorectal cancer. When comparing those with cancer to no neoplasia isopropyl alcohol was significantly more abundant in the colorectal cancer samples (p= < 0.0001, q=0.004), producing an AUROC curve of 0.76. When isopropyl alcohol is combined with butanoic acid, 3-methyl, the AUROC was 0.82, sensitivity 87.9% (95% CI 0.87-0.99) and specificity 84.6% (95% CI 0.65-1.0). Further logisitic regression analysis of VOC presence identified a three VOC panel (isopropyl alcohol, 2-Hexanone and butanoic acid,3-methyl-,ethyl ester) with an AUROC of 0.86: a person being 6 times more likely to have cancer if all 3 VOCs were present in their stool(p= < 0.0001). A number of the VOCs identified as important in those with colorectal neoplasia were also identified in the assessment of Fusobacterium nucleatum and Campylobacter showae, namely butanoic acid based compounds and isopropyl alcohol. VOCs emitted from urine failed to demonstrate any candidate biomarkers for colorectal neoplasia. With regards VOCs emitted from urine as biomarkers for HCC, AUROC comparing all those with and without HCC was 0.76 (Sensitivity 65% [95% CI 0.61-0.69] Specificity 74% [95% CI 0.69-0.78]). When assessing treatment naive HCC patients, 3 compounds were found to have significantly different abundance (p=<0.01), when combined and modelled these VOCs demonstrated a superior AUROC of 0.81 (Sensitivity 77% [95% CI 0.71-0.83], Specificity 75% [95% CI 0.71-0.79]). Of this treatment naive group patients defined by Barcelona Clinic Liver Cancer (BCLC) staging as having early disease and therefore potentially curative, demonstrated an AUROC of 0.82. VOC emitted from stool and urine show a clear ability to act as biomarkers for the diagnosis of colorectal cancer and hepatocellular carcinoma respectively.

Mestrado em Biologia Aplicada - Microbiologia Clínica e Ambiental
Fungal infections have greatly increased in risk populations, namely in immunocompromised patients, probabily because the diagnosis of fungal infections is delayed. Microbial metabolomics arises as a powerful feature screening the metabolites produced by microorganisms. It provides information regarding the state of biological organisms which can be used as a diagnostic tool for diseases through fungal metabolites pattern. Thus, this research aimed to in-depth study of the Aspergillus niger exometabolome, in order to establish a targeted metabolomic pattern that characterizes A. niger. A methodology based on headspace-solid phase microextraction combined with comprehensive two-dimensional gas chromatography coupled to mass spectrometry with a high resolution time of flight analyser (HS-SPME/GC×GC-ToFMS) was used. A. niger exometabolome was analysed in different growth conditions: temperature (25 and 37 °C), incubation time (3 and 5 days), and culture medium (solid and liquid medium). A. niger exometabolome included 430 metabolites, distributed over several chemical families, being the major ones alcohols, aldehydes, esters, hydrocarbons, ketones and terpenoids. Differences among volatile metabolites produced under different growth conditions were observed, being the major relative abundance determined for 5 days of growth, at 25 °C, using solid medium. These results indicated the high complexity of A. niger exometabolome. A subset of 44 metabolites, which were present in all previously tested growth conditions, was defined as the A. niger targeted metabolomic pattern. This pattern may be used in detection of fungal infections by this specie and be further exploited to fungal infections diagnosis. Furthermore, this subset of metabolites was compared with samples of Candida albicans (yeast) and Penicillium chrysogenum (filamentous fungi), and Partial Least Squares Discriminant Analysis (PLS-DA) was applied. The results clearly showed that this metabolites subset allowed the distinction between these microorganisms. In order to validate the PLS-DA model, permutation test was applied, and a statistically significant model for 44 metabolites was obtained with a predictive Q2 capability of 0.70 for A. niger. When the subset of compounds were reduced to 16 (obtained by Variables Importance in Projection (VIP) parameter), the obtained model had a predictive Q2 capability of 0.86 for A. niger, which was significantly higher, being more robust than the previous. The decrease of 44 to 16 metabolites, reduced the require analysis time and the conditions used were similar to the conditions used in clinical context, (solid medium, at 25 °C and ca. 1 week). However, in this study was possible to reduce the time for 3 days. In conclusion, these 44 volatile molecular biomarkers could be useful for diagnosis of fungal infections, and they can even be further exploited in clinical context.
As infeções fúngicas têm aumentado bastante em populações de risco, nomeadamente em pacientes imunocomprometidos, provavelmente devido a atrasos no diagnóstico das infeções fúngicas. A metabolómica microbiana surge como um poderoso recurso de triagem dos metabolitos produzidos por microrganismos. Esta fornece informações sobre o estado de organismos biológicos, que podem ser usados como uma ferramenta de diagnóstico para infeções fúngicas através de um padrão de metabolitos fúngicos. Assim, este trabalho teve como objetivo estudar em profundidade o exometaboloma de Aspergillus niger, a fim de estabelecer um padrão metobolómico alvo que caracterize o A. niger. Foi usada uma metodologia baseada em microextração em fase sólida no espaço de cabeça combinada com cromatografia de gás bidimensional abrangente acoplada a espectrometria de massa por tempo de voo (HS-SPME / GC×GC-ToFMS). O exometaboloma de A. niger foi analisado em diferentes condições de crescimento: temperatura (25 e 37 °C), tempo de incubação (3 e 5 dias) e meio de cultura (meio sólido e líquido). O exometaboloma do A. niger incluiu 430 metabolitos, distribuídos em várias famílias químicas, sendo os mais importantes os álcoois, aldeídos, ésteres, cetonas, hidrocarbonetos e terpenos. Observaram-se diferenças entre os metabolitos voláteis produzidos em diferentes condições de crescimento, sendo a maior abundância relativa determinada para os 5 dias de crescimento, a 25 °C, utilizando meio sólido. Estes resultados indicaram a alta complexidade do exometaboloma do A. niger. Um subconjunto de 44 metabolitos, que estavam presentes em todas as condições de crescimento testadas, foi definido como um padrão metabolómico alvo para o A. niger. Este padrão pode ser usado na deteção de infeções fúngicas por esta espécie e ser futuramente explorado para diagnóstico de infeções fúngicas. Além disso, este subconjunto de metabolitos foi comparado com amostras de Candida albicans (levedura) e Penicillium chrysogenum (fungo filamentoso), e a análise discriminante com método dos mínimos quadrados parciais (PLS-DA) foi aplicada. Os resultados mostraram claramente que este subconjunto de metabolitos permitiu distinguir estes microrganismos. Para validar o modelo do PLS-DA, o teste das permutações foi aplicado, e um modelo estatísticamente significante para os 44 metabolitos foi obtido com uma capacidade preditiva Q2 de 0.70 para o A. niger. Quando o subconjunto de compostos foi reduzido para 16 (obtidos pelo parâmetro Importância da Variável na Projeção (VIP)), o modelo obtido teve uma capacidade preditiva Q2 de 0.86 para o A. niger, que foi significantemente superior, sendo mais robusto que o anterior. A diminuição de 44 para 16 metabolitos, reduziu o tempo de análise necessário e as condições utilizadas foram semelhantes às condições utilizadas em contexto clínico, (meio sólido e 25 °C e aproximadamente 1 semana). No entanto, neste estudo, foi possível reduzir o tempo para 3 dias. Em conclusão, estes 44 biomarcadores moleculares voláteis poderão ser úteis para o diagnóstico de infeções fúngicas, e podem ser explorados em contexto clínico.

Introduction: Inflammatory bowel disease (IBD) is a chronic, remitting and relapsing disease of the gastrointestinal tract, the aetiology of which remains elusive. Diagnosis of IBD requires complex and invasive procedures. Endoscopy and histology of biopsy specimens remain the current "gold standard" for detecting and quantifying bowel inflammation. Due to similarity of symptoms of IBD and irritable bowel syndrome (IBS), invasive procedures are used excessively to differentiate the two conditions which have significant safety and economical implications. Currently available non-invasive serological markers (such as CRP and ESR) are unreliable due to their poor correlation with clinical and endoscopic findings. Volatile organic compounds (VOCs) are chemicals, which may be emitted from faeces, and other bodily fluids, and are responsible for their characteristic odours. Historically, changes in the smell of human excreta may be assigned to various illnesses; so understanding these changes in the smell by identifying its volatile signature can be of diagnostic value. Objective: To evaluate the faecal VOCs in the diagnosis of IBD and compare them with those from the patients with I BS and healthy controls Method: We studied samples from 356 individuals, 30 with diarrhoea predominant IBS, 217 with IBD (Crohn's disease=117, ulcerative colitis=100), and 109 healthy individuals. Faecal volatile were extracted using SPME (solid phase micro-extraction) technique and were analysed by GC-MS (gas chromatography-mass spectrometery). VOCs were identified using the NIST (National Institute of Science and Technology) library and manual visualization using the fragmentation pattern when appropriate. Results: From the 350 VOCs identified in these experiments, univariate analysis was used to identify those VOCs, which were statistically significant (p< 0.05) in discriminating between the groups. A forward stepwise discriminant function analysis was used to develop a predictive model, which showed significant value in separating active IBD cases from those with disease in remission, IBS and healthy controls (p<0.05). The model was able to differentiate active CD from inactive CD and from healthy controls. Similarly the model was able to differentiate active UC from inactive UC and healthy controls. The model also showed statistically significant value in discriminating IBS from active IBD and healthy controls. On cross-validation, the model remained stable: 90% of patients were correctly diagnosed. This model has overall sensitivity of 90% and specificity 80%. Conclusion: These results show that VOCs analysis has the potential to provide a non- invasive means of diagnosing IBD, monitoring the disease activity and to differentiate it from IBS. This technique is fast and convenient and opens up a promising area for use as a non- invasive diagnostic tool with advantages of providing a much-needed, reliable, real-time and point of care diagnosis and monitoring of various gastrointestinal disorders.

Exhaled breath is a rich and complex matrix containing many hundreds of compounds. Every breath offers the potential of a non-invasive measurement of the biochemical processes occurring in the human body and it is this notion that has led to the application of breath analysis for the detection of disease. With the majority of research in the field being focused on the detection of biomarkers, little has been presented on how the seemingly homeostatic matrix of breath varies during the course of normal life events. The research in this thesis describes how a subject's emotional state, physical state, and daily activities can alter the composition of exhaled breath.

Volatile organic compounds (VOCs) and lactoferrin have been studied as potential faecal biomarkers for ISO. Changes in VOC patterns in stool reflect both physiological and pathological processes within the gut. Lactoferrin, a protein present in neutrophils can be quantified in stool. This thesis studies the potential and efficacy of both urine and faecal VOCs and the efficacy of lactoferrin in differentiating ISO from ISS and healthy controls. CD was best differentiated from those with IBS by presence or absence of urine VOCs when compared to UC. Active UC versus IBS comparison, using the presence or absence of urine VOCs, yielded a higher sensitivity and specificity than when inactive UC was compared with ISS (Inactive UC vs ISS: 54.2%, 30.4% and Active uC vs IBS: 86%, 77% respectively). Active and inactive UC comparisons with IBS groups, based on peak area, resulted in significant urine VOCs with high sensitivity but moderate specificity (Inactive UC vs ISS: 83%, 52% and Active UC vs ISS: 100%, 78% respectively). Moderate sensitivity and specificity were recorded for faecal VOCs with comparisons based on peak area measures for inactive CD versus IBS (67%; 77%) and inactive UC versus ISS (100%; 59%). Faecal vacs in the active CD and ISS comparison recorded the be.st sensitivity and specificity (92%; 74%). Faecal vac profiles were better than urine for the group separation of active disease. Correct classification was much improved with peak area measure than the presence or absence of volatiles: Lactoferrin levels were considerably higher in 180 patients than those with IBS, while in IBS and healthy controls were similar. Lactoferrin levels were lower in inactive UC when compared with active UC. No such statistical difference was noted when active and inactive CD were compared. Lactoferrin levels in inactive IBD groups were higher compared to the healthy controls. Faecal and urinary biomarkers have potential to enhance management of patients with IBS and IBD.

Lung cancer is a leading cause of death from cancer worldwide. An early diagnosis and appropriate treatment are crucial in reducing mortality among people suffering from the disease. Therefore, one of the main focuses of lung cancer studies is on advances in its early detection. One of the most promising is the analysis of volatile organic compounds (VOCs). VOCs are a diverse group of carbon-based chemicals that are present in exhaled breath and biofluids, and may be collected from the headspace of these matrices. Different patterns of VOCs have been correlated with various diseases, cancer among them. Studies have shown that various cancer cells in vitro produce or consume specific VOCs that can serve as potential biomarkers to differentiate them from non-cancer cells. The present study aimed at the detection, identification and semi-quantification of VOCs released or consumed by the adenocarcinoma human alveolar A549 cell line. For this purpose, gas chromatography with mass spectrometric detection was combined with two pre-concentration techniques: monolithic material sorptive extraction (MMSE) or extraction of thermal desorption (TD) sorbent tubes with an Easy-VOC^TM pump as a sample loading tool. MMSE is a new technique for the extraction of VOCs from various samples and it is used for the first time for the analysis of VOCs from cell culture medium. TD-GC-MS is a popular technique for VOCs analysis and it has been used for the first time here with Bio-VOC^TM pump in the studies of VOCs in vitro. The project also aimed at the comparison of the A549 VOC level trends to the trends of the normal human lung fibroblasts NHLF (MMSE experiment) and normal human bronchial BEAS-2B cells (TD experiment). In addition, the VOC patterns between the growing and confluent cells of the same cell line were compared for the first time. In the MMSE experiment, seven VOCs were produced and 14 VOCs metabolised exclusively by the cancer cells. Among the released compounds were methylated hydrocarbons (2,4-dimethyl-1-heptene; 4-methylundecane; 2,3,6,7-tetramethyloctane 2,3,5-trimethylhexane and 2,3,5-trimethyldecane) and alcohols (cyclohexanol and 3- heptanol). The metabolised analytes were alcohols (4-decanol; 6-dodecanol; 2- ethylhexanol; 1-octanol), aldehydes (dodecanal; tetradecanal), ketones (acetophenone; cyclohexanone; 2-tetradecanone), phenols (phenol and 2-nitrophenol); an ether (2-methoxydiphenylmethane), an ester (pentanoic acid, 2,4-dimethyl-3-oxo-, methyl ester) and a hydrocarbon (tetradecane). 2,4-Dimethylheptane; 2,6-di-tert-butyl-1,4- benzoquinone; 1-phenylethanol and 2-pentadecanone were released by both A549 and NHLF cell lines. The cancer cells were observed, however, to emit the VOCs at a higher level than the fibroblasts. Benzaldehyde; 2-ethylhexanol; hexanal and 1-nonanol were found to be consumed by both the cancer and NHLF cells, however, at a greater rate by the former. In the TD experiment, 2,3,5-trimethylhexane and tert-butanol were produced exclusively by the A549 cells, while ethyl acetate solely by the BEAS-2B cells. Acetophenone, benzaldehyde and 2-methylbutanal were metabolised and acetone was produced at higher levels by the cancer cells than by the BEAS-2B cells. The possible use of the analysed VOCs as potential biomarkers of lung cancer is discussed, along with the suggestion and discussion of the possible metabolic pathways leading to the uptake and release of these VOCs by the analysed cells. Also the discussion of poor correlation between different in vitro studies, as well as between in vivo and in vitro studies of VOCs as potential biomarkers of cancer, is undertaken.

The work presented in this thesis was initiated in order to develop a non-invasive real-time gas phase analytical technique, based on selected ion flow tube mass spectrometry (SIFT-MS), for monitoring the progression of human cell cultures and the detection of microbial contamination in such cultures by monitoring and quantifying the emitted volatile compounds. Fundamental SIFT experiments were performed to characterise the reactions of the SIFT-MS precursor ions (H3O+, NO+ and O2 +●) with several volatile compounds of potential value to biological research; a necessity for their quantification. The work has resulted in new methods for the quantification acetaldehyde and CO2 in gaseous samples. The compounds present in the headspace of sealed cultures of six human cell types were analysed by SIFT-MS, the key finding being their consumption of the toxic volatile compound acetaldehyde from the media. Further experiments involved the addition of the enzyme aldehyde dehydrogenase inhibitors diethylaminobenzaldehyde and disulfiram to cultures of hepG2 (hepatocellular carcinoma) cells, when it was observed that consumption of acetaldehyde from the cultures/headspace was reduced, and in some cases, acetaldehyde was even produced due to the actions of the cellular alcohol dehydrogenase enzyme. Furthermore, the solvent dimethylsulphoxide was reduced to imethylsulphide by the cells, which is known to occur via the enzyme methionine sulphoxide reductase. This process was retarded by the ALDH inhibitors. The use of SIFT-MS for the detection of microbial infection in mammalian cell cultures was also explored. The volatile compounds emitted by E. coli (strain JM109), into the gas phase above two different culture media, were analysed using SIFT-MS. Further, the progression of a culture of this bacterium was monitored continuously over a 4-hour period. The findings of this research were then applied to the study of human cell cultures intentionally infected by E. coli bacterium, including cultures contained in a 1L bioreactor.

Human scent has been the focal point of diverse scientific interests and research initiatives for the past several years. The knowledge gained about its composition has favored the advancement of multiple disciplines, and promoted the development of a wide variety of applications. Among these applications is the use of human scent as a resource for Forensic investigations, where scent profiles are often used as evidence to associate individuals to the scene of a crime. The characteristic nature of individual human scent has enabled this type of evidence to be used as a biometric tool for the differentiation of subjects. Nevertheless, the present study discusses a new perspective towards human scent's role and application in Forensic investigations. The foundation of this new perspective consists of employing human scent’s biometric quality to classify individuals using common traits. In this research study, underarm and hand odor samples were collected from Caucasian, Hispanic and East Asian individuals, of both genders. Subjects were also organized into 3 different age groups: 18-30, 35-50 and 55+ years. Headspace Solid Phase Micro-extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC-MS) was used to create individual scent profiles for the evaluation of subject classification by age, gender and race/ethnicity. Individual classification was assessed through the identification of qualitative and quantitative patterns in the volatile organic compound (VOC) constituents that characterize human scent. Principal Component and Linear Discriminant analyses of the collected scent profiles, led to the identification and validation of characteristic VOC marker combinations for age, gender and race/ethnicity. Statistical analysis facilitated group classification and differentiation on the basis of these traits. Moreover, this study also evaluated the use of solvent extraction as a complementary technique to HS-SPME for human scent analysis. Findings from this assessment revealed that the simultaneous consideration of data from both extraction techniques favors an enhancement of the classification of subjects by means of human scent. The discoveries achieved in this study represent a significant step for human scent as a forensic tool. The outcome of this research has cleared a new path for further human scent investigation, and highlighted its further relevance to forensic applications.

There is limited scientific knowledge on the composition of human odor from different biological specimens and the effect that physiological and psychological health conditions could have on them. There is currently no direct comparison of the volatile organic compounds (VOCs) emanating from different biological specimens collected from healthy individuals as well as individuals with certain diagnosed medical conditions. Therefore the question of matching VOCs present in human odor across various biological samples and across health statuses remains unanswered. The main purpose of this study was to use analytical instrumental methods to compare the VOCs from different biological specimens from the same individual and to compare the populations evaluated in this project. The goals of this study were to utilize headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC/MS) to evaluate its potential for profiling VOCs from specimens collected using standard forensic and medical methods over three different populations: healthy group with no diagnosed medical or psychological condition, one group with diagnosed type 2 diabetes, and one group with diagnosed major depressive disorder. The pre-treatment methods of collection materials developed for the study allowed for the removal of targeted VOCs from the sampling kits prior to sampling, extraction and analysis. Optimized SPME-GC/MS conditions has been demonstrated to be capable of sampling, identifying and differentiating the VOCs present in the five biological specimens collected from different subjects and yielded excellent detection limits for the VOCs from buccal swab, breath, blood, and urine with average limits of detection of 8.3 ng. Visual, Spearman rank correlation, and PCA comparisons of the most abundant and frequent VOCs from each specimen demonstrated that each specimen has characteristic VOCs that allow them to be differentiated for both healthy and diseased individuals. Preliminary comparisons of VOC profiles of healthy individuals, patients with type 2 diabetes, and patients with major depressive disorder revealed compounds that could be used as potential biomarkers to differentiate between healthy and diseased individuals. Finally, a human biological specimen compound database has been created compiling the volatile compounds present in the emanations of human hand odor, oral fluids, breath, blood, and urine.

People spend most of their time indoors and mostly in the dwelling. It is therefore important to investigate associations between indoor exposure in dwellings and health. Symptoms that may be related to the indoor environment are sometimes referred to as the "sick building syndrome" (SBS). SBS involves symptoms such as eye, skin and upper airway irritation, headache and fatigue. Three longitudinal studies and one prevalence study on personal and environmental risk factors for SBS in adults were performed. The prevalence study included measurements of indoor exposures in the dwellings. The longitudinal studies, with 8-10 years follow-up time, showed that smoking and indoor paint emissions were risk factors for SBS. Moreover, building dampness and moulds in dwellings were risk factors for onset (incidence) of general symptoms, skin symptoms and mucosal symptoms. In addition subjects living in damp dwellings have a lower remission of general symptoms and skin symptoms. Hay fever was a risk factor for onset of skin symptoms and mucosal symptoms, and asthma was a risk factor for onset of general and mucosal symptoms. Biomarkers of allergy and inflammation (bronchial reactivity, total IgE, ECP and eosinophil count) were predictors of onset of SBS symptoms, in particular mucosal symptoms. In the prevalence study, any SBS-symptom was associated with some individual volatile organic compounds of possible microbial origin (MVOC) e.g. 2-pentanol, 2-hexanon, 2-pentylfuran and 1-octen-3ol. Moreover, there were associations between indoor levels of formaldehyde and the plasticizer Texanol and any SBS. The result from the study indicates that individual MVOC are better indicators of SBS than the total value of MVOC. A final conclusion is that smoking, dampness and moulds and emissions from indoor painting may increase the onset of SBS. The indoor environment in dwellings over time has improved, but there is still a need for further improvements of the indoor environment in dwellings. More longitudinal SBS studies are needed.

Studies have shown that some canines have the ability to predict seizures in people with epilepsy, and that canines can be trained to recognize changes in humans before an epileptic seizure and make these predictions. It is not known with any certainty to what the canines are alerting. However, canines’ exceptional sense of smell and their ability to discriminate human scent is well established. Therefore, it is possible that the canines could be responding to an olfactory cue, such as the release of some volatile organic compounds (VOCs) prior to the onset of a seizure. Individuals release a wide array of VOCs, both odorous and non-odorous, from their bodies. The odorous VOCs collectively make up human scent and a number of these VOCs have been identified as biomarkers of different diseases. Evidence suggests that canines can perceive these biomarkers, leading to early detection of underlying physical ailments before individuals are aware of their own symptoms. The main purpose of this study was to use headspace solid phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS) to analyze hand odor, saliva and breath samples from epileptic with and without seizure activity to determine if the human scent profiles resulting from a seizure event differs from the scent profiles in the absence of seizure activity. the HS-SPME-GC-MS method was also used to analyze and compare hand odor, saliva and breath samples of healthy individuals and epilepsy patients to determine if the profiles can be differentiated. Comparison of the VOCs in each specimen from healthy individuals and epileptic patients revealed compounds that could be used as potential biomarkers to differentiate between healthy and epileptic individuals. Comparison of the VOCs in each specimen from epileptic patients with and without seizure activity revealed compounds that could be used as potential biomarkers for epileptic seizures. Finally, canine trials were used to verify that these compounds are indeed biomarkers.

Ein nicht-invasiver Atemtest zur Lungenkrebsdetektion setzt Kenntnis über lungenkrebsspezifische Substanzen voraus. Die Identifizierung von Lungenkrebsbiomarkern in der Atemluft war das Ziel dieser Arbeit. Leichtflüchtige organische Substanzen (VOC) wurden als Zielkomponenten ausgewählt. Für die VOC-Analytik wurde eine SPME-GC-MS-Methode entwickelt und sowohl auf Modellsysteme als auch auf Realproben angewendet. Drei Lungenadenokarzinomzelllinien wurden in-vitro untersucht. Die VOC-Analyse wurde mit drei verschiedenen Probenahmestrategien durchgeführt und es war ein deutlicher Hintergrundeinfluss der eingesetzten Einwegzellkulturflaschen auf das analysierte VOC-Profil feststellbar. Trotzdem konnten signifikante Unterschiede zwischen Tumorzellen und zellfreien Nährmedien beobachtet werden: 1-Propanol wurde von den Zellen produziert, während der Gehalt einiger Aldehyde sank. Die eingeschränkte Ähnlichkeit des gewählten Zellkulturmodells mit realen Atemluftproben bedingt eine geringe Eignung dieser Ergebnisse für die Biomarkerableitung. Ein Gasmodell auf Basis angefeuchteter, synthetischer Luft wurde als Grundlage für die qualitätsgesicherte, quantitative VOC-Analyse der realen Atemluftproben konzipiert. Diese Modellluft wurde mit 24 Zielsubstanzen (Alkane, Aromaten, sauerstoffhaltige Spezies) sowie 3 Matrix-VOC mit starker Dominanz in den Atemluftproben (Isopren, Aceton, 2-Propanol) angereichert. In Kooperation mit zwei Berliner Kliniken wurden 37 Atemluftproben von Lungenkrebspatienten und 23 Proben von Gesunden gesammelt. Die Anwendung von 1-Butanol als univariater Marker erlaubt eine Erkennung von Lungenkrebs mit einer Sensitivität von 92% und Spezifität von 78%. Durch lineare Diskriminanzanalyse konnte ein Set aus 4 VOC (1-Butanol, 2-Butanon, 2-Pentanon, n-Hexanal) ermittelt werden, welches ebenfalls eine Sensitivität von 92% und mit 87% eine höhere Spezifität aufwies. Gegebenenfalls handelt es sich bei diesen Substanzen jedoch nur um allgemeine Krankheitsmarker.
A non-invasive breath test for lung cancer detection would be favorable but knowledge on lung cancer specific substances is required. This work aims at the identification of potential lung cancer biomarkers in breath. Volatile organic compounds (VOC) were chosen as targets and a SPME-GC-MS method was developed to analyze the VOC profiles of model systems and real samples. Three lung adenocarcinoma cell lines were investigated in-vitro. The VOC analysis, carried out with 3 different sampling strategies, was influenced by the VOC background of the used disposable culture vessels. Changes in the VOC profiles of cell lines compared to cell-free culture media were obvious: 1-propanol was released by the tumor cells whereas the content of some aldehydes was diminished. The similarity of this model system with real breath samples of lung cancer patients was seen to be insignificant. Consequently, these cell cultures were not suitable for biomarker identification. A gaseous model consisting of humidified synthetic air was developed. It was fortified with 24 target VOC (alkanes, aromatics and oxygenated species) as well as 3 matrix compounds (isoprene, acetone and 2-propanol) dominating patients’ VOC profiles in breath. This model was used for the quality assured quantitative VOC analysis in real breath samples. In cooperation with two hospitals 37 single mixed expiratory breath samples from lung cancer patients and 23 from healthy controls were collected. Applying 1-butanol as an univariate biomarker patients and controls were discriminated with a sensitivity of 92% and a specificity of 78%. Linear discriminant analysis displayed a set of 4 VOC (1-butanol, 2-butanone, 2-pentanone, n-hexanal) with similar sensitivity but higher specificity of 87%. However, these potential biomarkers might rather be a consequence of illness in general.

Les égoutiers sont exposés à une grande variété de mélanges de produits chimiques beaucoup ce sont des substances génotoxiques ou cancérigènes. Le but de cette étude transversale était d'explorer des biomarqueurs intégrés d’exposition et des effets précoces chez les égoutiers afin de mieux apprécier leur risque cancérogène (article I-protocole). L'article II expose l'étape de validation préalable effectuée sur la lignée cellulaire pour nos objectifs. Avec un recueil en 10 mois et sur une base hebdomadaire, nous avons, a) évalué l'exposition personnelle des égoutiers (n=34) et administratifs (n=30) (groupe témoin) à des hydrocarbures aromatiques polycycliques (HAP) et composés organiques volatils (COV) dans l'air des lieux de travail, b) évalué la génotoxicité de leurs extraits organiques urinaire par des tests in vitro : tests des comète et des micronoyaux sur les cellules Hep G2, c) évalué la réponse à un stress oxydatif de l'ADN par la mesure du 8 oxo 2' deoxyguanosine urinaire (8 oxodG) dans les urines de 24h (Article III). Les tests ont montré la présence de plus de génotoxiques dans les urines des égoutiers (P <0,001). La moyenne du 8 oxodG urinaire chez les égoutiers était plus élevée (non significatif, P = 0,28) que les administratifs et ses valeurs moyennes ont été associées (P = 0,04) avec les années de travail dans le système des égouts. Les concentrations des HAP et COV dans l'atmosphère professionnelle des égoutiers étaient élevées par rapport à celles des administratifs (P <0,01) et il a été montré une augmentation de risque du cancer en utilisant les facteurs d'équivalence de toxicité et de risque unitaire de cancer. En conclusion, les biomarqueurs intégrés urinaire d’exposition et non spécifiques montrent que les égoutiers sont professionnellement exposés aux mélanges des substances génotoxiques. Il n’est pas possible de relier nos résultats avec l’accroissement du risque cancérogène chez les égoutiers
Sewage workers are exposed to a wide variety of mixtures of chemicals many were shown to be genotoxicants or carcinogens. The aim of this cross sectional study was to explore integrated biomarkers of exposure and of early effects among sewage workers in order to better assess their carcinogenic risk (Paper I-protocol). Paper II, describes the validation step performed prior to choose the cellular line that meet our objectives. Over 10 months and on weekly basis, we, a) assessed the personal exposure of sewage (n=34) and office (n=30) workers (control group) to polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs) in the air of workplaces, b) evaluated the genotoxicity of organic urine extracts by in vitro comet and micronucleus assays on Hep G2 cells, c) assessed the DNA oxidative stress by the 24h urinary 8 oxo 2' deoxyguanosine (8 oxodG) (Paper III). The tests showed greater genotoxicity in the urine extracts of sewage workers (P<0.001). The 24hr urinary 8 oxodG mean level in sewage workers was higher (nonsignificant, P=0.28) than office workers and its mean values were associated (P=0.04) with working years in sewage system. Workplace air concentrations of PAHs and VOCs were elevated in sewage compared to office workplaces (P<0.01) and resulted in an increased lifetime cancer risk using toxicity equivalent factors and inhalation unit risk of cancer. In conclusion, the applied integrated and non-specific urinary biomarkers show that sewage workers experience exposure to multiple genotoxicants at the workplace. It is not possible to simply link our results with the increased cancer risk in sewage workers

L’objectif principal de ce travail de thèse est de contribuer au développement d’un outil de diagnostic miniaturisé permettant d’identifier et de quantifier une combinaison de composés organiques volatils (COVs) présents dans l’haleine et qui sont considérés comme des marqueurs chimiques du cancer du poumon. Les principaux verrous scientifiques de ce projet sont liés aux très faibles concentrations de ces composés cibles (de l’ordre de quelques ppb) et également à la présence de nombreux autres composés chimiques qui sont naturellement présents dans l’haleine. La voie de développement proposée dans ce projet est d’utiliser un micro-capteur résistif à base de SnO2 associé à un micro-préconcentrateur et une micro-colonne chromatographique afin d’aboutir à un dispositif sélectif et présentant des limites de détection très basses. Dans un premier temps, plusieurs adsorbants ont été caractérisés en vue d’être utilisés dans le micro-préconcentrateur afin de concentrer les marqueurs du cancer du poumon. Les résultats ont permis de sélectionner deux types de zéolites (DaY et NaY) ainsi que des microsphères de carbone W5. Par la suite, les unités de préconcentration et de séparation des COVs ont été développées en s’appuyant sur la technologie silicium/verre disponible en salle blanche. La dernière étape de cette étude a concerné l’évaluation des performances du système d’analyse alors assimilable à un micro-chromatographe en phase gazeuse. Après avoir déterminé les conditions optimales d’élution et de préconcentration des COVs, le système miniaturisé a permis d’analyser une haleine artificielle constituée de trois COVs présents à des concentrations proches des celles mesurées dans l’haleine (toluène (24 ppb), propanol (21 ppb) et o-xylène (5 ppb)) même en présence des interférents majeurs de l’haleine (vapeur d’eau et dioxyde de carbone)
The main goal of this research is to develop a miniaturized diagnostic equipment in order to identify some volatile organic compounds present in exhaled breath and referred as lung cancer biomarkers. The main scientific and technical obstacles of this project are linked to the very low concentrations of these chemical compounds and the presence of high concentrations of H2O and CO2 naturally present in exhaled breath. To address these issues, we suggest to use a SnO2-based gas sensor combined with a micro-preconcentrator and a chromatographic micro-column in order to engineer a low detection limit system. First, some specific adsorbents have been characterized with a view to concentrate chemical biomarkers trough the micro-preconcentrator. In accordance with research findings, two types of zeolites (DaY and NaY) and one type carbonaceous microspheres (W5) have been selected. Then micro-preconcentrators and chromatographic micro-columns have been developed on silicon wafers by using clean room facilities. The last step of this study was to evaluate the performances of the analytical device. After determining optimal elution and pre-concentration conditions of each VOCs, the miniaturized system achieved the analysis of an artificial breath constituted of toluene (24 ppb), 1-propanol (21 ppb) and o-xylene in presence of high concentrations of water vapors and carbon dioxide

Malgré la prise de conscience écologique et sanitaire, la consommation mondiale de pesticides est en augmentation. Étant donné que ces produits chimiques présentent de nombreux effets néfastes sur la santé humaine et l'environnement, des mesures doivent être prises afin de limiter leurs effets. Les produits de biocontrôle sont proposés comme une solution alternative aux produits synthétiques. En effet, ces « biopesticides » sont présumés être moins nocifs et relativement moins persistants. Toutefois, cet a priori doit être examiné et une évaluation stricte des risques de ces nouvelles substances doit être envisagée.Le développement de solutions de biocontrôle passe d'abord par les protocoles proposés pour étudier leur activité, leur devenir et leur impact environnemental. Actuellement, le temps de demi-vie (t½) est utilisé pour évaluer le devenir environnemental des pesticides synthétiques. Cependant, l'approche t½ ne donne que des informations sur la persistance des pesticides dans l'environnement, mais aucune indication concernant la formation de produits de dégradation ou son impact sur la biodiversité n'est apportée. De plus, les produits de biocontrôle sont des mélanges (bio)chimiques complexes. La t½ n'est pas applicable pour ces produits complexes. Par conséquent, de nouvelles approches analytiques doivent être envisagées afin de surmonter ces difficultés.Une nouvelle approche basée sur la méta-métabolomique et la Spectrométrie de Masse; « Empreinte Métabolique Environnementale » (EMF), a été récemment introduite. Elle offre un nouveau proxy universel et intégratif; le « temps de résilience », dédié à l'évaluation du devenir environnemental et de l'impact des (bio)pesticides complexes dans des matrices environnementales (ex. sol, sédiments). Néanmoins, le développement d'une telle approche de méta-métabolomique non ciblée basée sur la Spectrométrie de Masse doit être étudié en profondeur. Plusieurs tâches doivent alors être abordées: 1) les protocoles d'extraction performants et les méthodes analytiques basées sur la GC/LC-(HR)MS doivent être mis en place, 2) le traitement de données et les outils chimiométriques appropriés doivent être développés pour maitriser la complexité des ensembles des données générées, 3) l'impact de la complexité du xénométabolome sur les analyses basées sur la MS doit être évalué, et 4) l'étude des résidus volatiles doit être envisagée et nécessite donc le développement de nouvelles méthodologies analytiques.Le travail a été mené sur 3 axes principaux. Le premier axe portait sur 1) le développement de protocoles d'extraction et des méthodes LC-HRMS pour analyser à la fois les xénométabolites des pesticides et les endométabolites du sol, et 2) le développement d'une nouvelle approche chimiométrique pour évaluer les performances d'extraction. De nouveaux protocoles d'extraction se sont avérés optimaux pour l'EMF, et l'approche chimiométrique a donc été validée. Le deuxième axe a évalué l'impact de la complexité du xénométabolome sur la détermination des biomarqueurs environnementaux. La suppression d'ion a été révélée et une stratégie pragmatique a donc été élaborée pour surmonter son influence. Le troisième axe visait à mettre en place une nouvelle méthodologie pour analyser les résidus volatils de pesticides complexes. Des analyses HS-SPME-GC-MS ont été couplées à la chimiométrie afin de réaliser des études cinétiques et de suivre la transformation des résidus volatils. Le workflow chimiométrique a prouvé sa fiabilité pour expliquer la transformation du pesticide et de nouveaux xénométabolites et sous-produits ont été identifiés.En conclusion, une avancée significative a été apportée à l’EMF. Elle a été consolidée pour les applications en laboratoire et sur le terrain qui doivent être étudiées afin d'améliorer le proxy et de le valider comme une approche fiable pour l'évaluation des risques des pesticides
Despite the ecological and sanitary awareness, worldwide consumption of pesticides is increasing. As these chemical products represent several adverse effects on human health and environment, measures should be taken in order to limit their impacts. Biocontrol products are proposed as an alternative solution of the synthetic products. In fact, these “biopesticides” are presumed to be less harmful and relatively less persistent. However, this a priori must be examined and strict risk assessment of those new substances should be considered.The development of biocontrol solutions proceeds first of all through the proposed protocols to study their activity and their environmental fate and impact. Currently, half-life (DT50) is used in order to evaluate the environmental fate of synthetic pesticides. However, DT50 approach gives only information about pesticides' persistence in the environment, but no indications concerning the formation of degradation products or its impact on biodiversity are provided. Furthermore, biocontrol products are complex (bio)chemical mixes. The DT50 is not applicable for such complex products. Therefore, novel analytical approaches should be considered in order to overcome these difficulties.A novel approach based on meta-metabolomics and Mass Spectrometry; the “Environmental Metabolic Footprinting” (EMF), was recently introduced. It affords a novel universal and integrative proxy; the “resilience time”, dedicated to assess the environmental fate and impact of complex (bio)pesticides in environmental matrices (e.g. soil, sediment). Nonetheless, the development of such Mass Spectrometry-based untargeted meta-metabolomics approach needs to be in-depth studied. Several tasks should be addressed: 1) performant extraction protocols and GC/LC-(HR)MS-based analytical methods should be set up, 2) suitable data processing and chemometric tools should be developed to deal with the complexity of the generated datasets, 3) the impact of xenometabolome complexity on MS-based analyses should be assessed, and 4) the study of the volatile residues should be considered and thus needs new analytical methodologies to be developed.The work was carried out following 3 main axes. The first axis addressed 1) the development of extraction protocols and LC-HRMS methods to analyze both pesticides xenometabolites and soil endometabolites, and 2) the development of a novel chemometric approach to assess the extraction performance. Novel extraction protocols have been proven optimal for the EMF, and the chemometric approach was thus validated. The second axis assessed the impact of xenometabolome complexity on the determination of environmental biomarkers. Ion suppression was revealed and thus a pragmatic strategy has been developed to overcome its influence. The third axis aimed to set-up a novel methodology in order to analyze the volatile residues of complex pesticides. HS-SPME-GC-MS analyses were coupled to chemometrics in order to perform kinetics studies and to follow the transformation of the volatile residues. The chemometric workflow proved its reliability to explain pesticide’s transformation and novel xenometabolites and by-products were identified.In conclusion, significant advances were carried to the EMF. It has been consolidated for laboratory and field applications that must be investigated in order to improve the proxy and to validate it as a reliable approach for pesticides risk evaluation

abstract: One out of ten women has a difficult time getting or staying pregnant in the United States. Recent studies have identified aging as one of the key factors attributed to a decline in female reproductive health. Existing fertility diagnostic methods do not allow for the non-invasive monitoring of hormone levels across time. In recent years, olfactory sensing has emerged as a promising diagnostic tool for its potential for real-time, non-invasive monitoring. This technology has been proven promising in the areas of oncology, diabetes, and neurological disorders. Little work, however, has addressed the use of olfactory sensing with respect to female fertility. In this work, we perform a study on ten healthy female subjects to determine the volatile signature in biological samples across 28 days, correlating to fertility hormones. Volatile organic compounds (VOCs) present in the air above the biological sample, or headspace, were collected by solid phase microextraction (SPME), using a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coated fiber. Samples were analyzed, using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). A regression model was used to identify key analytes, corresponding to the fertility hormones estrogen and progesterone. Results indicate shifts in volatile signatures in biological samples across the 28 days, relevant to hormonal changes. Further work includes evaluating metabolic changes in volatile hormone expression as an early indicator of declining fertility, so women may one day be able to monitor their reproductive health in real-time as they age.
Dissertation/Thesis
Masters Thesis Biomedical Engineering 2018

Indiana University-Purdue University Indianapolis (IUPUI)
Researchers have shown links between various hydrocarbons and carbonyl compounds and diseases, such as cancer using exhaled breath analysis through gas chromatography/mass spectroscopy (GC/MS) analysis of volatile organic compounds (VOCs). Trained canines can detect these VOCs and can differentiate a patient suffering from cancer from a healthy control patient. In this project, an attempt has been made to develop highly sensitive sensors for the detection of low concentrations of aldehyde VOCs, such as nonanal, using conductive polymer composites (CPCs) and functionalized gold nanoparticles (f-GNPs). Facile methods have been used to enhance the sensitivity and cross-selectivity of the fabricated sensors towards nonanal. Interdigitated electrodes (IDEs) are fabricated through a photolithography process. Sensors of PEI/carbon black (CB) composite were developed via spin-coating of the material followed by the heat treatment process. Sensors of 1-Mercapto-(triethylene glycol) methyl ether functionalized GNPs are developed via drop-casting of nanomaterial and f-GNP/PEI sensors are fabricated by spin casting PEI film on top of f-GNPs. Fourier Transform Infrared (FTIR) analysis, X-Ray Diffraction (XRD) analysis, contact angle measurement, and Field Emission Scanning Electron Microscopy (FESEM) analysis was conducted to characterize the fabricated devices. The fabricated sensors have been tested with a low concentration of nonanal, nonanone, dodecane, and 1-octanol in dry air. Multiple sensors are fabricated to ensure sensors reproducibility. The sensors have been exposed repeatedly to the targeting VOC toxiv assess the repeatability of the sensors. PEI/CB sensor degradation was studied over a period of 36 days. The fabricated PEI/CB film could detect (1-80 ppm) of nonanal with higher selectivity, than the f-GNPs. The sensor0s sensitivity to nonanal was over fourteen times higher than 2-nonanone, 1-octanol, and dodecane. This shows the high selectivity of the fabricated sensor toward nonanal. In addition, the proposed sensor maintained its sensitivity to nonanal over time showing minimal degradation. The sensor response to nonanal at a relative humidity (RH) of 50% and 85% dropped less than 13% and 32% respectively. The Response of f-GNP sensors to nonanal (400 ppb - 15 ppm), dodecane (5 - 15 ppm), 1-octanol (5 - 15 ppm), and 2-nonanone (5 - 15 ppm) presented a sensitivity (∆R=R0) of 0.217%, 0.08%, 0.192% and 0.182% per ppm of the VOCs respectively. Despite the high sensitivity to the targeting VOCs, the fabricated sensors were damaged in an environment with relative humidity (RH) at 45%. A thin layer of PEI over the film was developed to ensure the sensor could tolerate longtime exposure to water vapor in an environment with RH up to 85% and enhance the sensor selectivity towards nonanal. The f-GNP/PEI sensors with nonanal (400 ppb- 15 ppm), dodecane (100 -200 ppm), 1-octanol (5 - 15 ppm) and 2-nonanone (5 - 15 ppm) presented sensitivity (∆R=R0) of 0.21%, 0.017%, 0.0438% and 0.0035% per ppm of the VOCs respectively.
2021-04-24

碩士
國立成功大學
環境醫學研究所
89
Most people spend the majority of their time in indoor environment as the modern society becomes industrialized. Consequently, the application of new building materials, and air conditioning systems has increased. In addition, the function and use of indoor environments has become even more diverse. All the above factors have, in part, contributed to the rising quantities and varieties of volatile organic compounds (VOCs) identified indoors. It is therefore important to better characterize these common indoor VOCs, and establish the analysis method of VOCs in air and blood of low concentrations. Moreover, questionnaire have been applied to determine the overall health status and prevalence of sick building syndrome (SBS) in office workers. The results suggest that SBS were frequently observed in office workers of our investigation. Most of them reported that symptoms associated with eyes, nose, or central nervous system improved when they were away from work, and office workers in business district reported greater frequency and severity than in industrial district. The most common symptom was sensory irritation in eyes, nose, or throat. This study also established the analytical method for VOCs in air and blood of low concentration using ATD-400, GC/MS and purge-and-trap concentrator, GC/FID/ECD. The R2 of standard curve of benzene, toluene, ethylbenzene, m-/p-xylene, o-xylene could be up to 0.995, and the method detection limit, accuracy and precision were in good control as well. Environmental sampling in an office building and biological monitoring for its staff was performed afterwards. The result reveals that contaminant level was low in that building and the concentration of serum VOCs were also correspondingly low. In addition, the serum VOCs appeared to be an ideal way to reflect the real outside exposure when the indoor exposure in low. The serum VOCs would therefore considered a relatively good biomarker to estimate the low level condition of outside environment.

碩士
國立陽明大學
環境與職業衛生研究所
100
Background/objectives In recent years, there has been a sustainable increase of industrial and science parks due to the economic development of Taiwan. Volatile organic compounds (VOCs), such as benzene and toluene, are emitted during the industrial manufacturing process. When the emission of VOCs is not well-treated by various control devices, it leads to a contaminated environmental quality near the industrial park and causes adverse effects on residents. The objective of this study is two-fold: (1) to measure the urinary metabolite levels and VOCs levels via personal and environmental sampling and (2) further assess the relationships between exposure to VOCs levels and various respiratory symptoms among residents living near industrial areas. Our study will be a reference for the controlled emission of VOCs from industrial areas. Materials and methods The exposure group was selected as two elementary schools’ students and their parents living nearby industrial and science parks in Nantun District of Taichung. The control group was chosen from another site, which does not have an industrial park—in the Eastern District of Taichung. We measured each participant in the two groups, measuring repertory symptoms using a structured questionnaire, measuring VOCs levels by personal passive samplers, and measuring urinary hippuric acid (HA) and cotinine via UV and HPLC. Respiratory symptoms in schoolchildren and their parents were assessed using a questionnaire from American Thoracic Society (ATS). Results The VOC concentrations by personal sampling in the exposure group were significantly higher than those in the control group. Average levels for exposure group in students were 6.01 ppb in benzene, 21.96 ppb in toluene, and 34.74 ppb in total VOCs, significant higher than those of the control group: 2.47 ppb in benzene, 12.22 ppb in toluene, and 25.27 ppb in total VOCs. In addition, the average levels of parents in the exposure group were 7.06 ppb in benzene, 33.63 ppb in toluene, 0.7 ppb in ethylbenzene, and 50.74 ppb in total VOCs, higher than the control group parents, which were 3.21 ppb in benzene, 17.72 ppb in toluene, N.D. in ethylbenzene, and 33.14 ppb in total VOCs. The urinary hippuric acid concentrations in the exposure group were also higher. Average levels for the exposure group’s students and their parents were 642.62 and 682.04 mg/g cre. higher than 627.1 and 599.86 mg/g cre. in the control group, but there was no significant difference in the two groups. The VOCs concentrations by personal sampling were significant positive correlations with outdoor VOCs concentrations, which the exposure group in students was found correlation coefficient (r) is 0.616 in benzene, 0.413 in xylene and 0.571 in total VOCs, respectively. The personal sampling for parents in exposure group had significant moderate positive correlation with outdoor toluene concentrations (r = 0.419). The personal sampling for students in the control group was correlated with outdoor levels of benzene (r = 0.891), toluene (r = 0.615), xylene (r = 0.713), total VOCs (r = 0.734), however, only found in total VOCs concentrations (r = 0.65, p < 0.05) in the control group of parents. The significant correlations between personal sampling of VOCs and concentrations of biological monitoring were found among students and their parents. Levels of benzene, xylene and total VOCs significant positively correlated with urinary hippuric acid concentrations in the exposure group (r = 0.557, 0.433, and 0.552, respectively). It were similar trends in the control group were significant positive correlation (r = 0.713, 0.804, and 0.734, respectively). Using multiple linear regressions adjusted for covariates, of urinary HA levels for students and their parent in the exposure group were 46.755 mg/g cre. and 34.632 mg/g cre. higher than the control group, but there were no significant difference in the two groups. Using multiple logistic regression adjusted for covariates, the risk suffering from cough and children and their parent in the exposure group were 1.57 and 1.25 times higher than in the control group, but there were no significant difference in the two groups. Conclusion Residents living near Taichung industrial park were found high VOCs levels in indoor and outdoor air, and personal exposure to VOCs concentrations explained by the emission from various sources of industries. In addition, people exposed to long-term VOCs from emitting from industrial or science park causes high risk of respiratory symptoms among schoolchildren and residents. Therefore, the environmental authorities should continually reduce emissions of VOCs and other pollutants sources in industrial and science parks, and to establish the isolation zone between communities and industrial parks in order to reduce adverse effects among residents living near industrial area.

Understanding complex Earth systems is challenging for scientists and students alike, because of the characteristics (e.g. bifurcations, self-organization, chaotic response) that are associated with these systems. This research integrates two research strands which contribute to the scientific and pedagogical understanding of complex Earth systems. In the first strand, a method that characterizes volatile organic compounds (VOCs) as ecological proxies of soil microbial ecosystems was validated. Unlike other measures of microbial community structure (e.g. Biolog and FAME), VOCs are advantageous because they are non-destructive and can provide temporal and spatial data. Additionally they are rich sources of information that describe the microbial metabolism, community structure, and organic carbon substrates utilized by soil microorganisms. Statistical results indicate that the detected and identified VOCs were significant (p < 0.05) indicators of microbial community composition shift in soil microcosm studies. Geographical information systems (GIS) illustrates that VOCs varied with space and time in south Texas soils. The second strand focuses on a geoscience education study exploring student conceptual model development of complex Earth systems. The efficacy of multiple representations and inquiry was tested as the pedagogical strategy in upper and lower level undergraduate courses to support students’ conceptual model development of complex Earth systems. Comparisons in student performance were based on prior knowledge (low and high) and on exposure to the implemented pedagogy (control and experimental groups). Results indicate that an inquiry-based learning model coupled with the use of multiple representations had significant positive performance impacts on students’ conceptual model development and content knowledge. This dissertation model integrates science and education research and is particularly useful for graduate students who intend to pursue a career in academia and envision teaching as part of their professional duties. It allows for synergy between teaching and research to be achieved where the classroom becomes a laboratory for research. Ultimately, the research conducted in the classroom informs pedagogy and enhances scholarship. Graduates learn to bridge the gap between education and science departments where they become leaders in science who conduct cutting-edge scientific research and also value making a broader impact on society through enhancing public education.

碩士
國立成功大學
地球科學系
107
Black carbon plays an important role in the global carbon cycle. Black carbon is composed of a variety of mostly incompletely-combusted products from biomass or fossil fuel. Normally considered chemically recalcitrant, black carbon may be stabilized within the natural environments for a long time, through interfacial chemical complexation of surface functional groups and minerals, and subsequent physical protection. This study is to investigate the molecular characteristics and the long-term fate of black carbon through degradation in natural environment and lab-mimicking condition. Using synchrotron-based X-ray near-edge absorption spectroscopy and high resolution time-of flight (TOF) mass spectrometry, we study the chemical structure and properties of semi-volatile compounds from black carbon made at different temperatures and from different biomass sources. We aim to seek a biomarker for the ill-defined black carbon, and probe the chemistry and functionality of small degradation products in the form of semi-volatile compounds over time. A lower level of oxidation and lower proportion was found for the semi-volatile compounds from black carbon made at higher-temperature (700C). For black carbon samples made at high temperature (700C) and buried in soil for six years, signal of biological degradation was observed. Anaerobic degradation happened to black carbon made at both low (500C) and high temperatures (700C) within the bioreactor. Chemical complexation and mineral coating was observed on the interface of black carbon and short-range-order minerals. A biomarker was found in all black carbon samples as V-type lignin component (MW166＆168, C9H10O3＆C8H8O4). The degradation products and path of black carbon are suggested similar to lignin. Our research provided insight on the molecular characteristics and degradation of black carbon, and contribute to the understanding of its long term fate, stabilization and functionality in the natural environment.

6 Abstract This thesis focuses on the identification and quantification of volatile metabolites in the exhaled breath that might be used as possible biomarkers of Gastroesophageal Reflux Disease. Animal tissue samples were exposed to conditions simulating the gastric environment to identify specific volatile compounds that would be chosen for real-time quantification in exhaled breath of GERD patients and healthy controls using selected ion flow tube mass spectrometry. Solid phase microextraction, was used in combination with gas chromatography mass spectrometry, for qualitative analyses of the headspace of these samples. Only acetic acid was significantly elevated and so it has been elected for the quantitative analysis in the breath of the patients. The median concentration of acetic acid measured by SIFT-MS in the exhaled breath of twenty-two GERD patients was found to be higher (85 ppbv) in comparison to the control group (31 ppbv). The results show that breath acetic acid could be valuable marker for GERD diagnosis and monitoring.

As doenças neurodegenerativas (NDDs) são um grupo heterogéneo de perturbações caracterizadas pela degeneração progressiva da estrutura e função do sistema nervoso central ou periférico. De entre as NDDs, as doenças de Alzheimer e Parkinson são as mais prevalentes e a sua incidência está a crescer simultaneamente com a esperança média de vida. Uma vez que ainda não existem biomarcadores (BMs) fiáveis para a maioria das NDDs, o diagnóstico depende essencialmente dos sintomas clínicos. Contudo, a neurodegeneração começa muito antes do paciente manifestar sintomas. Assim sendo, há um grande interesse no diagnóstico precoce das NDDs. Isto permitirá a antecipação do tratamento e a mitigação dos efeitos negativos da neurodegeneração. A caracterização da composição dos metabolitos orgânicos voláteis (VOMs) de biofluídos humanos está a ser explorada como uma ferramenta não-invasiva promissora para descobrir BMs para diagnóstico de doenças e infeções. Os perfis de VOMs fornecem informações importantes, particularmente acerca de alterações metabólicas relacionadas com o desenvolvimento de diferentes patologias, incluindo NDDs. Neste contexto, o objetivo deste trabalho foi o desenvolvimento de uma técnica utilizando membranas de polidimetilsiloxano (PDMS) e um método de microextração em fase sólida do headspace (HS-SPME) seguido de análise por cromatografia gasosa acoplada a espectrometria de massa (GC-MS) para a descoberta de possíveis BMs de NDDs. A técnica desenvolvida permitiu-nos identificar 82 VOMs – 67 no braço, 68 na orelha e 69 na nuca. Seria necessário, porém, analisarmos um maior número de amostras de forma a consolidar os resultados obtidos no grupo controlo antes de prosseguir com a amostragem de pacientes. Este trabalho sugere assim que a análise dos perfis voláteis da pele poderá ser uma ótima ferramenta para a identificação de possíveis BMs voláteis para diferentes NDDs. Tais BMs teriam um grande potencial para serem facilmente integrados em dispositivos de teste rápido (POCT) a serem utilizados em ambientes clínicos.
Neurodegenerative diseases (NDDs) are a heterogeneous group of disorders characterized by progressive degeneration of structure and function of the central or peripheral nervous system. Among NDDs, Alzheimer's and Parkinson's diseases are the most prevalent and their incidence is increasing concomitantly with average life expectancy. While there aren’t yet reliable biomarkers (BMs) for most NDDs, their diagnosis relies essentially on the clinical symptoms. However, neurodegeneration begins long before the patient experiences any symptoms. Therefore, there is an obvious interest in the early diagnosis of NDDs. This would allow the anticipation of the treatment and mitigation of the negative effects of neurodegeneration. The characterization of volatile organic metabolites (VOMs) composition of human biofluids is being explored as a promising and non-invasive tool to unveil BMs for the diagnosis of human diseases and infections. The VOMs profiles can provide important metabolic information, particularly about metabolic changes caused by different clinical conditions, including NDDs. In this context, the aim of this work was to develop a technique using polidimetilsiloxane (PDMS) membranes and a method of headspace solid phase microextraction (HS-SPME) followed by gas chromatography coupled to mass spectrometry (GC-MS) analysis to discover possible BMs for NDDs. The developed method allowed us to identify 82 VOMs – 67 in the arm, 68 in the ear and 69 in the nape of control subjects. However, it would be necessary to analyse a larger group of subjectsto improve the technique and obtain more robust data before introducing the NDDs patients sampling. This work suggests that the analysis of volatomic profiles of the skin can be an important approach to identify potential volatile BMs for NDDs. Ultimately, this can be a seamless tool for the identification of putative volatile BMs of different NDDs with a great potential to integrate in point-of-care testing (POCT) devices for the clinical environment.

L’évaluation de l’exposition aux composés organiques volatils (COV) recourt couramment à l’analyse des métabolites urinaires en assumant qu’aucune interaction ne survient entre les composés. Or, des études antérieures ont démontré qu’une inhibition de type compétitive survient entre le toluène (TOL), l’éthylbenzène (EBZ) et le m-xylène (XYL). Le chloroforme, qui est également un solvant métabolisé par le CYP2E1, se retrouve souvent en présence des autres COV dans les échantillons de biosurveillance. La présente étude visait donc à évaluer si le chloroforme (CHL) peut lui aussi interagir avec ces COV et évaluer ces interactions au niveau de l’excrétion des biomarqueurs urinaires associés, soit l’o-crésol, l’acide mandélique et l’acide m-méthylhippurique pour TOL, EBZ et XYL respectivement. Afin d’obtenir des données humaines, cinq volontaires ont été exposés par inhalation à différentes combinaisons de COV (seuls et mélanges binaires ou quaternaires) où la concentration de chacun des composés était égale à 1/4 ou 1/8 de la valeur limite d’exposition (VLE) pour une durée de 6h. Des échantillons d’air exhalé, de sang et d’urine ont été récoltés. Ces données ont ensuite été comparées aux modèles pharmacocinétiques à base physiologique (PCBP) existants afin de les ajuster pour l’excrétion urinaire. Certaines différences ont été observées entre les expositions aux solvants seuls et les coexpositions, mais celles-ci semblent majoritairement attribuables aux remplacements de participants à travers les différentes expositions. Les valeurs de Vmax pour EBZ et CHL ont été optimisées afin de mieux prédire les niveaux sanguins de ces COV. À l’exception du modèle pour EBZ, tous les paramètres pour l’excrétion urinaire ont été obtenus à partir de la littérature. Les modèles adaptés dans cette étude ont permis de simuler adéquatement les données expérimentales.
Evaluation of volatile organic compounds (VOC) exposure commonly resorts to urinary metabolite analyses, assuming that no interaction occur between coexposed chemicals. However, previous studies have reported competitive inhibition between toluene (TOL), ethylbenzene (EBZ) and m-xylene (XYL). Chloroform, which is also metabolized by CYP2E1, is also often found in human biomonitoring samples along with the mentioned VOCs. The goal of the present study was to evaluate if chloroform (CHL) can interact with previous VOC and to evaluate those interactions at the urinary biomarker excretion level for corresponding metabolites, namely o-cresol, mandelic acid and m-methylhippuric acid for TOL, EBZ and XYL respectively. To obtain human data, five male volunteers were exposed by inhalation to different VOC combinations (single and binary or quaternary mixtures) where concentration of each chemical was equal to 1/4 or 1/8 of the threshold limit value (TLV) for 6h. Exhaled air blood and urine samples were collected. These data were then compared with existing physiologically based pharmacokinetic (PBPK) model predictions for adjustment for urinary excretion. Some differences were observed between single and mixed exposures but they may be mainly related to volunteer replacements throughout experiments. Vmax values for EBZ and CHL were optimized to better fit blood data. Except for EBZ model, all urinary excretion parameters were taken from the literature. Models adapted in the present study adequately simulated experimental data.