Indiana University-Purdue University Indianapolis (IUPUI) / The aim of this study is to identify metabolic transformations in breast cancer through urinary volatile organic compounds in mammary pad or bone tumor mice models. Subsequently, it focuses on investigating the efficacy of therapeutic intervention through identified potential biomarkers. Methods for monitoring tumor development and treatment responses have technologically advanced over the years leading to significant increase in percent survival rates. Although these modalities are reliable, it would be beneficial to observe disease progression from a new perspective to gain greater understanding of cancer pathogenesis. Analysis of cellular energetics affected by cancer using bio-fluids can non-invasively help in prognosis and selection of treatment regimens. The hypothesis is altered profiles of urinary volatile metabolites is directly related to disrupted metabolic pathways. Additionally, effectiveness of treatments can be indicated through changes in concentration of metabolites. In this ancillary experiment, mouse urine specimens were analyzed using gas chromatography-mass spectrometry, an analytical chemistry tool in identifying volatile organic compounds. Female BALB/c mice were injected with 4T1.2 murine breast tumor cells in the mammary fat pad. Consecutively, 4T1.2 cells were injected in the right iliac artery of BALB/c mice and E0771 tumor cells injected in the tibia of C57BL/6 mice to model bone tumor. The effect of two different modes of treatment: chemical drug and mechanical stimulation was investigated through changes in compound profiles. Chemical drug therapy was conducted with dopamine agents, Triuoperazine, Fluphenazine and a statin, Pitavastatin. Mechanical stimulation included tibia and knee loading at the site of tumor cell injection were given to mice. A biological treatment mode included administration of A5 osteocyte cell line. A set of potential volatile organic compounds biomarkers differentiating mammary pad or bone confined tumors from healthy controls was identified using forward feature selection. Effect of treatments was demonstrated through hierarchical heat maps and multivariate data analysis. Compounds identified in series of experiments belonged to the class of terpenoids, precursors of cholesterol molecules. Terpene synthesis is a descending step of mevalonate pathway suggesting its potential role in cancer pathogenesis. This thesis demonstrates the ability of urine volatilomics to indicate signaling pathways inflicted in tumors. It proposes a concept of using urine to detect tumor developments at two distinct locations as well as to monitor treatment efficacy.

Indiana University-Purdue University Indianapolis (IUPUI) / In this work we developed paper spray mass spectrometry methods to obtain lower detection limits for pharmaceuticals and drugs of abuse. The second chapter investigates blood fractionation membranes for their ability to obtain lysis free plasma from whole blood without changing the drug concentration relative to centrifugation. We presented a device capable of obtaining and analyzing plasma samples from whole blood and obtaining quantitative results similar to traditional methods. In the third chapter the properties of the paper substrate are investigated systematically for their impacts on ionization efficiency and recovery in combination with the solvent choice. The fourth and fifth chapters detail a simple method for lowering detection limits using a method called paper strip extraction. In this method biofluids are wicked through either sesame seed oil or solid phase extraction powder on a paper strip to concentrate and preserve (in the case of THC) analytes out of biofluids. The use of 3D printing for rapid prototyping and how it potentially impacts paper spray MS sensitivity is outlined in the final chapter.

Indiana University-Purdue University Indianapolis (IUPUI) / In Headspace Solid-Phase Microextraction (Headspace SPME), a sample is heated to encourage a portion of the analyte into the headspace of a vial. A coated fiber is introduced into the sample headspace and the analyte is adsorbed onto the fiber coating. Total Vaporization Solid-Phase Microextraction (TV-SPME) is a technique that is derived from this technique. In TV-SPME, liquid samples are completely vaporized allowing for better adsorption and fewer matrix effects. This method does not require any sample preparation, utilizes minimal supplies and can be automated, making it both an efficient and cost-effective method. Chapter 1 will discuss the theory of SPME and TV-SPME. In Chapter 2, the detection of ɣ-hydroxybutyric acid (GHB) and ɣ-butyrolactone (GBL) in beverages is discussed. The detection of these compounds in beverages is of importance because these drugs may be used to facilitate sexual assault. This crime utilizes substances that cause sedation and memory loss. The derivatization of GHB as well as the properties that make GHB difficult to detect will be discussed. Chapter 3 will discuss the detection of methamphetamine and amphetamine (as their trifluoroacetyl derivatives), GBL, and the trimethylsilyl derivative of GHB in human urine. Amphetamine is a metabolite of methamphetamine, therefore, both drugs should be identified within biological samples. GHB and GBL are metabolites of one another and interconvert when in aqueous solution. This interconversion will be discussed. Chapter 4 will cover method optimization of the Total Vaporization Solid-Phase Microextraction method. Analytes of interest for these analyses were methamphetamine, amphetamine, GHB, and GBL. The optimal extraction temperature ranging from 60-160°C of each drug will be discussed as well as why higher temperatures may not be suitable for this method. A limit of detection study for methamphetamine and amphetamine will also be covered. Chapter 5, the future work chapter, will discuss future analyses using the Total Vaporization Solid-Phase Microextraction method including the analysis of powder materials, plant material, and toxicological samples. Powder material will include the analysis of individual powdered drugs as well as realistic drug mixtures. Some analyses on individual powder samples has already been completed and will be shown. Plant material will include the analysis of naturally occurring compounds found in marijuana plants as well as synthetic cannabinoids. Toxicological samples will expand on previously mentioned urine samples to include drugs such as benzoylecgonine and THC-COOH.

Indiana University-Purdue University Indianapolis (IUPUI) / Gas-phase ion/ion covalent modification was coupled to ion mobility/mass spectrometry analysis to directly correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross-section (CCS) distributions were measured prior to ion/ion reactions to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Ubiquitin ions were electrosprayed from aqueous and methanolic solutions yielding a range of different charge states that were analyzed by ion mobility and time-of-flight mass spectrometry. Aqueous solutions stabilizing the native state of ubiquitin generated folded ubiquitin structures with CCS values consistent with the native state. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous solutions were covalently modified preferentially at the lysine 29 and arginine 54 residues, indicating that elements of secondary structure, as well as tertiary structure, were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase.

A rapid method was developed for the quantitative analysis of N, N'hexamethylenebisacetamide (HMBA) and its deacetylated metabolites, N-acetyl-1, 6- diaminohexane (NADAH) and 1, 6-diaminohexane (DAH). The method used flowinjection liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)selected ion monitoring at low/high resolving power (LR/HR-SIM)-mass spectrometry (MS). Samples were extracted from murine erythroleukemia (MEL) cell culture and were analyzed without chemical derivatization. The limits of detection (LOD) for HMBA, NADAH and DAH measured by HR-SIM were 0.05, 0.1 and 0.2 picomole, respectively. Calibration curves were constructed by using tetradeuterated DAH as an internal standard for both SIM methods. The ratio of the analyte concentration in the extract of a MEL cell culture treated with HMBA for 120 hours was found to be 1. 7, 27 and 17 times that of the control for DAH, NADAH and HMBA, respectively. The HRSIM that used a lock-mass technique helped eliminate chemical interferences that had mlz values similar to the one of interest. Although this technique was developed using HMBA, NADAH and DAH, it is expected to work for natural polyamines and their acetylated metabolites. Qualitative analysis was performed by both positive electrospray (ES)- and APCIMS under full mass range scanning. The mass spectra of polyamine standards were characterized. HMBA exhibited an ability to form mono- and bi-molecular adducts with metal ions, itself and with other polyamine molecules in ES-MS. The LODs estimated by ES-MS were consistently higher than the APCI-SIM methods that is described here.

Spiders are able to spin a variety of silk types for various purposes, each with their own unique properties. The mechanical properties of spider silk out-perform the mechanical properties of many man-made materials we use today, including tensile steel, KevlarTM, and nylon. To further understand the proteins the silks are made of and how they are synthesized in the silk glands, transcriptional and proteomic analysis was conducted. Transcriptional regulation of silk genes was investigated to determine how and why several silk proteins are transcribed into mRNA products together in the same gland. The tubuliform gland is one of the major contributors of egg case silk production. The mRNA of major ampullate spidroins 1 and 2 (MaSp1, MaSp2) and tubuliform spidroin 1 (TuSp1) is found in the tubuliform glands, but not all are translated into proteins for egg case silk purposes. To understand why not all of the transcribed mRNA products are not being translated into proteins, the promoter sequences of MaSp1, MaSp2, and TuSp1 were aligned and found to contain an E-Box site. Several constructs containing the cDNA of the promoter sequences and cDNA of bHLH transcription factors were built to test transcriptional regulation of MaSp1, MaSp2, and TuSp1. Proteomic analysis of egg case silk and the tubuliform glands was also conducted to identify further proteins synthesized in the tubuliform glands and to determine which of these proteins are ultimately incorporated into the egg case silk fibers by MS/MS analysis. Multiple silk proteins were identified within the tubuliform glands and incorporated into the egg case fibers, suggesting silks are composite fibers of multiple spidroins.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19552号 / 医博第4059号 / 新制||医||1012(附属図書館) / 32588 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 藤田 潤, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Our study focuses on the application of volatile metabolomics and chemometrics in plant pathology. Specifically, volatile metabolites or volatile organic compounds (VOCs) from the American chestnut tree (Castanea dentata) and its pathogenic fungus Cryphonectria parasitica have been investigated. The American chestnut was once a dominant tree species in the eastern forests of the United States. However, it was nearly devastated by the fungal pathogen C. parasitica. The loss of this tree species has significantly impacted the ecosystem. Therefore, preservation and restoration of American chestnut are crucial. Chapter one provides an overview of mass spectrometry based volatile metabolomics and their implementation in the investigation of plant pathology. The study of volatile metabolites profiles from virulent and hypovirulent strains of C. parasitica are presented in chapter two. The microbial volatile organic compounds (MVOCs) profiles were analyzed via nondestructive sampling method, headspace solid phase microextraction (HS-SPME), combined with gas chromatography (GC)-mass spectrometry (MS). The results indicate that the MVOCs profiles emitted from these two strains are significantly different. In general, compared with its hypovirulent strains, high emissions of sesquiterpenes were observed in the virulent strains. Furthermore, the study explored MVOCs differences associated with hypovirulence processes. The study found that both hypovirulence and aging can alter the virulent strains' MVOCs, and the process can be observed via their volatile metabolites. Chapter three describes the effects of aging, cultivation medium, and pH on fungal volatile metabolite profiles, all of which can change the strength of MVOCs emission and their composition. An acidic environment favors fungal bioactivity and therefore enhanced MVOCs emission. However, due to the inherently low MVOCs production from hypovirulent strains, the pH effect was less apparent in the hypovirulent isolates. The strength of MVOCs emission was highly correlated to the fungal expansion in virulent strains for the first 14 days. The overall emission from hypovirulent strains was relatively steady during the 28-day observation. Finally, the cultivation media are critical to the fungal MVOCs production. Among the tested media, cornmeal was least favorable for MVOCs production for both strains. Finally, Chapter Four presents a study of the total constitutive phenolic content estimation and volatile organic compounds identification from four species of chestnut tree leaf tissues. Folin Ciocalteu reagent assay with UV/Vis spectrophotometry was applied to estimate the total phenolic content in leaf tissues of American chestnut (Castanea dentata), Chinese chestnut (Castanea mollissima), and their backcross breeding generations (B3F2 and B3F3). The results from leaf tissue extraction in methanol/water (95:5 v/v), pH 2, and analyzed under the UV/Vis at 765 nm show that the variations among these tree species are significant (ANOVA, p < 0.05). The kinetics of phenolic compound solid-liquid extraction was elaborated using Peleg, second order, and power law models. Moreover, the analysis of VOCs collected from these species indicated that the distinction of American and Chinese chestnut could be archived via their VOCs, while the hybrids’ leaf VOCs are different from their parents’.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13561号 / 論医博第2290号 / 新制||医||1067(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 武藤 学, 教授 今中 雄一, 教授 村川 泰裕 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM