Dissertations / Theses on the topic 'Identification of transformation products'

For bioactive chemical classes, it is often assumed that environmental transformation eliminates associated ecosystem risks. However, for endocrine-active steroid hormones, modest changes in structure can have a significant influence on biological activity and thus, subtle environmental transformations can yield products with conserved, enhanced, or activity across different biological endpoints. The aim of this work was to explore the environmental fate of high potency, endocrine-active steroid hormones during natural or engineered water processes in order to test the hypothesis that steroid transformation products generated during these processes are likely to contribute to residual bioactivity often reported in water resources. Specifically, laboratory experiments were used to simulate chemical disinfection (e.g., chlorination) or natural processes (e.g., photolysis) to: (i) determine the rate and extent of steroid transformation, (ii) isolate and identify products that are formed, and (iii) evaluate products or product mixtures for biological activity. These experimental results can be used to help guide occurrence studies for any products of concern in the environment and also guide computational predictions or rationalizations of chemical reactivity. Ultimately, the goal is to expand upon our awareness and understanding of how these potent endocrine ligands behave in the environment and how they potentially affect ecosystem health. Chapter 2 discusses the reaction of glucocorticoids (GC)s with aqueous chlorine (effectively, HOCl) to simulate their fate during engineered drinking water and wastewater chemical disinfection. Numerous transformation pathways were unveiled, including interconversion of GCs (e.g., endogenous cortisol to synthetic prednisolone), production of known androgens in the adrenosterone class, and chlorination of GCs (e.g., formation of 9-chloro-prednisone). We also showed that other advanced processes (e.g., oxidation via ozonation) result in more complete degradation of such pollutants, and may be better alternatives to chemical disinfection at eliminating bioactive steroidal product formation. In Chapter 3, results of the direct photolysis of dienogest (DNG), a widely prescribed oral contraceptive agent, are presented to simulate its fate in natural sunlit surface waters and engineered photochemical treatment systems (e.g., UV disinfection systems). The major products (~ 80% of the converted mass in neutral aqueous solutions) were identified to be photohydrates resulting from photochemical-induced incorporation of water into parent DNG. These products were found to be prone to dehydration in the dark, and thus, a source of substantial DNG regeneration (~ 65% after 72 h in neutral solutions). Other minor, non-revertible products were also identified, including two known estrogens. Although minor in initial yield, these estrogens are likely to accumulate over time through repeated cycling between DNG and its photohydrates, and thus, dominate DNG long-term fate. It was also found that DNG undergoes an unusual photochemical rearrangement to produce a minor product with a novel tetracyclic ring system--the subject of Chapter 4. Further, the generality of this unique photorearrangement process was explored through extension to the photolysis of two other dienone pharmaceutical steroids (e.g., the androgens methyldienolone and dienedione). Surprisingly, despite the significant change in core steroidal structure, the rearrangement products retain some progesterone receptor (PR) and androgen receptor (AR) bioactivity (i.e., low-µM to sub-nm EC50 values). Again, these represent other non-revertible, minor products that are likely to accumulate over time, with likely adverse ecological consequences. Chapter 5 covers results arising from the direct photolysis of trenbolone acetate (TBA) metabolites in the presence of model nucleophiles (e.g., sodium azide, sodium thiosulfate, ammonium hydroxide, hydroxylamine, and humic acid), some of which would be expected to be present, along with TBA metabolites, in agriculturally-impacted water resources. Previous studies by our group revealed that TBA metabolites undergo photohydration-thermal dehydration cycling, like that described above for DNG photolysis. The objective of this study was to determine if other nucleophiles would outcompete water for photochemical incorporation across the TBA metabolite extended conjugation system. It was found that TBA metabolite photolysis results in photochemical (and at times thermal) addition of the nucleophile to the TBA metabolites. It was also found that the addition products undergo thermal elimination in the dark and contribute to TBA metabolite regeneration, and therefore, are expected to increase TBA metabolite persistence in water resources. Finally, Chapter 6 discusses the reactions of various trienone and dienone steroids with aqueous chlorine to simulate their fate during engineered drinking water and wastewater chemical disinfection. Single-step transformation pathways were unveiled for each steroid class, including 4-chlorination (trienones) and 9,10-epoxidation (dienones). Chlorination at position C-4 is known to enhance anabolic potency of androgenic steroids and the 9,10-epoxy products were found to undergo acid- or base-catalyzed ring-opening and aromatization to yield known estrogenic products. In addition, Chapter 7 provides conclusions and future directions, while Chapter 8 details the experimental methods and procedures used throughout this thesis. Collectively, the results presented herein confirm our overall hypothesis that steroid transformation products would be expected to contribute to residual biological activity often detected in water resources. Furthermore, the results indicate that the transformation of high potency pharmaceuticals does not automatically equate with reduction or elimination of hazards to exposed organisms, especially in cases where such compounds have potential to form products exhibiting diverse biological endpoints. More holistic approaches to risk assessment of such high potency environmental contaminants are needed in order to accurately assess the fate and effects of such emerging pollutant classes and their bioactive transformation products.

Pharmaceuticals which are used worldwide are designed to facilitate the life for the human society and have an important role in treatment and prevention of disease for both humans and animals. They are ubiquitous in the aquatic environment and are mainly derived from municipal wastewater treatment plants (WWTPs) due to their low removal rate. Therefore, their presence in the environment is directly linked to the human impact. Various biological and abiotic processes in the environment can transform them to transformation products (TPs). In many cases, transformation is already initiated in the human body by a variety of drug-metabolizing enzymes. The metabolites formed through human metabolism present some modifications in their chemical structures that can differ in physicochemical properties to their parent compound. Once they are excreted from the human body, both the unmetabolised parent drug and their metabolites enter WWTPs by means of the sewer system. Since the WWTPs are not designed to remove completely pharmaceutical residues, the fraction not removed after the treatment will eventually end up in the receiving water bodies. Consequently, due to pharmaceutical transformations in the human body, biotransformations in WWTPs and phototransformations in surface water, they can potentially produce a high number of TPs in real world samples which makes their identification a challenge. In this thesis, two different approaches (TPs profiling and suspect screening) based on high resolution mass spectrometry (HRMS) for the detection and identification of TPs of pharmaceuticals were investigated. TPs profiling approach was applied for the identification of phototransformation products of an antiviral zanamivir (ZAN) in batch reactors filled with surface water. On the other hand, suspect screening approach was applied for evaluation of transformation, prioritization and identification of photoTPs of six iodinated contrast media in surface water. Finally, a combination of suspect and TPs profiling approach was applied for the detection of TPs of an anticonvulsant lamotrigine and its main human metabolite lamotrigine N2-glucuronide which were formed as the result of their degradation in both activated sludge and pH dependent hydrolysis. The TPs profiling approach for evaluation of these transformations is illustrated in the example of photodegradation of an antiviral ZAN with identification of its TPs in surface water (Chapter 3.). Here a set of lab- scale experiments was performed in order to determine the susceptibility of ZAN towards photodegradation under simulated and natural sunlight. The identification of the TPs was performed using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry (HILIC-HRMS) where four photoTPs were tentatively identified and their proposed structures were rationalized by photolysis mechanisms. Kinetic experiments showed that photodegradation kinetics of ZAN in surface waters would proceed with slow kinetics since upon exposure of aqueous solutions of surface water (20 μg L-1) to simulated sunlight, ZAN was degraded with t1/2 of 3.6 h. Under natural sunlight irradiating surface water, about 30 % of the initial concentration of the antiviral disappeared within 18 days. However, when ZAN and its TPs were retrospectively screened from surface water extracts, neither the parent nor the TPs were detected. The results of this TP profiling used for the identification of TPs of ZAN, although straightforward, suggests that it is not suitable when dealing with a considerably elevated number of TPs formed in batch experiments. However, time and effort needed to be optimised for the structure elucidation of 108 photoTPs of six iodinated contrast media (ICM) (Chapter 3.). Again, the photodegradation study was performed in surface water spiked with the ICMs using a sunlight lab-scale simulator. 108 TPs were generated and each photoTP was characterised by its unique exact mass of the molecular ion and retention time and added to a suspect list. Once the suspect list was generated, the photoTPs were searched in thirteen surface water samples which were extracted using a generic solid- phase extraction method (four cartridges of different chemistries in order to retain ample number of compounds with different chemical properties). Based on their detection frequency (those TPs with the frequency higher than 50 % were deemed important), eleven TPs were prioritized and their structures elucidated by HRMS and NMR (when possible). Out of the eleven prioritised TPs, ten were formed as the result of deiodination (either by deiodination, oxidative deiodination or intramolecular elimination). In the real surface water samples, median concentration of parent compounds was 110 ngL-1 reaching up to 6 µgL-1 for iomeprol while TPs were found at median concentration of 8 ngL-1, reaching up to 0.4 µgL-1 for iomeprol TP651-B. Here detection-based prioritization served as a crucial step to reduce the number of TPs to be identified and thereby reducing costs and time for the subsequent target analysis. This time-effective approach not only guaranties that the degradation products elucidated would be found, but also that they are environmentally relevant. In summary, the proposed screening approach facilitates the evaluation of the degradation of polar compounds at a real scale with a fast detection of TPs without prior availability of the standards. Approach used for detection and identification of TPs of ICM in Chapter 3 was an example of suspect screening where the suspect list of TPs was generated at lab-scale, In Chapter 4, the work started with a suspect screening of lamotrigine (LMG) and related compounds (its human metabolites, synthetic impurities and photoTPs) which were listed from the literature and searched in wastewater and surface water samples. As the result of suspect screening, LMG, three human metabolites and a LMG synthetic impurity (OXO-LMG) were detected in the screened samples. Preliminary results showed significantly higher concentrations of OXO-LMG in wastewater effluent, suggesting its formation in the WWTPs. However, biodegradation reactors amended with mixed liquor at neutral pH showed that LMG is resistant to biodegradation with only about 5 % elimination after 6 days. Since LMG is extensively and predominantly metabolised by phase II metabolism to its N2-glucuronide, this metabolite (LMG-N2-G) was degraded following the same experimental setup. Results showed that this metabolite was the actual source of the TP detected. Additionally, in batch experiments, LMG-N2-G was transformed, following pseudo-first kinetics, to three TPs as a result of i) deconjugation (to LMG), ii) oxidation of the glucuronic acid (to LMG-N2-G-TP430) and iii) amidine hydrolysis in combination with deconjugation (to OXO-LMG). In order to further rationalize the formation of the TP OXO-LMG, the stability of LMG-N2-G and related compounds was studied as a function of pH in the range of 4 – 9. Same as during biodegradation, LMG was stable across the entire pH range tested. However, LMG-N2-G was transformed to three TPs at neutral – basic pH. They were identified as TPs formed after hydrolysis of amidine and guanidine moieties. The third TP detected was an intermediated in the guanidine hydrolysis reaction. Kinetic experiments in wastewater samples at different concentration (20 and 200 nM) and pH (pH 6.5, 7, 8, 8.5 and 9) demonstrated that while the degradation constants were concentration independent, at higher pH, LMG-N2-G degraded at higher rate. The pH-dependent stability experiments of related compounds with different nitrogen N2-substituents on the 1,2,4-triazine ring showed that reaction of amidine and guanidine hydrolysis depends on imine tautomer equilibrium whose formation depends directly on the N2-supsitutent. LMG- N2-G major abiotic TP (amidine hydrolysis TP) was detected in hospital effluent and WWTP influent samples. Having in mind the concentrations of both biotic and abiotic TPs detected, a total mass balance at two- concentration levels batch reactors was closed at 86% and 102%, respectively. In three WWTPs total mass balance of LMG-N2-G ranged from 71-102%. Finally, LMG-N2-G and its TPs were detected in surface water samples with median concentration ranges of 23–186 ngL-1. The work presented in this chapter gives a new insight into the behaviour of glucuronides of pharmaceuticals, suggesting that they might also be sources of yet undiscovered, but environmentally relevant TPs.
Els productes farmacèutics, l'ús dels quals s'estén a nivell mundial, estan dissenyats per millorar la qualitat de vida de la societat i juguen un paper clau en el tractament i la prevenció de malalties, tant en homes com en animals. Aquests compostos químics es troben de forma ubíqua en el medi ambient. Això es deu principalment a les estacions depuradores d'aigua residual (EDARs), les quals no són capaces d'eliminar de manera eficient aquest tipus de compostos, ja que no estan dissenyades amb aquesta finalitat. Per tant, la presència de fàrmacs en el medi ambient està directament relacionada amb l'activitat humana. Un cop al medi ambient, l'estructura d'aquests compostos pot ser modificada per diferents processos biològics i abiòtics, generant-se així els que es coneixen com a productes de transformació (PTs). De fet, la transformació dels fàrmacs pot iniciar-se en alguns casos en el cos humà, després de la seva administració a causa de l'activitat metabòlica dels diferents enzims que posseeix l'home. Els metabòlits formats en aquests processos presenten algunes modificacions en les seves estructures químiques pel que fa al compost original, i, en conseqüència, unes propietats fisicoquímiques diferents. Un cop excretats, tant el fàrmac original no metabolitzat com els seus metabòlits arriben a les EDARs mitjançant la xarxa de sanejament municipal d'aigües residuals. La fracció d'aquests compostos que no s'elimina en els diferents tractaments realitzats en l'EDAR, es descarrega juntament amb l'efluent de la planta als aigües receptores. El gran nombre de transformacions que poden experimentar els fàrmacs en el seu cicle de vida a causa del seu metabolisme en el cos humà, la seva biotransformació per microorganismes i la seva fototransformació per llum solar, pot generar un nombre molt elevat de PTs en el medi ambient, i, per tant, la identificació dels mateixos, necessària per avaluar el destí dels fàrmacs en el medi ambient, és un desafiament. En el desenvolupament d'aquesta tesi es van aplicar dues aproximacions analítiques diferents: a)avaluació de perfils de PTs generats en experiments a escala de laboratori i b) anàlisi qualitativa dirigida suspect screening en mostres reals, tots dues basades en espectrometria de masses d'alta resolució (HRMS) per a la detecció i identificació de PTs de productes farmacèutics. L'aproximació d'avaluació de perfils de PTs en reactors a escala de laboratori es va aplicar per identificar productes de fototransformació (fotoPTs) de l'antiviral zanamivir (ZAN) en aigua superficial. L'aproximació de suspect screening es va aplicar per prioritzar i identificar fotoPTs de sis mitjans de contrast radiològics iodats (ICM) en aigua superficial. Finalment, una combinació de les dues aproximacions es va aplicar per detectar PTs de l’anticonvulsiu lamotrigina (LMG) i del seu principal metabòlit humà, el lamotrigina-N2-glucurònid (LMG- N2-G), resultants de la seva degradació tant en fangs activats com a reaccions d'hidròlisi a diferents valors de pH.

Development and application of target and non-target techniques for routine analysis, identification of transformation products, and characterization of unknown compounds in water matrices using liquid-chromatography high-resolution mass spectrometry (HRMS) were explored in this dissertation. A novel analytical method based on online-SPE-LC-HRMS was developed for the detection of 18 drugs of abuse (DOAs) in raw sewage water from a college campus. Results showed the presence of 14 DOAs for which amphetamine and 11-nor-9-carcoxy-THC (THC metabolite) were the most prevalent and had the highest potential consumption rates. A second study dedicated to the identification of transformation products (TPs) generated from DOAs was conducted using a combination of HR-MS/MS and metabolic identification and structural elucidation software. Findings confirmed the presence of multiple phase I and II DOA TPs (n=35) in raw sewage influents. Concentrations of all TPs were estimated based on the parent DOAs response factors, and used to calculate the percent mole fraction contributions of each TP to the parent concentrations. High abundance and frequency (compared to the parent drug) was determined for 9 of the TPs coming from drugs like oxycodone, morphine, codeine, methadone, LSD, cocaine, and MDEA. Non-target analysis using HRMS was explored as a tool to characterize, and compare a series of interconnected water matrices along a river system. Several thousands of formulae were generated using automated heuristic rules from the full-scan acquisition at 140,000 resolution. Samples were part of a trajectory covering upstream, effluent, effluent mixing zone, downstream, drinking water intake, and treated drinking water locations. Graphical representations of the data were used to evaluate commonalities among the system. Using this approach, a total of 64 recalcitrant components were identified throughout the samples downstream of the effluent release point. Using a combination of MS/MS and computer-aid software techniques 4 out of the 64 compounds were tentatively confirmed. In addition, comparison of drinking water intake and finalized treated drinking water sites showed the presence of 1,152 chemical entities that were common to both locations; and 1,857 that were unique to the treated drinking water. Therefore, this non-target technique could be used to identify the potential formation of treatment byproducts.

Aujourd’hui, la présence de pesticides dans les eaux fait l’objet d’une forte préoccupation car ils constituent un risque majeur pour la qualité des eaux. Ce travail s’est intéressé à étudier la transformation de deux herbicides de la famille des béta-tricétones, la tembotrione et la sulcotrione, lors de réactions de photolyse appliquées à leur devenir dans l’environnement et lors de l’utilisation du chlore et de l’ozone dans les filières de production d’eau potable. Dans un premier temps, la photolyse directe a été étudiée en condition de lumière polychromatique puis des expériences ont été réalisées en présence d’ions nitrate et de peroxyde d’hydrogène afin d’obtenir les constantes de réaction des molécules avec le radical hydroxyle. Dans un second temps, les constantes de réaction avec le chlore et l’ozone ont été déterminées afin de prédire leur cinétique de transformation. Des sous-produits de transformation ont été identifiés par LC-MS/MS et des mécanismes réactionnels ont été proposés. Des tests de toxicité utilisant la mesure de l’inhibition de la luminescence de Vibrio fischeri ont été réalisés en parallèle du suivi des sous-produits de transformation. Une augmentation de la toxicité a été observée lors de la photolyse et de la chloration de ces deux composés, tandis qu’une diminution a été notée au cours de l’ozonation. Enfin, une augmentation de la biodégradabilité des solutions de pesticides, déterminée par le test OCDE 301D, a été observée en cours d’ozonation et pour les premiers taux de photodégradation
Today, the presence of pesticides in water is a major concern and a threat to water quality. This work has focused on the behavior of two béta-triketones herbicides, tembotrione and sulcotrione, under photolysis with direct applications for their behavior in surface water resources, and also during chlorination and ozonation in drinking water treatment plant. First, direct photolysis was studied under polychromatic irradiations, and then experiments were conducted in the presence of nitrates and hydrogen peroxide in order to determine the rate constants of molecules with hydroxyl radical. Secondly, chlorination and ozonation kinetic rate constants were determined in order to predict their transformation kinetics. Transformation by-products were identified by LC-MS/MS and reaction mechanisms were proposed. Toxicity tests performed by using measurement of Vibrio fischeri luminescence inhibition were compared with the evolution of transformation by-products. An increase in toxicity was observed for both compounds during photolysis and chlorination, while it decreased under ozonation. Finally, an increase in biodegradability of pesticide solutions, determined by using the OCDE 310D test, was observed during ozonation and for the first yields of photolysis

L'objectif de ce travail était les composés odorantes clés de l'arôme du camenbert et de rechercher ceux qui dont susceptibles d'induire des compotements d'attrait ou de rejet chez les consommateurs. Un nouvel outil de CPG-O à huit voies synchrones a permis de repérer et d'identifier plus de 27 composés volatils odorants majeurs dans 9 fromages représentatifs de la diversité aromatique du camenbert. Un sous-ensemble de ces composés a été sélectionné pour appréhender les préférences qu'ils sont susceptibles d'induire chez des consommateurs de camenbert. Pour cela, nous avons réalisé deux études sur deux panels d'environ 180 consommateurs répartis selon trois "segments de préférence", selon qu'ils appréciaient les camenberts "peu affinés", "typés", ou "corsés". Ces études ont permis d'acquérir des informations sur les préférences induites "hors contexte produits" et en "contexte produit" et de mettre en evidence 8 composés "préférences actifs"

De nos jours, l’évaluation de la qualité d’un milieu naturel repose traditionnellement sur des analyses ciblant des molécules déjà connues pour être biologiquement actives. Cependant ces molécules ne reflètent que partiellement les effets biologiques observés et la complexité des matrices environnementales. Par ailleurs, les contaminants dans l’environnement subissent des phénomènes de dégradation (hydrolyse, biodégradation, photodégradation). Ces processus peuvent conduire à la génération de produits de transformation qui sont encore trop peu pris en compte dans l’évaluation du risque, alors qu’ils sont susceptibles d’occasionner des effets toxiques plus importants que les molécules mères. L’impact écotoxicologique de ces mélanges nécessite préalablement l’obtention d’une vision globale quant à la présence et au devenir de ces molécules.Dans ce contexte, ces travaux appliquent une méthodologie pluridisciplinaire afin d’identifier des molécules préoccupantes et pertinentes à surveiller. Pour cela, ce travail s’appuie sur des analyses ciblées ultra-traces, des outils bioanalytiques, et des analyses non ciblées, qui ont été appliqués sur des échantillons environnementaux ou issus d’études en laboratoire mimant l’évolution des matrices environnementales. Au final, cette étude a permis : (1) d’identifier des molécules préoccupantes en estuaire de Seine sur la base de leur occurrence et de leur persistance, (2) d’identifier de nouveaux produits de transformation et (3) d’identifier les molécules responsables d’activités biologiques dans des matrices très complexes
To date, environmental risk assessment is based on a restricted number of molecules assessed by targeted chemical analyses. However, this approach give a partial picture of co-occurrence of known and unknown compounds. Moreover, in the aquatic environment, chemicals are not completely mineralized and are subject to abiotic and biotic processes. Transformation products (TPs) can be more toxic and more persistent than the parent compound. However, TPs are not typically included in classical monitoring and risk assessment. Because of complexity and variability of these matrices and the restricted number of molecules focused in targeted chemical analyses, selection of relevant molecules for environmental monitoring is often laborious.In this context, an integrative approach was used in order to identify chemicals of concern for a classical monitoring. This work has been realized on environmental complex samples and on laboratory-generated samples. A combination of targeted, non-targeted analyses and in vitro bioassays was performed and allowed to: (1) identify polar chemical of concern in the Seine estuary on the basis of occurrence and persistence, (2) identify new transformation products and (3) identify compounds responsible of biological activity observed in complexes matrices

Thesis (M. Arch.)--Massachusetts Institute of Technology, Dept. of Architecture, 1992.
Includes bibliographical references (p. 102-103).
This thesis outlines a process of observation and transformation using formalized concepts about space, territory, and position as well as transfonnational rules implicit in these concepts. A set of references is analyzed and explained using a common graphical language which serves to locate the references within a continuum of spatial configurations. Transformational rules are developed which allow for the generation of new configurations. Some proposals are made about inhabiting these configurations and the process of formal observation and trtransformation is placed within the larger context of design.
by Edward C. Reifenstein.
M.Arch.

The aging processes of ink dyes in ballpoint inks were determined using modem chromatographic and spectrometric methods with their possible application. Two main methods were applied IP–HPLC method with photodiode array detection for ink dyes separation and LC–MS method for the identification of ink degradation products as a function of age, for this purpose the two methods were developed and validated. The aging processes of standard dyes as reference substances at different conditions (light and heat) were investigated. These processes were used then on ink entries on paper. Dye components and their degradation products were successfully separated and identified by their retention time, UV-visible absorption profiles and mass spectra. Different blue ballpoint inks were identified and discriminated by comparing their relative peaks areas (RPA) to obtain the aging profiles. Moreover, other types of ink (gel pens, fibre tips and different colours) were also analysed and discriminated successfully by the developed method. In addition, several months' entries from multiple inks entries exposed to normal sunlight and samples stored in the dark were compared. A typical and expected degradation of ink dyes was characterized by the de-alkylation process (loss of CH2– or CH2–CH3 groups). Crystal violet and Victoria blue dye groups, two major dyes used in ballpoint ink industries were thoroughly investigated. In this work, mass spectrometric methods were developed and validated to describe the ageing mechanism of ballpoint ink dyes. Furthermore, factors influencing the aging process (heat, light, natural aging factors) were studied. For real forensic cases the storage conditions of the paper and type of pen used should be known.

Thesis (M.S. in Software Engineering)--Naval Postgraduate School, March 2003.
Thesis advisor(s): Norman Schneidewind, Richard Riehle. Includes bibliographical references (p. 162-168). Also available online.

Malaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater. Screening of natural product libraries is known to have higher hit rates than synthetic chemical libraries. This elevated hit rate is somewhat attributed to the greater biodiversity available in natural products. Marine life is the most biodiverse system on the planet, containing 34 of the 36 known phyla of life, and is expected to be a rich source of novel chemotypes. In collaboration with the Harbor Branch Oceanographic Institute in Ft. Pierce we have screened a library of over 2,800 marine macroorganism peak fractions against Plasmodium falciparum using the SYBR green I fluorescence-based assay. In this screening process we have identified six compounds from five novel chemical scaffolds all of which have low micromolar to submicromolar IC50 values and excellent selectivity indices. Additionally, one of these chemical scaffolds, the bis(indolyl)imidazole, was selected for further in vitro pharmacological and structure-activity relationship (SAR) profiling, key steps in the challenging process of identifying a new antimalarial drug lead compound.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology

The work presented in this thesis is based on a growing interest in new technology and new business models in forestry that will contribute to increased revenue and greater acceptance for forest utilization while at the same time ensuring sustainability. Thus, the aim of the research presented in this thesis included development of methods, strategies and tools that support technological advancements in the area of forestry, as well as understanding the effects of introducing Functional Products in this field. The research has been conducted through analysis of the current forestry situation followed by development of a future scenario where Functional Products are used in forestry. The gap between the current situation and the future scenario was analysed and used as a base for the technology development. The results include opportunities and challenges that would arise if a Functional Product were introduced to the field of forestry, the foundation to enable clearcut obstacle identification and the theoretical possibilities for increasing site preparation effectiveness in case where ideal obstacle identification has already been conducted on the site. Future work includes development of methods and technology to enable clearcut identification, as well as addressing challenges of Functional Products in forestry.By introducing new ways of doing business through Functional Products, as well as recognising the benefit of obstacle avoidance in silvicultural operations such as site preparation or wood extraction, some of the rather urgent needs for sustainability are addressed. Paper A illustrates the opportunities and challenges that would arise if a Functional Product were introduced to the field of forestry.Paper B is the foundation for the future development of techniques enabling obstacle detection at clearcuts. It communicates the different aspects of clearcut obstacles that must be considered for obstacle detection at clearcuts to work efficiently during site preparation.Paper C shows the theoretical possibilities for increasing site preparation effectiveness in cases where ideal obstacle identification has already been conducted.
The work presented in this thesis is based on a growing interest of new technology and new business models in forestry to increase economic revenue, find higher acceptance for forest utilization and at the same time ensure sustainability. Thus, the aim for the research presented in this thesis included development of methods, strategies and tools that support technological advancements in the area of forestry, as well as understanding the effects of introducing Functional Products in this field. The research has been conducted through analysis of the current forestry situation followed by development of a future scenario where Functional Products are used in forestry. The gap between the current situation and the future scenario was analysed and used as a base for the technology development.. The results include opportunities and challenges if a Functional Product would be introduced to the field of forestry, the foundation to enable clearcut obstacle identification and the theoretical possibilities to increase site preparation effectiveness if ideal obstacle identification already has been conducted on the site. Future work include development of methods and technology to enable clearcut identification, as well as addressing challenges of Functional Products in forestry.
Godkänd; 2013; 20130920 (haklid); Tillkännagivande licentiatseminarium 2013-10-25 Nedanstående person kommer att hålla licentiatseminarium för avläggande av teknologie licentiatexamen. Namn: Håkan Lideskog Ämne: Datorstödd maskinkonstruktion/Computer Aided Design Uppsats: Clearcut Obstacle Identification and Functional Products Promoting Sustainable Forestry Examinator: Associate Professor Magnus Karlberg, Institutionen för teknikvetenskap och matematik, Luleå tekniska universitet Diskutant: Associate Professor Ola Lindroos, Sveriges lantbruksuniversitet, Umeå Tid: Fredag den 15 november 2013 kl 09.00 Plats: E632, Luleå tekniska universitet
Fastelaboratoriet - VINNEXC

Mestrado em Métodos Biomoleculares
Cardiolipina (CL) é um fosfolípido de estrutura complexa, que se encontra quase exclusivamente na membrana interna da mitocôndria, onde se encontra associada a diferentes complexos da cadeia respiratória. Pela sua localização, este fosfolípido desempenha um importante papel no metabolismo energético mitocôndrial. Alterações na cardiolipina nomeadamente por modificações oxidativas têm sido relacionadas com a apoptose celular e com várias condições patológicas, particularmente nas doenças neurodegenerativas. A estrutura da CL apresenta quatro cadeias de ácidos gordos que podem diversificar no comprimento e no grau de insaturação, é muito susceptível a danos oxidativos por espécies reactivas de oxigénio (ROS). A sua localização, na mitocôndria aumenta a probabilidade desta sofrer oxidação, uma vez que existe uma considerável produção de ROS na membrana interna mitocôndrial. ROS estão envolvidas no stress oxidativo em diferentes biomoleculas, nomeadamente nos lípidos, levando a modificações na sua estrutura e consequentemente, à perda da sua função. Apesar da importância da oxidação da CL e das suas possíveis consequências biológicas, existe um conhecimento limitado em relação aos produtos de oxidação formados em condições de stress oxidativo. Neste estudo, a espectrometria de massa e a espectrometria de massa acoplada à cromatografia liquida (LC-ESI-MS) foram utilizados para identificar modificações oxidativas especificas da etra-linoleoil CL induzidas pelo radical hidroxilo através da reacção de Fenton (H2O2 and Fe2+). Os resultados obtidos neste estudo permitiram identificar pela primeira vez diversos produtos de oxidação de cadeia curta e também outros de cadeia longa durante a oxidação da CL. Os produtos de oxidação de cadeia curta resultam de uma clivagem do ácido gordo oxidado, conduzindo á formação de ácido gordos de cadeia curta com terminal aldeído, e ácidos dicarboxilico, observados em MS na forma de iões [M-H]- e [M-2H]2-. Estes resultados permitiram detectar alguns destes produtos de oxidação na mitocôndria dos rins dos ratos tratados com gentamicina, com nefropatia confirmada. A identificação detalhada da fragmentação dos produtos de cadeia longa por LC-MS/MS permitiu a diferenciação de isómeros, e a identificação do local de oxidação para CL com 2, 4, 6 e 8 átomos de oxigénios. Este trabalho permitiu identificação de iões produto específicos que permitem a sua atribuição inequívoca, que será fundamental para a identificação destas espécies em amostras biológicas. ABSTRACT: Cardiolipin (CL) is a phospholipid with a complex structure, found almost exclusively in the inner mitochondrial membrane, where it is found associated with several different complexes of the respiratory chain. Because of its localization, this phospholipid performs an important role in the mitocôndrial energetic metabolism. Alteration of CL namely by oxidative modifications has been related with various pathological conditions, particularly in neurodegenerative diseases. CL structure bears four chains of fatty acids that can diversify in length and degree of unsaturation; it is susceptible to oxidative damage by reactive oxygen species (ROS). Their location, in the mitochondria, makes them even more likely to be oxidized, since that there is a considerable production of ROS in the inner mitochondrial membrane. ROS are involved in oxidative stress modifications of different biomolecules, namely lipids, leading to changes in their structure and in consequence, loss of their function. In spite of the importance of CL oxidation and it biological consequence, there is a limited knowledge of the oxidation products of CL formed under oxidative stress. In this study, mass spectrometry and mass spectrometry coupled with liquid chromatography (LC-MS) was used to identify the specific oxidative modifications of tetra-linoleoyl CL induced by the hydroxyl radical generated under Fenton reaction conditions (H2O2 and Fe2+). The results obtained with this study allowed us to identify, for the first time, several short chain oxidation products along with some long chain products during the oxidation of CL. Short-chain oxidation products resulted from a cleavage of the oxidized fatty acid, leading to the formation of short chain fatty acids with aldehyde terminal, and dicarboxylic acids, observed in MS in the form of the ions [M-H]- and [M- 2H]2-. These results allowed us to detect some of these oxidation products in the mitochondria of kidneys obtained from rats treated with gentamicin, with confirmed nephropathy. The detailed identification of the fragmentation of the long chain products by LC-MS/MS allowed us to differentiate isomers, and the identification of the oxidation location for the CL with 2, 4, 6 and 8 oxygen atoms. This work allowed the identification of specific product ions which allow their unequivocal assignment that will be paramount for the identification of these species in biological samples.

Sous des conditions de croissance particulières, certaines bactéries sont capables d'entrer en état de « compétence » pour la transformation naturelle, c'est-à-dire d'exprimer un ensemble de gènes nécessaires à la mise en place d'un système d'import d'ADN exogène dont l'intégration conduit à une transformation génétique et phénotypique. C'est le cas de Legionella pneumophila, bactérie environnementale et agent étiologique de la légionellose. La transformation naturelle a potentiellement participé à l'évolution du génôme de L. pneumophila.Ainsi, l'objectif premier de cette thèse était de décrire les composants principaux du système de transformation naturelle de L. pneumophila, ainsi que son activation et rôle potentiel dans la relation de la bactérie avec ses hôtes. Des méthodes d'analyse transcriptomique et de mutagénèse dirigée ont permis d'identifier les principaux gènes impliqués dans la mise en place du système de transformation naturelle qui, de façon cohérente avec un rôle adaptatif, ne semble pas impliqué dans la virulence bactérienne. Le système inclut un pilus de transformation, structure fréquemment observée chez les espèces naturellement transformables. Le rôle de la protéine structurale MreB dans le mécanisme de transformation naturelle a également été étudié. En proposant un premier modèle du système de transformation naturelle de L. pneumophila, ces travaux ouvrent la voie à une analyse plus détaillée de la dynamique du système et, plus généralement, à une meilleure compréhension des mécanismes de la transformation naturelle chez les bactéries Gram-négatives
Under certain growth conditions, some bacteria are able to develop a « competence » state for natural transformation, that is, to express a panel of genes involved in the assembly of a DNA uptake system that allows bacteria to take up and recombine free exogenous DNA, leading to a genetic and phenotypic transformation. Natural transformation may have played a role in the evolution of the L. pneumophila genome.Thus, the main objective of this work was to describe the main components of the L. pneumophila DNA uptake system and to investigate its role regarding the host-pathogen interaction. Transcriptomic analysis and directed mutagenesis permitted to identify the main components of the system which is not involved in bacterial virulence. The system include a transformation pilus that is a structure frequently found in transformable species. The role of the structural protein MreB has also been investigated.By describing a first model of the natural transformation system of L. pneumophila, this work paves the way to a deeper analysis of the system dynamics and, more generally, to a better understanding of natural transformation in Gram-negative species

The measurement of materials properties and the control of processing parameters is important for both materials development and quality control. Determination of these parameters is sometimes advantageous if done by means of microstructural characterisation as here additional information may be gained from the sample. These values, e.g. the grain size or the volume fraction of the present microconstituents, may allow the correlation with mechanical properties or processing properties. Unfortunately, the diffusional and displacive transformation products, martensite and bainite, exhibit very fine microstructures with a low contrast, so that conventional microstructural image analysis cannot be applied readily to distinguish these structures. As an alternative microstructural characterisation technique texture analysis based on Haralick parameters calculated from second order grey value statistics was successfully applied. It has been shown that the analysis of large sample areas can be done automatically enabling the correlation of the texture data with the respective local microhardness using a neural network. The analysis is limited due to the dependence of the texture parameters on preparation and imaging conditions. A more detailed understanding of the individual Haralick parameters will be the basis to extend the method to a correlation with other properties of the sample that may be not easily accessible by physical testing, such as toughness.

3,6,7-trihydroxycoumarin C11 (2.14) was first isolated from the green alga Dasycladus vermicularis in 1983. C11 and 3,7,8-trihydroxycoumarin C21 (2.15), alongside their precursors C12 (2.18) and C22 (2.20), were synthesized for a target-based screen for anti-HCV drugs, where ideal hits eliminate fluorescence signals by inhibiting the proteolytic activity of HCV NS3pro/Pep4A against a synthetic peptide “BS-IQFS”. With C12 and C22 serving as negative controls, C11 and C21 inhibited the NS3pro/Pep4A activity in vitro. The IC₅₀’s of C11 and C21 were 3.07 μM and 2.10 μM, respectively. A bioassay-guided fractionation identified sintokamides A – E (3.11 – 3.15) from extracts of the sponge Dysidea sp. in 2008. In a phenotypic screen, the chlorinated dipeptides showed strong to modest inhibition of luciferase activity caused by AR NTD transactivation in LNCaP cells. Larger quantities of sintokamides A and B were isolated from the sponge for further biological study. After developing a practical synthetic route, a comprehensive SAR of the sintokamides was available from the in vitro activities of 29 synthetic analogues/precursors based on a 1,17-dinorsintokamide skeleton. LPY26 [(4R,10R)-3.233] showed the best biological activity in the synthetic analogues prepared to date and it was selected as a drug lead. Mechanism of action study using synthetic probes LPY30 (4.7) and LPY31 (4.8) revealed that the hexachlorinated 1,17-dinorsintokamides covalently bound to the AR, but not to the same AF1 region in the AR NTD as EPI-001 (3.8). The structure of latonduine A (5.1) isolated from the sponge Stylissa carteri and its total synthesis were published in 2003. Later, latonduine A was shown to be active in a phenotypic screen to find drug leads for the treatment of cystic fibrosis caused by the F508del mutation. Latonduine A could efficiently correct immunofluorescent F508del-CFTR trafficking from the endoplasmic reticulum to the plasma membrane in the engineered cells. Synthetic latonduine A and N-biotinylated latonduine A (5.17) were prepared to support biological studies aimed at identifying its cellular protein target(s). These studies culminated in the finding that latonduine A is an inhibitor of PARP-3 with an EC₅₀ of 400 pM in CFBE41o­- cells.
Science, Faculty of
Chemistry, Department of
Graduate

Flux of ferrous iron (FeII) to the estuarine environment, whether from bottom sediments or via groundwater seepage, has been identified as a potentially important source of iron required for the development and sustenance of nuisance blooms of the toxic cyanobacteria Lyngbya majuscula in Moreton Bay, Queensland. However the rapid oxidation of FeII in seawater imparts importance to the resultant form of ferric iron (FeIII). Oxidation of FeII in the presence of natural organic matter (NOM) results in a mixture of FeIII-NOM complex and amorphous ferric oxides (AFO). The fate of these oxidation products has implications for the supply of iron to L. majuscula where transformations over time scales of hours are likely to be important. In this thesis the process of oxidation of FeII in seawater in the presence of NOM and the subsequent transformations of the products of oxidation are investigated. UV and visible spectroscopic techniques were used to monitor the production of organically complexed FeIII for both NOM and a model organic compound. Kinetic modelling of data facilitated the examination of key reactions, especially those involving AFO. Controls on the reactivity and aging of AFO were investigated using two different dissolution reactions to measure reactivity. Light scattering techniques were used to probe the structure of AFO and X-ray absorption spectroscopy was used to examine the coordination environment of Fe centres within AFO. Analysis of a kinetic model of iron transformations parameterised using the best available knowledge revealed large uncertainty surrounding the role of ligand classes in complex formation and dissociation and the role of AFO in both formation of oxidation products and the subsequent decay of organically complexed FeIII. Laboratory studies demonstrated that, within a wide range of initial concentrations, unstable mixtures of FeIII-NOM and AFO are produced from the oxidation of FeII in seawater containing NOM and that the organic complexes immediately commence transformation to AFO. Simulation using numerical kinetic modelling of the processes investigated indicated that AFO has a significant role in the processes of formation of oxidation products and dissociation of organically complexed FeIII. The rapid aging that AFO was recognised to undergo was successfully incorporated into the model though whether the aging was due toeither 1) increased coordination of Fe centres or 2) decreased Fe centre accessibility due to aggregation could not be ascertained from the model results. However, together with information regarding the coordination environment of the Fe centres and the particle and fractal structure of the aggregates, aggregation was considered most likely to be the factor responsible for the observed and modelled decreases in AFO reactivity.

Advanced line pipe steels are microalloyed with Nb to promote the formation of complex microstructures leading to the required mechanical properties. In addition to its role during thermo-mechanical processing Nb in solution affects significantly the austenite decomposition kinetics and the resulting microstructure. A systematic study has been carried out to quantify the influence of Nb on the austenite decomposition in a commercial X80 line pipe steel containing 0.06C- 0.034Nb- 0.24Mo- 0.012Ti- 0.0005N (in wt. %) for a variety of austenite grain sizes and cooling rates that are relevant for the heat affected zone. To quantify the influence of Nb on transformation kinetics, two distinct amounts of Nb in solution were obtained through carefully designed reheat treatments prior to continuous cooling transformation tests conducted with a Gleeble 3500. The amount of Nb in solution was quantified based on ageing experiments. To investigate the combined influence of Nb and Mo on austenite decomposition two laboratory cast low-carbon steels containing 0.06 wt. % Nb and 0.045 wt. % Nb and 0.145 wt. % Mo, respectively, were compared with the X80 steel. The obtained transformation products include irregular ferrite, upper and lower bainite and martensite/ austenite. Electron backscatter diffraction (EBSD) was used to distinguish upper and lower bainite based on their orientation relationship with the prior austenite and to quantify microstructural features which are relevant for the tensile properties. Based on the quantitative measures obtained from the EBSD analysis structure-property relationships were developed to predict the yield strength, uniform elongation and ultimate tensile strength of the studied X80 line pipe steel. An effective grain size was defined including martensite/ austenite to consider grain refinement and the kernel average misorientation was used to quantify dislocation strengthening. A phenomenological model was applied and modified to capture the austenite decomposition of the X80 steel considering the effect of prior austenite grain size, amount of Nb in solution and cooling rate. The amount of martensite/ austenite depends on the surrounding matrix microstructure and is predicted as a function of the transformation start temperature with an empirical fit.
Applied Science, Faculty of
Materials Engineering, Department of
Graduate

An estimated 500 million cases of malaria occur each year. The increasing prevalence of drug resistant strains of Plasmodium in most malaria endemic areas has significantly reduced the efficacy of current antimalarial drugs for prophylaxis and treatment of this disease. Therefore, discovery of new, inexpensive, and effective drugs are urgently needed to combat this disease. Marine biodiversity is an enormous source of novel chemical entities and has been barely investigated for antimalarial drug discovery. In an effort to discover novel therapeutics for malaria, we studied the antimalarial activities of a unique marine-derived peak fraction library provided by Harbor Branch Oceanographic Institute (HBOI). Within this unique library, we have screened 2,830 marine natural product (MNP) peak fractions through a medium throughput screening effort utilizing the SYBR Green-I fluorescence based assay, and have identified 253 fractions that exhibit antimalarial activity. From those inhibiting fractions we have identified twenty species of marine organisms that inhibit Plasmodium falciparum growth, from which thirty-five fractions were selected for further study. Among those thirty-five, eighty-three percent were also found to inhibit the chloroquine resistant strain of P. falciparum, Dd2. The most potent inhibitors were then screened for their cytotoxic properties using the MTT cell viability assay. Among the samples that exhibited potent inhibition of P. falciparum growth were fractions derived from a sponge of the genus Spongosorites sp.. This genus of sponge has been reported to contain the nortopsentin and topsentin class of bis-indole imidazole alkaloids. Nortopsentin A inhibited the parasite growth at the trophozoite stage with an IC[sub]50 value of 1.6micrometer]. This is the first report of antimalarial activity for this class of compound.
ID: 030423269; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2010.; Includes bibliographical references (p. 56-61).
M.S.
Masters
Burnett School of Biomedical Sciences
Medicine

This work presents a systematic approach to the identification of possible by-products in process synthesis. The objective is to discover all possible reactions that might take place in a reaction system, in addition to already known main reactions. These side reactions may generate species in minor quantities which can be difficult to separate and therefore be into the recycle system. These species can thus remain in the process and accumulate. A literature survey indicated that generic languages for chemical reactions have been developed recently by other workers. These have adopted the concepts of fundamental chemistry with the aim of identifying major chemical reactions. For the purpose of the present work, strict interpretation of chemistry was not considered necessary since the aim was to generate minor species which might be formed. The work covers aspects of molecular representation, reaction generation and thermodynamic evaluation for screening. Molecular properties are estimated using group contribution methods. Unlikely species are ruled out using both stoichiometric and thermodynamic tests. The approach was first tested by comparison with experimental work, on the partial wet oxidation of p-coumaric acid and alkylation of toluene with ethanol. This shows favourable results. Process case studies were then undertaken including vinyl chloride monomer and the preparation of R-134a and R-22. These processes have been used to study the effect of by-product build-up. The study involved separation simulation to investigate the separability of the components. This confirmed that many potential by-products would be difficult to separate and might tend to accumulate in recycles. The results show that the approach has a promise for identifying possible side reactions, by-products and the problems that they will cause in a continuous process. Thus in the long term the approach affects the possibility of reducing and eliminating pilot plant studies in moving from laboratories to full scale processes.

Following the application of pesticides during normal agricultural practice these compounds can degrade to form transformation products. When assessing the risks posed by pesticides it is important to include any additional risks posed by these compounds. Current guidance within the EU suggests that data requirements for transformation products during the risk assessment do not necessarily need to be addressed with experimental studies but alternative techniques can be explored and used. Therefore the aim of this research was to investigate and develop pragmatic approaches for assessing the fate and effects of transformation products in the absence of experimentally determined data. Approaches designed to provide information on the physico-chemical properties, environmental parameters, ecotoxicology and toxicology of pesticide transformation products are explored and evaluated, and recommendations made on how to obtain the most appropriate estimates of these factors. Hydrophobicity, dissociation constant, soil sorption, daphnid aquatic ecotoxicology and rat oral lethality can all be estimated with confidence. Moreover, approaches were developed to I) indicate whether a transformation product may exhibit pesticidal activity and subsequently estimate its acute aquatic ecotoxicity in the absence of experimental data, 2) combine well known techniques and experimental data to obtain estimates of transformation product mutagenicity with limited risk of obtaining false negatives and 3) prioritise transformation products of most concern to drinking water supplies and its consumers. Overall, recommendations are made throughout this thesis on appropriate approaches and methods for generating estimates of transformation product properties, ecotoxicity and toxicity for use in risk assessment and prioritisation frameworks.

This thesis represents a continuation of the work on the isolation and structure elucidation of potential drug leads from terrestrial fungal sources that the natural products group at the University of Canterbury has been engaged in. Capillary NMR spectroscopy was involved in the research as the main tool for dereplication and elucidating the structures of novel bioactive compounds as well as for biosynthetic studies. Eleven new compounds including five cyclic peptides, four related pyrones and two diketopiperazines were isolated from the extract of Aspergillus sp. of endophyte collected from Malaysia. The five peptides F8268-A-1 to F8268-A-5 showed excellent P388 (HCT116 (ATCC CCL-247) and human breast cancer, MCF7 (ATCC HTB-22)) activities. Two of the peptides F8268-A-3 and F8268-A-5 were 4,000 times more active when compared with commercial drugs (fluorouracil, cisplatin and tamoxifen). The partial stereochemistries of F8268-A-2 and F8268-A-3 were established by Marfey’s method. Four related pyrones isolated from the same extract were also shown to have good P388 activities. They are related to the known compound NF00659A3. The relative stereochemistries were established from NOSEY experiments and the energy-minimised (MM2) model created using CHEM 3D software. Two new diketopiperazines, F7474-D3 and F7474-D11, also isolated from the Aspergillus extract did not show activity in the P388 assay. F7474-D11 contained the amino acid Me-kynurenine which is the first report of this from a natural source. The absolute stereochemistry of F7474-D11 was elucidated by Marfey’s method. The other diketopiperazine F7474-D3 was similar to the known compound lumpidin, and combined use of ROESY NMR and Marfey’s method established that the constituent amino acids had the unusual R configuration. Dereplication has been greatly improved by the application of capillary NMR. For example, the HPLC analysis and UV library searching of compounds from extracts F8095 and F7855 suggested they contained related compounds belonging to the lasiodiplodin family. However, CapNMR spectroscopic analysis and AntiMarin database searching revealed that the compounds from F8095 were all known polyesters while the compounds from F7855 did belong to the lasiodiplodin family. Two new lasiodiplodins were found in the F7855 extract, (3R,4R)-4-hydroxy-de-O-methyl-lasiodiplodin (F7855-4) and (E)-9-etheno-de-O-methyl-lasiodiplodin (F7855-6). The relative stereochemistries were elucidated from NMR coupling constant analyses. Two new dimers (F7090-A and F7090-B) were elucidated from a New Zealand fungal endophyte. The differences between these two dimers was their stereochemistries. F7090-A had the same stereochemistries for the three stereocentres in both parts, while the stereochemistry of F7090-B was different in the two parts of the dimer. Biosynthetic studies were also carried out using CapNMR methodology. A known compound tetrahydrofuran A and a new compound tetrahydrofuran B from an unidentified New Zealand fungus were used for this study. For the first time an INADEQUATE NMR experiment was successfully carried out using CapNMR spectroscopy, thus demonstrating the capability for carrying out biosynthetic studies on a very small scale (<200 μg of ¹³C-labelled compound). The implementation of efficient dereplication procedures with CapNMR methodology is paramount for those working in the field of natural products. The recent advances that have been made in the dereplication process in the natural products group at the University of Canterbury are given using examples from this research and where necessary from other group members.

In the first of the metalforming operations considered, that of plane-strain side-pressing, the finite-element predicted deformation behaviour of 60-40 brass and 7075 aluminium alloy has been compared with experiment. Both visioplasticity and hardness studies have been performed. The macrohardness survey was found to be the most appropriate in validating the use of the finite-element technique. The numerical calculations of the local stress and strain distributions are then used with a number of previously published continuum fracture criteria to predict the fracture initiation sites. For certain of the successful criteria the level of deformation at fracture has also been predicted using critical values of fracture found experimentally from an axisymmetric tensile test. For the operation of plane-strain side-pressing, very good agreement with both the experimental fracture initiation site, and the level of deformation at fracture, is reported for the criterion of a critical value of generalised plastic strain at fracture, and the equivalent criterion of generalised plastic work. The second metalforming operation considered is simple upsetting. The fracture initiation behaviour of 60-40 brass has been examined for specimens of the four initial aspect ratios of 0.5, 1.0, 1.5 and 2.0. Again, the generalised plastic strain/work criterion has successfully predicted the experimental fracture initiation sites in all cases. However, satisfactory levels of deformation at fracture predictions have only been found for the two lower aspect ratio specimens. A small range of strip compression and tension operations on 60-40 brass compose the third type of metalforming operation considered in this thesis. Yet again the generalised plastic strain/work criterion has successfully predicted the fracture initiation site found experimentally but not the level of deformation at fracture. Finally, the axisymmetric extrusion of 60-40 brass and 7075 aluminium alloy is examined. For the brass using the generalised plastic strain/work criterion, good agreement with experiment has been found for both the fracture initiation site and level of deformation at fracture found experimentally. However, for the aluminium alloy only the correct fracture initiation site has been found. In summary, it appears that the fracture criterion of a critical value of generalised plastic strain/work has successfully predicted the fracture initiation site found experimentally in all the operations considered in this thesis but has been unable to consistently predict the correct level of deformation at fracture. These differences in the predicted and experimental level of deformation at fracture cannot be explained with reference to the finite-element calculated levels of hydrostatic stress. Further work is necessary to explain this difference.

Doctor of Philosophy
Department of Statistics
Gary L. Gadbury
High-throughput metabolite analysis is an approach used by biologists seeking to identify the functions of genes. A mutation in a gene encoding an enzyme is expected to alter the level of the metabolites which serve as the enzyme’s reactant(s) (also known as substrate) and product(s). To find the function of a mutated gene, metabolite data from a wild-type organism and a mutant are compared and candidate reactants and products are identified. The screening principle is that the concentration of reactants will be higher and the concentration of products will be lower in the mutant than in wild type. This is because the mutation reduces the reaction between the reactant and the product in the mutant organism. Based upon this principle, we suggest a method to screen the possible lipid reactant and product pairs related to a mutation affecting an unknown reaction. Some numerical facts are given for the treatment means for the lipid pairs in each treatment group, and relations between the means are found for the paired lipids. A set of statistics from the relations between the means of the lipid pairs is derived. Reactant and product lipid pairs associated with specific mutations are used to assess the results. We have explored four methods using the test statistics to obtain a list of potential reactant-product pairs affected by the mutation. The first method uses the parametric bootstrap to obtain an empirical null distribution of the test statistic and a technique to identify a family of distributions and corresponding parameter estimates for modeling the null distribution. The second method uses a mixture of normal distributions to model the empirical bootstrap null. The third method uses a normal mixture model with multiple components to model the entire distribution of test statistics from all pairs of lipids. The argument is made that, for some cases, one of the model components is that for lipid pairs affected by the mutation while the other components model the null distribution. The fourth method uses a two-way ANOVA model with an interaction term to find the relations between the mean concentrations and the role of a lipid as a reactant or product in a specific lipid pair. The goal of all methods is to identify a list of findings by false discovery techniques. Finally a simulation technique is proposed to evaluate properties of statistical methods for identifying candidate reactant-product pairs.

This thesis is about double product integrals with pseudo rotational generator, and aims to exhibit them as unitary implementors of Bogolubov transformations. We further introduce these concepts in this abstract and describe their roles in the thesis's chapters. The notion of product integral, (simple product integral, not double) is not a new one, but is unfamiliar to many a mathematician. Product integrals were first investigated by Volterra in the nineteenth century. Though often regarded as merely a notation for solutions of differential equations, they provide a priori a multiplicative analogue of the additive integration theories of Riemann, Stieltjes and Lebesgue. See Slavik [2007] for a historical overview of the subject. Extensions of the theory of product integrals to multiplicative versions of Ito and especially quantum Ito calculus were first studied by Hudson, Ion and Parthasarathy in the 1980's, Hudson et al. [1982]. The first developments of double product integrals was a theory of an algebraic kind developed by Hudson and Pulmannova motivated by the study of the solution of the quantum Yang-Baxter equation by the construction of quantum groups, see Hudson and Pulmaanova [2005]. This was a purely algebraic theory based on formal power series in a formal parameter. However, there also exists a developing analytic theory of double product integral. This thesis contributes to this analytic theory. The first papers in that direction are Hudson [2005b] and Hudson and Jones [2012]. Other motivations include quantum extension of Girsanov's theorem and hence a quantum version of the Black-Scholes model in finance. They may also provide a general model for causal interactions in noisy environments in quantum physics. From a different direction "causal" double products, (see Hudson [2005b]), have become of interest in connection with quantum versions of the Levy area, and in particular quantum Levy area formula (Hudson [2011] and Chen and Hudson [2013]) for its characteristic function. There is a close association of causal double products with the double products of rectangular type (Hudson and Jones [2012] pp 3). For this reason it is of interest to study "forwardforward" rectangular double products. In the first chapter we give our notation which will be used in the following chapters and we introduce some simple double products and show heuristically that they are the solution of two different quantum stochastic differential equations. For each example the order in which the products are taken is shown to be unimportant; either calculation gives the same answer. This is in fact a consequence of the so called multiplicative Fubini Theorem Hudson and Pulmaanova [2005]. In Chapter two we formally introduce the notion of product integral as a solution of two particular quantum stochastic differential equations. In Chapter three we introduce the Fock representation of the canonical commutation relations, and discuss the Stone-von Neumann uniqueness theorem. We define the notion of Bogolubov transformation (often called a symplectic automorphism, see Parthasarathy [1992] for example), implementation of these transformations by an implementor (a unitary operator) and introduce Shale's theorem which will be relevant to the following chapters. For an alternative coverage of Shale's Theorem, symplectic automorphism and their implementors see Derezinski [2003]. In Chapter four we study double product integrals of the pseudo rotational type. This is in contrast to double product integrals of the rotational type that have been studied in (Hudson and Jones [2012] and Hudson [2005b]). The notation of the product integral is suggestive of a natural discretisation scheme where the infinitesimals are replaced by discrete increments i.e. discretised creation and annihilation operators of quantum mechanics. Because of a weak commutativity condition, between the discretised creation and annihilation operators corresponding on different subintervals of R, the order of the factors of the product are unimportant (Hudson [2005a]), and hence the discrete product is well defined; we call this result the discrete multiplicative Fubini Theorem. It is also the case that the order in which the products are taken in the continuous (non-discretised case) does not matter (Hudson [2005a], Hudson and Jones [2012]). The resulting discrete double product is shown to be the implementor (a unitary operator) of a Bogolubov transformation acting on discretised creation and annihilation operators (Bogolubov transformations are invertible real linear operators on a Hilbert space that preserve the imaginary part of the inner product, but here we may regard them equivalently as liner transformations acting directly on creation and annihilations operators but preserving adjointness and commutation relations). Unitary operators on the same Hilbert space are a subgroup of the group of Bogolubov transformations. Essentially Bogolubov transformations are used to construct new canonical pairs from old ones (In the literature Bogolubov transformations are often called symplectic automorphisms). The aforementioned Bogolubov transformation (acting on the discretised creation and annihilation operators) can be embedded into the space L2(R+) L2(R+) and limits can be taken resulting in a limiting Bogolubov transformation in the space L2(R+) L2(R+). It has also been shown that the resulting family of Bogolubov transformation has three important properties, namely bi-evolution, shift covariance and time-reversal covariance, see (Hudson [2007]) for a detailed description of these properties. Subsequently we show rigorously that this transformation really is a Bogolubov transformation. We remark that these transformations are Hilbert-Schmidt perturbations of the identity map and satisfy a criterion specified by Shale's theorem. By Shale's theorem we then know that each Bogolubov transformation is implemented in the Fock representation of the CCR. We also compute the constituent kernels of the integral operators making up the Hilbert-Schmidt operators involved in the Bogolubov transformations, and show that the order in which the approximating discrete products are taken has no bearing on the final Bogolubov transformation got by the limiting procedure, as would be expected from the multiplicative Fubini Theorem. In Chapter five we generalise the canonical form of the double product studied in Chapter four by the use of gauge transformations. We show that all the theory of Chapter four carries over to these generalised double product integrals. This is because there is unitary equivalence between the Bogolubov transformation got from the generalised canonical form of the double product and the corresponding original one. In Chapter six we make progress towards showing that a system of implementors of this family of Bogolubov transformations can be found which inherits properties of the original family such as being a bi-evolution and being covariant under shifts. We make use of Shales theorem (Parthasarathy [1992] and Derezinski [2003]). More specifically, Shale's theorem ensures that each Bogolubov transformation of our system is implemented by a unitary operator which is unique to with multiplicaiton by a scalar of modulus 1. We expect that there is a unique system of implementors, which is a bi-evolution, shift covariant, and time reversal covariant (i.e. which inherits the properties of the corresponding system of Bogolubov transformation). This is partly on-going research. We also expect the implementor of the Bogolubov transformation to be the original double product. In Evans [1988], Evan's showed that the the implementor of a Bogolubov transformation in the simple product case is indeed the simple product. If given more time it might be possible to adapt Evan's result to the double product case.

Orientadores: Albina Messias de Almeida Milani Altemani, Marinus Antonius Jacobus Hermsen
Texto em português e inglês
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-24T20:49:21Z (GMT). No. of bitstreams: 1 Costa_AnaFlaviadeMattos_D.pdf: 73992592 bytes, checksum: 3b90deaed579b13c568eeda812a10ef2 (MD5) Previous issue date: 2014
Resumo: O carcinoma adenóide cístico pode raramente sofrer desdiferenciação, um fenômeno também referido como transformação para alto grau. Contudo, alguns casos de carcinoma adenóide cístico foram descritos mostrando transformação para adenocarcinomas que não são pobremente diferenciados, indicando que a transformação para alto grau pode não refletir necessariamente um estágio mais avançado da progressão tumoral, mas sim uma transformação em uma outra forma histológica, que pode abranger um amplo espectro de carcinomas em termos de agressividade. Inicialmente, investigamos a expressão das proteínas reguladas pela hipóxia (HIF-1?, VEGF, GLUT-1 e CD105), dado que a hipóxia contribui para a agressividade tumoral e, pode, também, promover um fenótipo desdiferenciado em certos tipos de câncer. Em seguida, analisamos um importante ponto de interesse em relação ao carcinoma adenóide cístico com transformação para alto grau, o seu pior prognóstico, que é sugerido ser comparável ou até pior do que o subtipo sólido. Para isso, comparamos as alterações genéticas do carcinoma adenóide cístico com transformação para alto grau com o subtipo sólido e, com os aspectos clínicos e patológicos de ambos os tumores. Além disso, em outro trabalho, usamos a hibridização genômica comparativa em microarranjo para comparamos o perfil genético de ambos os componentes histológicos do carcinoma adenóide cístico com transformação para alto grau. Atenção especial foi dada à expressão da proteína e à translocação cromossomal do gene MYB, que está sendo considerado o maior evento precoce e oncogênico do carcinoma adenóide cístico clássico. Nossos resultados mostraram que o carcinoma adenóide cístico com transformação para alto grau pode apresentar uma complexidade genética similar ao subtipo sólido e, também, que o processo de transformação não é sempre acompanhado pelo acúmulo de alterações genéticas, o que indica uma progressão paralela de ambos os componentes do carcinoma adenóide cístico transformado. Em contrapartida à expressão da proteína MYB, a translocação entre MYB/NFIB não é necessariamente um evento precoce e, bem como a hipóxia, não são fundamentais para o desenvolvimento destes tumores. Finalmente, o estudo advindo do carcinoma adenóide cístico com transformação para alto grau, também nos permitiu fazer uma revisão sobre o assunto. Neste outro estudo fizemos um panorama sobre os recentes conceitos na classificação histopatológica dos tumores de glândula salivar com desdiferenciação/transformação para alto grau descritos na literatura. Destaque também foi dado aos achados imuno-histoquímicos e genéticos que podem ajudar no diagnóstico de cada um destes tumores
Abstract: Adenoid cystic carcinomas can occasionally undergo dedifferentiation, a phenomenon also referred to as high-grade transformation. However, cases of adenoid cystic carcinomas have been described showing transformation to adenocarcinomas that are not poorly differentiated, indicating that high-grade transformation may not necessarily reflect a more advanced stage of tumor progression, but rather a transformation to another histological form, which may encompass a wide spectrum of carcinomas in terms of aggressiveness. The aim of this study was to gain more insight in the biology of this pathological phenomenon. Firstly, we investigated expression of proteins regulated by hypoxia (HIF-1?, VEGF, GLUT-1 and CD105), given that hypoxia contributes to aggressive tumor behavior and can also promote a dedifferentiated phenotype in certain types of cancer. Hereafter, we analyzed an important point of interest of adenoid cystic carcinoma with high-grade transformation that is its proposed poor prognosis to be comparable to or even worse than solid subtype. Therefore, we compared the genetic changes of transformed and solid subtype adenoid cystic carcinomas and correlated the results to their clinico-pathological features. In addition, in another work, we used microarray comparative genomic hybridization to compare the genetic profiles of both histological components of adenoid cystic carcinomas with high-grade transformation. Special attention was given to chromosomal translocation and protein expression of MYB, recently being considered to be an early and major oncogenic event in adenoid cystic carcinomas. Our data showed that transformed adenoid cystic carcinoma with high-grade transformation may present a genetic complexity similar to the solid subtype and, also that the process of high-grade transformation is not always be accompanied by an accumulation of genetic alterations; which indicate a parallel progression of the two histological components of transformed adenoid cystic carcinoma. In contrast to MYB protein expression, MYB/NFIB translocation is not necessarily an early event and, as hypoxia, not fundamental for the development of these tumors. Finally, the study that comes from of adenoid cystic carcinoma with high-grade transformation also allowed us to do a review about it. In this study we made an overview of the latest concepts in histopathological classification of salivary gland tumors with dedifferentiation / high-grade transformation described in the literature. Highlight was also given to immunohistochemical and genetic findings that can help in the diagnosis of each of these tumors
Doutorado
Ciencias Biomedicas
Doutora em Ciências Médicas

Els productes naturals derivats de plantes són una font abundant de compostos biològicament actius, molts dels quals han servit de base pel desenvolupament de productes nutracèutics i farmacèutics. El disseny de fàrmacs assistit per ordinador juga un paper essencial en la identificació de productes naturals bioactius, reduint els costos i esforços experimentals. La diabetis mellitus tipus 2 és considerada com "l’epidèmia del segle 21" i, en conseqüència, és un dels principals reptes en el descobriment de fàrmacs en l'actualitat. Per tant, aquesta tesi doctoral es centra en la identificació de nous compostos i extractes naturals que actuïn com a agents antidiabètics. En aquest sentit, PPARgamma i DPP-IV han estat les dianes estudiades, en les quals s’han desenvolupat i validat diferents protocols de cribatge virtual. Els compostos bioactius trobats poden ser de gran utilitat com additius en l’alimentació funcional adreçada en la prevenció o tractament de la diabetis.
Natural products derived from medicinal plants are an abundant source of biologically active compounds, many of which have formed the basis for development of nutraceuticals and pharmaceuticals compounds. Computer-aided drug design methods such as virtual screening workflows play an essential role in the identification of undescribed bioactivities of natural products by minimizing the synthetic and biological testing efforts. Type 2 diabetes mellitus (T2DM) is considered to be the “epidemic of the 21st century” and, consequently, is one of the main challenges in drug discovery today. There is a significant unmet medical need for better drugs to treat T2DM because some therapies suffer undesirable side effects. Therefore, this PhD thesis is focus on the identification of new natural compounds as antidiabetic agents. In that sense, PPARgamma and DPP-IV have shown to be appropriate targets for antidiabetic drugs, targeting them appears to have good prospects for a successful therapeutic approach in the T2DM.

Thesis (MScFoodSc)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: The regular consumption of probiotics is becoming a recognized trend in the food industry due to several reported health benefits. A probiotic is defined as a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance. A wide variety of probiotic food products are available on the South African market and comprise an assortment of fermented milks, as well as lyophilized preparations in tablet or capsule form. Strains of Lactobacillus acidophilus and Bifidobacterium species are mostly used as probiotic microbes in the industry due to their health enhancing effect. The survival of sensitive probiotic microbial species in food matrices are influenced by various factors such as oxygen concentration, pH levels and manufacturing and storage conditions. These should be considered and monitored as the South African food and health regulations stipulate that probiotic microbes should be present at a concentration of 10⁶ cfu.ml ̄ ¹' in order to exert a beneficial effect. Some health benefits are also correlated to specific microbial species and strains and these factors have resulted in the need for the rapid and accurate identification of probiotic microbes present in food products. The probiotic microbes present in probiotic yoghurts and supplements have in the past been identified using traditional methods such as growth on selective media, morphological, physiological and biochemical characteristics. However, even some of the most sophisticated cultural-dependant techniques are not always sufficient for the identification and classification of especially Bifidobacterium, as well as closely related Lactobacillus species. Molecular techniques are more often employed for the rapid and accurate detection, identification and characterization of microbial species present in food products. The aim of this study was to detect and identify the probiotic species present in various commercial South African yoghurts and lyophilized preparations using peR-based DGGE analysis. A 200 bp fragment of the V2-V3 region of the 16S rRNA gene was amplified and the peR fragments were resolved by DGGE. The unique fingerprints obtained for each product were compared to two reference markers A and B in order to identify the bands present. The results obtained were verified by species-specific peR, as well as sequence analyses of bands that could not be identified when compared to the reference markers. Only 54.5% of the South African probiotic yoghurts that were tested did contain all the microbial species as were mentioned on the labels of these products, compared to merely one third (33.3%) of the lyophilized probiotic food supplements. Some Bifidobacterium species were incorrectly identified according to some product labels, while other products contained various microbes that were not mentioned on the label. Sequence analysis confirmed the presence of a potential pathogenic Streptococcus species in one of the yoghurt products and in some instances the probiotic species claimed on the labels were non-scientific and misleading. The data obtained in this study showed that the various South African probiotic products tested were of poor quality and did not conform to the South African regulations. peR-based DGGE analysis proofed to be a valuable approach for the rapid and accurate detection and identification of the microbial species present in South African probiotic products. This could help with future implementation of quality control procedures in order to ensure a reliable and safe probiotic product to the consumer.
AFRIKAANSE OPSOMMING: Die gereelde inname van probiotiese produkte is besig om In erkende tendens in die voedselindustrie te word, as gevolg van verskeie gesondheidsvoordele wat daaraan gekoppel word. In Probiotika word gedefinieer as In voedingsaanvulling wat uit lewendige mikrobes bestaan en wat In voordelige effek op mens of dier het deur In optimale mikrobiese balans in die ingewande te handhaaf. In Wye verskeidenheid probiotiese voedselprodukte is tans beskikbaar op die Suid- Afrikaanse mark. Hierdie bestaan hoofsaaklik uit verskeie gefermenteerde melkprodukte asook 'n reeks tablette en kapsules wat probiotiese mikrobes in gevriesdroogde vorm bevat. Lactobacillus acidophilus tipes en Bifidobacterium spesies word die algemeenste in die voedselindustrie gebruik aangesien hierdie spesifieke mikrobes bekend is om goeie gesondheid te bevorder. Die oorlewing van sensitiewe probiotiese mikrobiese spesies in voedsel matrikse word beïnvloed deur faktore soos suurstof konsentrasie, pH-vlakke en vervaardigings- en opbergings kondisies. Hierdie faktore moet in aanmerking geneem word en verkieslik gemonitor word aangesien die Suid-Afrikaanse voedsel en gesondheids regulasies stipuleer dat probiotiese mikrobes teen In konsentrasie van 10⁶ kolonie vormende eenhede per ml teenwoordig moet wees om In voordelige effek te toon. Sommige gesondheidsvoordele word direk gekoppel aan spesifieke mikrobiese spesies en spesie-tipes. Hierdie faktore het gelei tot In groot aanvraag na vinnige en akkurate metodes vir die identifikasie van probioties mikrobes in voedselprodukte. Die probiotiese mikrobes teenwoordig in probiotiese joghurts en ook die gevriesdroogde vorms in tablette en kapsules, was al geïdentifiseer deur gebruik te maak van tradisionele metodes soos groei op selektiewe media, morfologiese, fisiologiese en biochemiese eienskappe. Selfs van die mees gesofistikeerde kultuur-afhanklike tegnieke is egter nie altyd voldoende vir die identifikasie en klassifikasie van veral Bifidobacterium en na-verwante Lactobacillus spesies nie. Molekulêre metodes word dikwels aangewend vir die vinnige en akkurate deteksie, identifikasie en karakterisering van mikrobes teenwoordig in voedselprodukte. Die doel van hierdie studie was om die probiotiese mikrobes teenwoordig in verskeie Suid-Afrikaanse joghurts en gevriesdroogde aanvullings, te identifiseer deur gebruik te maak van polimerase kettingreaksie (PKR)-gebaseerde denaturerende gradiënt jelelektroforese (DGGE) analise. 'n PKR fragment van 200 bp van die V2-V3 gedeelte van die 16S ribosomale RNS (rRNS) geen is geamplifiseer, en die PKR fragmente is geskei met behulp van DGGE. Die unieke vingerafdrukke wat verkry is vir elke produk is teen twee verwysings merkers A en B vegelyk om die bande teenwoordig in die profiele te identifiseer. Die resultate is bevestig deur spesies-spesifieke PKR en ook deur die ketting volgordes van die DNS fragmente te bepaal wat nie geïdentifiseer kon word deur vergelyking met die verwysings merkers nie. Slegs 54.5% van die Suid-Afrikaanse probiotiese joghurts wat getoets is het al die mikrobiese spesies bevat soos aangedui was op die etikette van hierdie produkte, teenoor slegs 'n derde (33.3%) van die gevriesdroogde voedingsaanvullings. Sekere Bifidobacterium spesies is verkeerd geïdentifiseer op sommige van die produk etikette, terwyl ander produkte verskeie mikrobes bevat het wat nie op die etiket aangedui was nie. 'n Potensiële patogeniese Streptococcus spesie is in een van die joghurt produkte gevind soos bevestig deur DNS kettingvolgorde bepalings. In sommige gevalle was die probiotiese spesienaam wat aangedui is op die etiket onwetenskaplik en misleidend. Die resultate wat uit hierdie studie verkry is dui aan dat die Suid-Afrikaanse probiotiese produkte wat getoets is van 'n swak gehalte is en nie aan die Suid- Afrikaanse regulasies voldoen nie. Daar is getoon dat PKR-gebaseerde DGGE analise 'n waardevolle tegniek kan wees vir die akkurate deteksie en identifisering van die mikrobiese spesies teenwoordig in probiotiese produkte. Dit kan help met die toekomstige implementering van kwaliteitskontrolerings prosedures om 'n mikrobiologiese betroubare en veilige produk aan die verbruiker te verseker.

Thesis (MSc Food Sc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia. Cronobacter infections have been reported in all age groups, however, immunocompromised infants are more susceptible to these infections. Furthermore, Cronobacter strains have been reported to show differences in sensitivity to antibiotics and virulence. These differences led to the reclassification of Cronobacter and currently the genus contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this reclassification was only accepted recently, there are not many typing methods optimised for differentiation between the five Cronobacter species. Typing of Cronobacter strains are important as the species may be diverse regarding their virulence. Cronobacter strains have been isolated from infant formula milk (IFM), the environment of an IFM processing facility and fresh produce in South Africa. However, little is known about the phylogeny and prevalence of these strains. The aim of this study was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii) and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide variety of isolation sources. Other studies have also found that irrespective of the isolation source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South African strains were found to be phylogenetically closely related. However, two distinct clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group based on the 16S rRNA gene analysis. Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr. sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %. Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not sufficient to distinguish between all the Cronobacter species. The sequence data of these two genes can be used to differentiate between the Cronobacter strains when used in combination with malonate utilisation analysis. A PCR-RFLP method was subsequently developed to facilitate the simultaneous differentiation between all five Cronobacter species. The PCR-RFLP assay was based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species were obtained and it was also possible to differentiate between Enterobacteriaceae and Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical analysis with malonate broth confirmed the identities of these two strains as Cr. malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures rapid differentiation between the five species of Cronobacter.
AFRIKAANSE OPSOMMING: Die Cronobacter genus (Enterobacter sakazakii) bevat opportunistiese patogene wat 'n ernstige vorm van neonatale meningitis, enterokolitis en septisemie kan veroorsaak. Cronobacter infeksies is al in alle ouderdomsgroepe aangemeld, maar immuungekompromitteerde babas is die meeste vatbaar vir hierdie infeksies. Verder toon Cronobacter spesies verskille in virulensie en sensitiwiteit vir antibiotika. Hierdie verskille het gelei tot die herklassifikasie van Cronobacter en tans bestaan die genus uit vyf afsonderlike spesies, naamlik Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis en Cronobacter muytjensii. Aangesien hierdie herklassifikasie slegs onlangs aanvaar is, is daar nie baie metodes wat geskik is vir onderskeiding tussen die vyf Cronobacter spesies nie. Onderskeiding tussen Cronobacter spesies is belangrik omdat die spesies verskillend kan wees met betrekking tot hulle virulensie. Cronobacter is geisoleer uit baba formule melk (BFM), die omgewing van 'n BFM fabriek en vars produkte in Suid-Afrika. Daar is egter nie baie bekend oor die filogenie en voorkoms van hierdie isolate nie. Die doel van hierdie studie was om 24 Suid-Afrikaanse Cronobacter stamme (voorheen geïdentifiseer as E. sakazakii) te klassifiseer en die filogenie van die isolate te evalueer gebaseer op die 16S ribosomale RNS (rRNS) en rpoA gene. Al 24 Suid-Afrikaanse stamme is geïdentifiseer as Cr. sakazakii ten spyte van 'n wye verskeidenheid isolasie bronne. Ander studies het ook gevind dat, ongeag die isolasie bron, die meerderheid van Cronobacter stamme as Cr. sakazakii geïdentifiseer word. In hierdie studie is gevind dat die Suid-Afrikaanse stamme filogeneties nou verwant is. Op grond van die 16S rRNA geen analise is die Cr. sakazakii stamme egter in twee afsonderlike groepe gedeel met 'n 93% vertrouens vlak. Dit was moontlik om stamme van Cr. sakazakii, Cr. dublinensis, Cr. turicensis en Cr. muytjensii van mekaar te onderskei met die DNS volgorde data van die 16S rRNA en rpoA gene, maar geen onderskeid tussen Cr. sakazakii en Cr. malonaticus stamme was moontlik nie. Die filogram gebaseer op die rpoA DNS volgorde data het aparte takke vir Cr. malonaticus en Cr. sakazakii stamme getoon, maar die twee takke is met ‘n lae vertrouens waarde van slegs 70 % geskei. Filogenetiese analise gebaseer op die rpoA en 16S rRNA gene is dus nie voldoende om te onderskei tussen al die Cronobacter spesies nie. Die DNS volgorde data van hierdie twee gene sou egter gebruik kon word om te onderskei tussen die Cronobacter spesies wanneer dit gebruik word in kombinasie met malonaatbenutting-analises. 'n Polimerase ketting reaksie (PKR) beperkings fragment lengte polimorfisme (BFLP) metode is ontwikkel om die gelyktydige onderskeiding tussen al vyf Cronobacter spesies te fasiliteer. Die PKR-BFLP metode is gebaseer op die vermeerdering van die rpoB geen gevolg deur die gesamentlike vertering met die beperkingsensieme, Csp6I en HinP1I. Unieke profiele vir elk van die vyf Cronobacter spesies is verkry en dit was ook moontlik om tussen Enterobacteriaceae en Cronobacter spesies te onderskei. Verder het twee stamme wat as Cr. sakazakii geïdentifiseer is met DNS volgordebepaling van die 16S rRNA en rpoA gene, PKR-BFLP profiele identies aan dié van Cr. malonaticus getoon. DNS volgordebepaling van die rpoB geen en ‘n addisionele biochemiese toets met malonaat sop het die identiteit van hierdie twee stamme as Cr. malonaticus bevestig. Hierdie PKR-BFLP is dus 'n akkurate metode wat vinnige onderskeid tussen die vyf spesies van Cronobacter kan verseker.

Endoxifen is an effective metabolite of tamoxifen, a commonly used chemotherapy drug, and has been detected at the final effluent of a municipal wastewater treatment plant (WWTP). Its presence in the environment could bring negative effects to aquatic lives. This research investigated the use of ultraviolet radiation (253.7 nm) and natural sunlight to photodegrade endoxifen in water and wastewater, the generation of photodegradation by-products (PBPs) and their toxicity. Endoxifen concentration in water was reduced by 99.1% after 35 seconds of UV light generating two toxic PBPs. Experiments in wastewater at UV light doses used for disinfection resulted in reduction of endoxifen by 30 to 71%. Endoxifen concentration in wastewater was reduced by at least 83% after 150 minutes of solar radiation generating eight PBPs. Seven of these PBPs are potentially more toxic than endoxifen itself. Therefore, highly toxic PBPs are potentially generated at WWTPs if endoxifen is present in wastewater.
North Dakota State University. Department of Civil and Environmental Engineering
Chulalongkorn University. Center of Excellence for Hazardous Substance Management
North Dakota State University. Environmental and Conservation Sciences Program
North Dakota Water Research Institute Fellowship

Advancement in digital technology is rapidly changing the contemporary landscape of business and associated networks for manufacturing firms. Many traditional physical products are now being embedded with digital components, providing them digital capability to become digitized products. The digitization of physical products has become an important driver for digital service innovation within manufacturing industries. Such digital service innovation transforms value networks of manufacturing firms in various industries. While digitization of products and digital service innovation can be observed in many manufacturing industries, this thesis focuses on the transformation of value networks within the vehicle industry. This thesis is a collection of papers and a cover paper. The thesis reports from a collaborative project in the vehicle industry. The project explored new digital services for vehicles based on remote diagnostics technology. The exploration and conceptualization of digital services is investigated in a collaborative manner with participants from the vehicle industry. The results reflect that there is a paradigm shift for manufacturing firms digitizing their products, and stretching the business scope from product to solution oriented business. This thesis contributes to the existing literature on digital innovation with insights on the transformation of value networks in the vehicle industry. The research question addressed in this thesis is: How are value networks of manufacturing firms transformed by digital service innovation? To answer the question, this thesis conceptualizes how the value creating pattern of digitized products transforms value networks of manufacturing firms. A model is presented that reflects how the symbiotic value relationship between the digitized product and digital services transforms the roles, relationships and exchanges in the value networks of manufacturing firms. The model can serve as an analytical tool to further advance the knowledge on business aspects in digital innovation. This thesis contributes to practice by providing an understanding of how manufacturing firms can leverage value of digitized products and digital services in value networks.
ReDi2Service

This study was conceptualised within the Aristotelian tradition of phronesis and dialectics (Eikeland, 2006; 2012) where the focus of the study was the identification of teacher's voice (Sutherland et al., 2010) using Songs of Praxis, an art-based reflexive account within a reflexivity paradigm of uncovering biographical perspective of identity (Alheit, 1992; 2009). This had two reflexivity contexts; within research (e.g. Rolfe, 2011a; 2011b; Rolfe, Jasper and Freshwater, 2011; Etherington, 2007; Simon, 2013; Ellis, 2004; 2007; 2011) and within critical reflective practice (e.g. Gardner, 2009; Gardner, 2014; Rolfe, Jasper and Freshwater, 2011). This reflexive process led to teacher voice development and transformation through a hybridity of creative pedagogy, teaching for creativity and creative learning (Lin, 2011; 2014; Sawyer 2004; 2011). This visualization was completed using reflexive accounts (referred to as Songs of Praxis) which created space for a reflexive relationship (Etherington, 2007; Ellis, 2004) allowing for emotional recall (understanding biographical identity) through to therapeutic inquiry by dealing with my Mask of Self-Hate (the name I use to describe my bipolarity). This facilitated my authentication and transformation as a scholar of learning and teaching. This study was conducted within an auto ethnographic action research and reflexive accounts methodology using cycles of critical reflection. The cycles of critical reflection are; teacher voice identification stage 1, the reimagination of professional voice framework leading to teacher voice identification stage 2, the multiplicity of voice prism. This led to teacher voice development stage1, the reflexive provocateur toolkit which developed into the Reflexive Classroom, teacher voice development stage 2. This led to the teacher voice transformation through the creation of reflexive spaces of creative pedagogic practices; digital reflexivity; and employability practices. This allowed for teacher voice identification, development and transformation which I illustrate within the 'portfolio' which I conceptualised around an 'Album', Songs of Praxis: Teacher Voice Identification, development and transformation'. This corroboration of the data (experience) has led to the contribution of this study to creative teaching practices, digital reflexivity and employability practices. I propose Songs of Praxis create reflexive space for the identification, development and transformation of teacher's voice.