L’étude physicochimique de trois lichens récoltés en Limousin, Usnea florida, Flavoparmelia caperata et Cladonia incrassata, a permis d’isoler et de caractériser 16 composés issus du métabolisme secondaire. Dans un premier temps, ces lichens ont été sélectionnés lors d’un criblage analytique préliminaire réalisé sur 17 espèces. L’activité antimicrobienne des extraits acétoniques a été évaluée, puis leur fractionnement bioguidé a été mis en œuvre. Les deux isomères de l’acide usnique, 3 dibenzofuranes, 4 depsides, 3 depsidones, un acide aliphatique, un phtalide et un stérol ont été isolés. Une xanthone trichlorée, la cladoxanthone A, a été extraite et identifiée pour la première fois à partir d’une source naturelle. L’effet de tous les composés a été évalué sur Staphyloccocus aureus par bioautographie. Pour les produits isolés en quantité suffisante, des tests de dilution en milieu liquide ont permis de déterminer leurs CMI, en comparaison avec le Phénonip®, conservateur couramment utilisé dans l’industrie cosmétique. Les acides didymique et condidymique en particulier s’avèrent être de bons candidats pour une utilisation en tant que conservateurs. Dans un second temps, compte tenu de leur potentielle application industrielle, nous avons exploré des voies de synthèse de l’acide usnique et de la cladoxanthone A. Ainsi, la préparation de l’intermédiaire clé pour l’accès à l’acide usnique a été optimisée et les premiers essais de couplage en présence d’une porphyrine sont encourageants. De même, les voies de synthèse proposées et mises en œuvre pour la cladoxanthone A sont prometteuses. / Phytochemical study of three lichens collected in Limousin, Usnea florida, Flavoparmelia caperata and Cladonia incrassata, led to the isolation and the structural identification of 16 secondary metabolites. First, these lichens were chosen after a preliminary screening performed on 17 species. Antimicrobial activity of acetone extracts was evaluated and bioassay-guided fractionation was performed. The two isomers of usnic acid, 3 dibenzofurans, 4 depsides, 3 depsidones, one aliphatic acid and one sterol were isolated. A trichlorinated xanthone, named cladoxanthone A, was extracted and identified for the first time in a natural source. A bioautographic protocol was used to evaluate antibacterial activities of these compounds on Staphylococcus aureus. MICs of the products obtained in sufficient amounts were determined by a broth microdilution method in parallel with Phenonip®, a preservative commonly used in cosmetic industry. Didymic and condidymic acids were found to be good candidates for use as preservatives. Secondly, owing to their potential industrial application, we explored synthetic routes for usnic acid and cladoxanthone A. Thus, the preparation of the key intermediate for access to usnic acid was optimized and the first coupling tests in the presence of a porphyrin were encouraging. Similarly, the proposed synthetic routes for cladoxanthone A are promising.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Erythropoietin is a sialoglycoprotein which promotes the increase of erythropoiesis. Clinically is used for the treatment of anaemia associated to chronic renal failure. Identification and separation of isoforms of recombinant human erythropoietin (rhEPO) in biopharmaceuticals of different origins, was carried out by isoelectric focusing (IEF) western blotting and, also by lectin binding with Triticum vulgaris, showing 4-7 isoforms distributed in the isoeletric range of 4.4 to 5.2. N-acetylneuraminic acid content was quantified by reversed-phase liquid chromatography method with fluorescence detection giving values higher than 108.74 ηg/μg. Biological activity was evaluated by the normocythaemic mice bioassay, and investigating the TF-1 cell line in vitro. The correlation of the results of both of the methods were significant, as calculated by the Pearson s coefficient (r = 0.9967). In addition, the content/potency of the biopharmaceutical products was assessed by validated reversed phase and size exclusion liquid chromatography methods, showing mean values 2.11% and 1.21% lower, respectively, related to the in vivo bioassay. Sample was degraded under UV light to generate deamidate/sulphoxide forms and treatment at 65ºC for 12 hours to produce dimeric and aggregated forms. The potencies were evaluated by the normocythaemic mice assay and the TF-1 cell culture assay giving mean reduction of 14.05% and 32.87%, respectively, related to the intact molecule. The alternative in vitro assay investigated in the context of the reduction or replacement of the animals, and the evaluation of the correlations between physicochemical and biological methods, represent improvements which can be applied to the production steps and for the quality control of rhEPO, contributing to ensure the batch-to-batch consistency of bulk and finished biological products. / A eritropoietina é uma sialoglicoproteína que promove o aumento da eritropoiese. Clinicamente é usada para o tratamento de anemias associadas à falência renal crônica. No presente realizou-se identificação e separação das isoformas de eritropoietina humana recombinante (rhEPO) em produtos biofarmacêuticos de diferentes origens, por focalização isoelétrica (IEF), seguida de imunodetecção, e também por ligação à lectina Triticum vulgaris, demonstrando a presença de 4 a 7 isoformas, distribuídas na faixa de ponto isoelétrico de 4,4 a 5,2. Quantificou-se o conteúdo de ácido N-acetilneuramínico por cromatografia líquida por fase-reversa e detecção por fluorescência obtendo teores acima de 108,74 ηg/μg. Avaliou-se a atividade biológica pelo bioensaio em camundongos normocitêmicos e pesquisou-se o ensaio alternativo baseado na cultura da linhagem celular TF-1 in vitro. Os resultados dos bioensaios apresentaram correlação significativa, conforme calculado pelo coeficiente de correlação de Pearson (r = 0,9967). Paralelamente, determinou-se o teor/potência dos produtos pelas metodologias validadas por cromatografia líquida por fase reversa e por exclusão molecular, que forneceram média de resultados 2,11% e 1,21% menores, respectivamente, em relação ao bioensaio in vivo. Submeteu-se amostra à degradação por luz UV para obter as formas desamidadas/oxidadas e tratamento a 65°C por 12 horas para as diméricas e agregadas. Efetuou-se a avaliação pelo bioensaio in vivo e in vitro, que apresentaram redução média de 14,05% e 32,87% respectivamente, em relação à molécula intacta. Desse modo, o ensaio biológico alternativo in vitro, pesquisado no contexto da redução ou substituição do uso de animais, e as avaliações de correlação entre métodos físico-químicos e biológicos, representam aprimoramentos aplicáveis para as etapas do processo de produção e para o controle de qualidade de rhEPO, contribuindo para garantir a consistência lote-a-lote da solução concentrada e dos produto biológicos acabados.

<div>Vibrational spectroscopic imaging has become an emerging platform for chemical visualization of biomolecules and materials in complex systems. For over a century, both Raman and infrared spectroscopy have demonstrated the capability to recognize molecules of interest by harnessing the characteristic features from molecular fingerprints. With the recent development of hyperspectral vibrational spectroscopy imaging, which records the chemical information without sacrificing the spatial-temporal resolution, numerous discoveries has been achieved in the field of molecular and cellular biology. Despite the ability to provide complimentary chemical information to Raman-based approaches, infrared spectroscopy has not been extensively applied in routine studies due to several fundamental limitations: 1). the poor spatial resolution; 2). inevitable strong water absorption; 3). lack of depth resolution.</div><div>Mid-infrared photothermal (MIP) microscopy overcame all the above mentioned problems and for the first time, enabled depth-resolved in vivo infrared imaging of live cells, microorganisms with submicrometer spatial resolution. The development of epi-detected MIP microscopy further extends its application in pharmaceutical and materials sciences. With the deployment of difference frequency generation and other nonlinear optical techniques, the spectral coverage of the MIP microscopy was significantly enhanced to enable chemical differentiation in complex systems across the broad mid-infrared region. In addition to the efforts to directly improve the performance of MIP microscopy, a novel quantitative phase imaging approach based on polarization wavefront shaping via custom-designed micro-retarder arrays was developed to take advantage of the highly sensitive phase measurement in combination with the photothermal effect. Besides, the extended depth-of-field and multifocus imaging enabled by polarization wavefront shaping could both improve the performance of MIP microscopy for volumetric imaging.</div>

M. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology / The present study includes the use of a green synthetic method to prepare copper and silver nanoparticles using chitosan, aqueous extracts of Camellia sinensis, Combretum molle and Melia azedarach linn leaves. This study aims to investigate the influence of capping and precursor concentration on the properties of silver nanoparticles with emphasis on the medicinal plants chosen. The effect of capping agent on the properties of copper nanoparticles is also investigated. The phytochemical properties of plant extracts and the antimicrobial activity of the synthesized particles were also studied; this was achieved by using microdilution bioassay. Decoction method was used to extract secondary metabolites from plant leaves. Preliminary phytochemical screening carried out on the aqueous extracts of the plant leaves showed the presence of tannins, proteins, flavonoids, phenols, and carbohydrates. The total phenolic and flavonoids content of the aqueous extract was determined using spectroscopic methods. The highest phenolic content was found in the aqueous extract of Combretum molle (135 mg/g), and the highest flavonoid content was found in the aqueous extract of Camellia sinensis (0.4 mg/g). Characterization was done by a combination of spectroscopic, microscopy and XRD techniques. Both the size and shape of the synthesized silver nanoparticles were dependent on the identity of the capping molecule, precursor and capping agent concentration as depicted from their TEM and XRD results. Silver nanoparticles were found to be predominantly spherical. The capping agent concentration was also found to influence the degree of agglomeration, with an increase in capping agent concentration giving lesser agglomeration. FTIR spectral analysis showed that silver nanoparticles interact with bioactive compounds found in the plants through the hydroxyl functional group. Other shapes including diamond were observed for the effect of precursor concentration. The XRD micrographs revealed a face-centered cubic geometry and the phase remained the same with an increase in precursor concentration. The synthesized silver nanoparticles were all blue shifted compared to the bulk material. The TEM results revealed that copper nanoparticles with different sizes and shapes were successfully synthesized. All the prepared copper and silver nanoparticles showed satisfactory antifungal and antibacterial activity against Candida albicans, Cryptococcus neoformans, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia and Pseudomonas aeruginosa. The capping molecules used in this study also showed some antibacterial and antifungal activity against the selected strains. However nanoparticles performed better than these capping molecules. Both silver and copper nanoparticles were found to be more active against gram-negative bacteria compared to gram-positive bacteria. Amongst all the prepared silver nanoparticles Combretum molle capped nanoparticles were found to be the most active nanoparticles. Also with copper nanoparticles, it was found that Combretum molle capped nanoparticles were the most active nanoparticles. Between the two metal nanoparticles, silver nanoparticles showed high antibacterial and antifungal activity compared to copper nanoparticles. The antioxidant activity of silver nanoparticles was assessed using 2.2-diphenyl-1-picrylhydrazyl. Silver nanoparticles were found to have some antioxidant activity. However, the capping molecules were found to be more active than the synthesized nanoparticles. This observation is attributed to the presence of some bioactive compounds in the plant extracts.

Im Rahmen der EU-Richtlinie REACH müssen industrierelevante Chemikalien bezüglich des Risikos für Mensch und Umwelt (neu) untersucht und bewertet werden, wobei die Anzahl an Tierversuchen zu minimieren ist und neue toxikologische Prüfmethoden, Bewertungsstrategien sowie alternative Testsysteme entwickelt und optimiert werden sollen. Ubiquitär vorkommende Testorganismen, welche ähnliche Eigenschaften und eine vergleichbare Sensitivität bezüglich äußerer Einflüsse wie komplexere Organismen (Fisch, Säugetier, Mensch) besitzen, sind hier besonders gefragt um die Belastung abzuschätzen und Trendaussagen über die Wirkung zu formulieren. Durch den Einsatz vieler dieser Testorganismen sowie durch die Ermittlung der chemischen Reaktivität und der Strukturmerkmale einer Verbindung kann ein globales Bild über die Wirkung erhalten werden. Ein Organismus, der diese Charakteristika aufweist, ist der eukaryotische Einzeller Tetrahymena pyriformis GL. In der vorliegenden Dissertation wird zum einen die jeweilige Toxizität elektrophiler Substanzen unterschiedlicher Stoffklassen und Reaktions-mechanismen (α,β-ungesättigte Carbonyl- und Carboxylverbindungen, heterozyklische Drei- und Vierringe, α-halogenierte Carbonyle, Harnstoffe und Thioharnstoffe, aromatische Disulfide sowie aliphatische und aromatische Nitroverbindungen) nach den Expositionszeiten 24 h, 48 h und 72 h mit Hilfe eines etablierten Wachstumshemmtests (Müller, 2001) bestimmt und Struktur-Toxizitäts-Beziehungen bzw. Strukturalarme abgeleitet. Neben der methodischen Optimierung werden die substanzspezifischen Eigenschaften, Flüchtigkeit und Sorptionsfähigkeit, quantitativ erfasst und durch neu aufgestellten Modellgleichungen für jede Substanz ermittelt. Mit der Bestimmung der Toxizitätserhöhung, Te, welche auch als ein Maß für die Reaktivität angesehen werden kann, werden Organismen-spezifische Narkose-Basis-Geraden generiert, exzesstoxische Verbindungen identifiziert und Strukturalarme für die jeweiligen Stoffklassen aufgestellt. Zum anderen werden binäre Mischungen untersucht, welche aus den Einzelstoffen bestehen, deren Toxizität im ersten Abschnitt der Arbeit ermittelt wurden und bei denen theoretische Annahmen über die Reaktionsmechanismen bestehen. Dieser Abschnitt identifiziert, ob gleiche bzw. unähnliche primäre Wechselwirkungen zweier Substanzen mit den biologischen Targets vorliegen und welcher Mechanismus, der reaktive Mechanismus exzesstoxischer Substanzen oder die Wechselwirkungen mit Membran-Bestandteilen, dominierend ist. Für die Auswertung der Mischungsergebnisse werden die etablierten biometrischen Modelle Konzentrations-Additivität und Unabhängigen Wirkung verwendet. Es wird dargestellt, dass die Mischungstoxizitäten der binären Mischungen ähnlich wirkender Substanzen zwar relativ genau mithilfe beider Modelle berechnet werden, jedoch keine eindeutige Charakterisierung der Mechanismen möglich ist. Die Ergebnisse der untersuchten binären Mischungen unterschiedlich wirkender Substanzen zeigen dagegen, dass die Mischungstoxizitäten unähnlicher Substanzen relativ genau mithilfe des Modells der Unabhängigen Wirkung berechnet werden und eine Charakterisierung der Mechanismen möglich ist.

In the area of forensic chemistry, improved or new analysis methods are continually being investigated. One common and powerful technique used in forensic chemistry is wall-coated open-tubular column (WCOT) gas chromatography with electron ionization single quadrupole mass spectrometry (GC-MS). Improvements to and effectiveness of alternatives to this instrumental platform were explored in an array of parallel inquiries. The areas studied included the column for the chromatographic separation, the universal detection method employed, and the fragmentation method used to enhance molecular identification. <br><br>Superfine-micropacked capillary (SFµPC) columns may provide an alternative to commercial packed GC columns and WCOT GC columns that combines the benefits of the larger sample capacity of packed columns and the benefits of the excellent separation capabilities and mass spectrometry (MS) flow rate compatibility of WCOT columns. SFµPC columns suffer from high inlet pressure requirements and prior reported work has required specialized instrumentation for their use. Fabrication of and chromatography with SFµPC GC columns was successfully achieved with typical GC-MS instrumentation and within the flow rate limit of a MS. Additionally, the use of higher viscosity carrier gasses was demonstrated to reduce the required inlet pressure for SFµPC GC columns.<br><br>Recently, a new vacuum ultraviolet spectrometer (VUV) universal detector has been commercialized for GC. The ability of VUV detectors to acquire absorbance spectra from 125 nm to 430 nm poses a potential alternative to MS. As such, GC-VUV provides an exciting potential alternative approach to achieving excellent quantitative and qualitative analysis across a wide range of analytes. The performance of VUV and MS detectors for forensic analysis in terms of quantitative and qualitative analysis was compared. Analysis of alkylbenzenes in ignitable liquids was explored, which can be important evidence from suspected arson fires and are difficult to differentiate with MS. The VUV detector was found to have superior specificity and comparable sensitivity to the MS detector in scan mode.<br><br>Addition of thermolysis (Th) as an orthogonal fragmentation pathway provides the opportunity to increase the differences between MS fragmentation patterns. Fragmentation has been widely established to aid in identification of molecules with MS by providing characteristic fragments at characteristic relative abundances. However, molecules with very similar structures do not result in sizable spectral differences in all cases with typical MS fragmentation techniques. A series of Th units were fabricated and integrated into GC-Th-MS instruments. Th-MS was conducted with the thermally labile nitrate esters across a range of instrumentation and thermal conditions.<br>

No description available.

<p>The rapid analysis of complex samples using mass spectrometry (MS) provides valuable information in both point-of-care (e.g. drug testing) and laboratory-based applications, including the generation of spectral libraries for classification of biosamples, the identification of biomarkers through large-scale studies, as well as the synthesis and bioactivity assessments of large compound sets necessary for drug discovery. In all these cases, the inherent speed of MS is attractive, but rarely fully utilized due to the widespread use of sample purification techniques prior to analysis. Ambient ionization methodologies can help circumvent this drawback by facilitating high-throughput qualitative and quantitative analysis directly from the complex samples without any need for work-up. For instance, the use of swabs or paper substrates allows for rapid identification, quantification, and confirmation, of drugs of abuse from biofluids or surfaces of forensic interest in a matter of minutes, as described in the first two chapters of this dissertation. Faster analysis can be achieved using an automated desorption electrospray ionization (DESI) platform which allows for the rapid and direct screening of complex-sample microarrays with throughputs better than 1 sample per second, giving access to rich spectral information from tens of thousands of samples per day. The development of the bioanalytical capabilities of this platform, particularly within the context of drug discovery (e.g. bioactivity assays, biosample analysis), is described across most other chapters of this dissertation. The use of DESI, a contactless ambient ionization method developed in our laboratory and whose 20 years of history are overviewed in the introduction of this document, provides an additional advantage as the secondary microdroplets generated through the DESI process act as reaction vessels that can accelerate organic reactions by up to six orders of magnitude, facilitating on-the-fly synthesis of new compounds from arrays of starting materials. Unique implications of this microdroplet chemistry in the prebiotic synthesis of peptides and spontaneous redox chemistry at air-solution interfaces, together with its practical applications to the synthesis of new drug molecules, are also overviewed. The success obtained with the first automated DESI-MS system, developed within the DARPA Make It program, led to increased interest in a new-generation platform which was designed over the past year, as overviewed in the last section of this dissertation, and which is currently being installed for validation prior to the transfer of the technology to NCATS, where we anticipate it will make a significant impact through the consolidation and acceleration of the early drug discovery workflow.</p>

<p>Canines can detect different diseases simply by smelling different biological sample types, including  urine,  breath  and  sweat.  This  has  led  researchers  to  try  and  discovery  unique  volatile  organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using  miniaturized  biosensors.  However,  the  identity  of  the  specific  VOC  biomarkers  must  be  demonstrated before designing and fabricating sensing systems. Through  an  extensive  series  of  experiments,  VOCs  in  urine  are  profiled  by  solid  phase  microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that  unique  classes  of  urinary  VOCs,  primarily  terpene/terpenoids  and  carbonyls,  are  potential  biomarkers  of  breast  cancer.  Through  implementing  chemometric  approaches,  unique  panels  of  VOCs  were  identified  for  breast  cancer  detection,  identifying  tumor  location,  determining  the  efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples.  VOC biomarker identification is an important step toward developing portable gas sensors, but  another  hurdle  that  exists  is  that  current  sensors  lack  selectivity  toward  specific  VOCs  of  interest.  Furthermore,  testing  sensors  for  sensitivity  and  selectivity  is  an  extensive  process  as  VOCs  must  be  tested  individually  because  the  sensors  do  not  have  modes  of  chromatographic  separation or compound identification. Another set of experiments is presented to demonstrate that SPME  fibers  can  be  coated  with  materials,  used  to  extract  standard  solutions  of  VOCs,  and  analyzed  by  GC-MS  to  determine  the  performance  of  various  gas  sensing  layers.  In  the  first  of  these  experiments,  polyetherimide  (PEI)  was  coated  onto  a  SPME  fiber  and  compared  to  commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity  for  urinary  VOC  extraction  relative  to  a  polydimethylsiloxane  (PDMS)  SPME  fiber.  These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers. </p>

<p dir="ltr">The beautiful complexity of our world is manifested in how macro- and even planetary-scale processes are essentially completely determined and regulated by chemical and physical transformations happening at the micro- and nanoscale. The introduction and subsequent development of optical microscopy methods have provided us with a unique opportunity to visualize, probe, and sometimes even control these processes that are too small to be seen by the human eye by their nature.</p><p dir="ltr">Among the great variety of truly impressive advances in microscopy instrumentation, two techniques stand out in their widespread and usefulness. First of them, fluorescence imaging has completely revolutionized the study of biological specimens and living systems due to its unprecedented single-molecule sensitivity and resolution combined with video-rate imaging capability. On the other hand, chemical imaging in the mid-infrared region provides an unmatched amount of chemical information enabling label-free mapping of the spatial distribution of various classes of biological molecules. However, each of these techniques falls short where the other excels. For example, despite its high resolution and sensitivity, fluorescence imaging does not carry direct chemical information and relies on labeling specificity, while infrared microscopy is diffraction-limited at the resolution of several micrometers and suffers from low penetration depth in aqueous solutions.</p><p dir="ltr">This dissertation introduces a novel imaging method designed to combine the advantages of fluorescence imaging and infrared spectroscopy. Fluorescence-detected photothermal mid-IR (F-PTIR) microscopy is presented in <b>chapter 1</b> as a technique enabling sub-diffraction chemically-specific microscopy by detecting local temperature-induced fluctuations in fluorescence intensity to inform on localized mid-infrared absorption. F-PTIR applications in targeted biological microspectroscopy (<b>chapter 1</b>) and pharmaceutical materials (<b>chapters 2 and 3</b>) analysis are demonstrated to highlight the potential of this new method. Furthermore, instrumentation developments relying on modern radiation sources such as dual-comb quantum cascade laser and synchrotron infrared radiation are shown to improve spectral acquisition speed (<b>chapter 4</b>) and spectral coverage (<b>chapter 5</b>), respectively, to extend the application range of F-PTIR.</p>