La résilience des agricultures subsahariennes face aux perturbations environnementales et socio-économiques repose en partie sur le maintien des diversités spécifique, variétale et génétique présentes au sein des agrosystèmes, mais aussi sur la préservation du droit pour les cultivateurs de reproduire et d'échanger librement les semences et les savoirs relatifs aux variétés. Pourtant, peu de données sont réellement disponibles sur la circulation effective des semences et les mécanismes modulant les flux de gènes et les introgressions génétiques entre variétés cultivées dans les agrosystèmes sahéliens. Chez le mil en particulier, il n’existe que des données très parcellaires sur les relations entre la diversité de la plante d’une part et la diversité socio-culturelle des agriculteurs d'autre part. Dans la première partie je me suis intéressé aux interactions entre l’organisation en groupes socio-culturels des agriculteurs et la structure génétique des populations de mil dans le bassin du lac Tchad. Les analyses de la diversité génétique suggèrent l’existence d’une barrière sociale à la diffusion des gènes sur l’ensemble de cette région. Néanmoins ces barrières ne sont pas suffisantes pour empêcher les introgressions génétiques entre populations cultivées par des agriculteurs appartenant à des groupes ethnolinguistiques différents. Dans le deuxième chapitre, je me suis intéressé au rôle des processus d’adaptation locale sur la circulation des gènes. Les résultats m’ont conduit à proposer l’hypothèse selon laquelle les flux de gènes entre types nommés précoces et tardifs sont plus importants dans les régions du nord où la pluviométrie est faible. Enfin dans le troisième chapitre j’ai quantifié la diversité biochimique des grains de mil de plusieurs variétés de mil, dont les usages culinaires varient selon les groupes ethnolinguistiques ou dont les qualités gustatives sont appréciées différemment. J’ai utilisé pour cela une approche combinée de protéomique et de métabolique. Parmi les 1072 spots protéiques quantifiés seulement 7 permettent de distinguer les types nommés photopériodiques des types nommés non ou peu photopériodiques. Les données de métabolomique suggèrent la présence de champignons, qui pourraient être des endophytes, dans certains des échantillons analysés. / The resilience of sub-Saharan farming systems to environmental and socioeconomic disturbances is partly based on the maintenance of agro-biodiversity, but also on preserving the right for farmers to reproduce and freely exchange seeds and related knowledge. However, few data are actually available on effective seed flow and on mechanisms modulating gene flow and genetic introgression between landraces grown in Sahelian farming systems. For pearl millet especially, there are only very few data on the relationship between diversity of this crop on the one hand and the socio-cultural diversity of farmers on the other hand. In the first part of my thesis, I was interested in assessing a potential relationship between ethnolinguistic diversity and population genetic structure of pearl millet in the Lake Chad Basin. Analysis of molecular polymorphisms suggests the existence of social barrier to seed flow among ethnolinguistic groups. However, these barriers are not sufficient to prevent genetic introgression between pearl millet populations cultivated by farmers belonging to different ethno-linguistic groups. In the second chapter, I focused on the role of local adaptation on gene flow. The results led me to propose the hypothesis that gene flow between early and late landraces are higher in the northern regions where rainfall is weak. Finally in the third part, I have quantified the biochemical compound of pearl millet seeds belonging to different varieties that show different uses or culinary preferences among farmers belonging to different ethnolingiuistic groups. I used a combined approach of proteomics and metabolics. Among the 1072 protein spots quantified only 7 distinguish the very photoperiodic non photoperiodic landrace. Metabolomics data suggest the presence of fungi, possibly endophytes, in some of the samples analyzed.

Background Prostate cancer (PCa) is the most frequently diagnosed malignant disease among adult males in the USA and the second leading cause of cancer deaths in men. Due to the lack of diagnostic tools that are able to differentiate highly malignant and aggressive cases from indolent tumors, overtreatment has become very common in the era of prostate specific antigen (PSA) screening. New diagnostic methods to determine biological status, malignancy, aggressiveness and extent of PCa are urgently needed. 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HRMAS MRS) can be used to establish PCa metabolomic profiles while preserving tissue architecture for subsequent histopathological analysis. Immunohistochemistry (IHC), as opposed to conventional histopathology methods, has the potential to provide objective, more accurate and quantitative knowledge of tissue pathology. This diagnostic- accuracy study sought to evaluate a novel approach to quantitatively identify metabolomic markers of PCa by exploring the potential of PCa immunomarkers to quantify metabolomic profiles established by 1H HRMAS MRS. Material and Methods 1H HRMAS MRS was performed on tissue samples of 51 prostate cancer patients using a 14.1 Tesla NMR spectrometer (BRUKER Biospin, Billerica, MA) with a rotor synchronized CPMG pulse sequence. Spectral intensities of 36 regions of interest were measured as integrals of curve fittings with Lorentzian-Gaussian line shapes. Immunohistochemistry (IHC) was carried out following the spectroscopy scan, using three prostate immunomarkers to identify cancerous and benign glands: P504S (Alpha-methylacyl-CoA-racemace), CK903 (high-molecular weight cytokeratin) and p63. The immunostaining quality following 1H HRMAS MRS was evaluated and compared to unscanned sections of the same sample, to verify the stability and accessibility of the proposed immunomarkers. IHC images were automatically and quantitatively evaluated, using a quantitative image analysis program (QIAP), to determine the percentage of cancerous and benign epithelia in the tissue cross- sections. The results of the program were validated by a correlation with the results of a quantitative IHC review and quantitative conventional histopathology analysis performed by an experienced pathologist. Ultimately, spectral intensities and the cancer epithelium percentage, obtained from quantitative immunohistochemistry, were correlated in order to validate PCa metabolomic markers identified by 1H HRMAS MRS. Patient outcomes and incidence of recurrence were determined by retrospective review of medical records five years after initial surgery. Categories of recurrence were correlated to spectral intensities to explore potential metabolomic markers of recurrence in the cohort. Results Immunostainings with P504S and CK903 showed excellent staining quality and accessibility following 1H HRMAS MRS, suggesting these markers to be suitable for the presented quantitative approach to determine metabolomics profiles of PCa. In contrast, the quality of p63 IHC was impaired after previously performed spectroscopy. IHC using the immunomarkers P504S and CK903 on adjacent slides was found to present a feasible quantitative diagnostic method to distinguish between benign and cancerous conditions in prostate tissue. The cancer epithelium percentage as determined by QIAP showed a significant correlation to the results of quantitative IHC analysis performed by a pathologist (p < 0.001), as well as to a quantitative conventional histopathology review (p = 0.001). The same was true for the benign epithelium percentage (p < 0.001 and p = 0.0183), validating the presented approach. Two metabolomic regions showed a significant correlation between relative spectral intensities and the cancer epithelium percentage as determined by QIAP: 3.22 ppm (p = 0.015) and 2.68 ppm (p = 0.0144). The metabolites corresponding to these regions, phosphocholine and citrate, could be identified as metabolomic markers of PCa in the present cohort. 45 patients were followed for more than 12 months. Of these, 97.8% were still alive five years after initial surgery. 11 patients (24.4%) experienced a recurrence during the follow- up time. The categories of recurrence showed a correlation to the spectral intensities of two regions, 2.33 – 2.3 ppm (p = 0.0403) and 1.28 ppm (p = 0.0144), corresponding to the metabolites phosphocreatine and lipids. Conclusion This study introduces a method that allows an observer-independent, quantitative analysis of IHC to help establish metabolomic profiles and identify metabolomic markers of PCa from spectral intensities obtained with 1H HRMAS NMR Spectroscopy. The immunomarkers P504S and CK903 have been found suitable IHC analysis following 1H HRMAS MRS. A prospective in vivo application of PCa metabolite profiles and metabolomic markers determined by the presented method could serve as highly sensitive, non- invasive diagnostic tool. This observer- independent, computer- automated, quantitative analysis could help to distinguish highly aggressive tumors from low-malignant conditions, avoid overtreatment and reduce risks and complications for cancer patients in the future. Further studies are needed to verify the identified PCa metabolomic markers and to establish clinical applicability.:Table of Contents Glossary 1 Introduction 1. 1 Prostate Cancer 1. 2 Detection of Prostate Cancer – State of the Art 1. 2. 1 Prostate- Specific Antigen Test and Digital Rectal Examination 1.2.2 Radiographic Methods in PCa Detection 1.2.3 Transrectal Core Biopsies and Histopathological Analysis 1.2.4 Histopathological Grading of Prostate Cancer: GLEASON Score 1.3 Challenges and Need for New Approaches in PCa Diagnostic Management 2 Scientific Background I: Nuclear Magnetic Resonance,1H HRMAS NMR Spectroscopy and Metabolomic Profiles 2.1 Nuclear Magnetic Resonance 2.1.1 Spin Precession 2.1.2 Magnetic Resonance 2.1.3 Chemical Shift and J- coupling 2.2 Nuclear Magnetic Resonance 2.2.1 Magic Angle Spinning and 1H HRMAS NMR Spectroscopy 2.2.2 MAS Spinning Rates and Spinning Side Bands 2. 3 Metabolomics, Metabolite Profiles and Clinical Utility 3 Scientific Background II: Immunohistochemistry of Prostate Cancer 4 Aims of the Study 5 Material and Methods 5.1 Prostate Tissue Samples and Patient Demographics 5.2 1H HRMAS NMR Spectroscopy 5.2.1 Sample Preparation 5.2.2 Spectroscopy Scan 5.2.3 Data Processing 5.3 Immunohistochemistry 5.3.1 Immunohistochemistry Material and Equipment 5.3.2. Immunohistochemistry Protocol 5. 3. 3 Prostate Immunomarker Stability after 1H HRMAS NMR Spectroscopy 5.3.4 Qualitative IHC Analysis 5. 3.5 Quantitative IHC Analysis 5.3.5.1 Quantitative IHC Slide Review 5.3.5.2 Computer-Automated Quantitative IHC Analysis 5.3 Quantitative Histopathology 5. 4 Identification of Prostate Cancer Metabolomic Markers 5. 5 Patient Outcomes and Recurrence Categories 5.6 Statistical Analysis 6 Results 6. 1 Patient demographics 6. 2 Spectroscopy Results 6. 3 Immunohistochemistry 6. 3. 1 Evaluation of Prostate Immunomarker Stability after 1H HRMAS MRS 6. 3. 2 Qualitative Immunohistochemistry 6. 4 Quantitative Immunohistochemistry 6. 4. 1 Quantitative IHC Slide Review 6. 4. 2 Computer-Automated Quantitative IHC Evaluation using QIAP 6. 5 Quantitative Histopathology 6. 6 Identification of Prostate Cancer Metabolomic Markers using QIAP 6. 7 Patient Outcomes and Recurrence 7 Discussion 8 Summary / Abstract 9 Zusammenfassung 10 References 11 Erklärung über die eigenständige Abfassung der Arbeit 12 Danksagung 13 Lebenslauf und Publikationsverzeichnis Appendix A.1 Immunostaining protocols A.2 Spectral Intensities Measured by 1H HRMAS MRS in 51 Samples A.3 Graphs for Correlations of Spectral Intensities and CaE% determined by QIAP in 34 Additional Regions of Interest / Einführung Prostatakrebs ist eine häufigsten Krebserkrankungen in den USA und die zweithäufigste malignom- assoziierte Todesursache männlicher Patienten weltweit. Seit der Einführung des Prostata- spezifischen Antigen (PSA)- Screeningtests wird diese Krebsart in früheren Stadien diagnostiziert und therapiert, wodurch die Mortalitätsrate in den letzten Jahren deutlich reduziert werden konnte. Da moderne diagnostische Methoden bislang jedoch nicht ausreichend in der Lage sind, suffizient zwischen hochmalignen und weniger aggressiven Varianten dieses bösartigen Krebsleidens zu unterscheiden, werden häufig auch Patienten aggressiv therapiert, deren niedriggradiges Prostatakarzinom keine klinische Relevanz gehabt hätte. Es besteht daher ein großes wissenschaftliches Interesse an der Entwicklung neuer diagnostischer Methoden zur akkuraten Bestimmung von biologischem Status, Malignität, Aggressivität und Ausmaß einer Prostatakrebserkrankung. \\\\\\\"1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy\\\\\\\" (1H HRMAS MRS) ist eine vielversprechende diagnostische Methode, welche es ermöglicht, metabolomische Profile von Prostatakrebs zu erstellen, ohne die Gewebsstruktur der analysierten Proben zu zerstören. Durch anschließende histopathologische Begutachtung lassen sich die erstellten Metabolitprofile validieren und evaluieren. Im Gegensatz zu konventionellen histopathologischen Methoden können durch immunhistochemische Verfahren dabei objektivere, akkuratere und quantifizierbare histopathologische Erkenntnisse gewonnen werden. Die vorliegende Studie präsentiert einen neuentwickelten diagnostischen Ansatz zur quantitativen Bestimmung von metabolomischen Markern von Prostatakrebs, basierend auf der Durchführung von 1H HRMAS NMR Spektroskopie und quantitativer Immunhistochemie. Material und Methoden Einundfünfzig Gewebsproben von Prostatakrebspatienten wurden mittels 1H HRMAS MRS an einem 14.1 T BRUKER NMR Spektrometer unter Einsatz einer CPMG-Pulssequenz untersucht. Spektrale Intensitäten in 36 Metabolitregionen wurden gemessen. Anschließend wurden die analysierten Gewebeproben mit drei Immunfärbemarkern für sowohl malignes (P504S, Alpha-methylacyl-CoA-racemase) als auch benignes (CK903, High-molecular weight cytokeratin, und p63) Prostatagewebe angefärbt und quantitativ mit Hilfe eines Bildanalyseprogramms (QIAP) ausgewertet. Die Anwendbarkeit und Auswertbarkeit der genannten Immunomarker nach Spektroskopie wurde evaluiert und mit der Färbungsqualität von nicht- gescannten Schnitten verglichen. Die Resultate der automatischen Auswertung durch QIAP konnten durch einen erfahrenen Pathologen in einer quantitativen Analyse der Immunfärbungen sowie konventioneller histologischer Färbungen derselben Gewebsproben validiert werden. Die spektralen Intensitäten aus den Messungen mit 1H HRMAS MRS wurden mit den korrespondierenden Ergebnissen der quantitativen Auswertung der Immunfärbungen korreliert, um metabolomische Marker von Prostatakrebs zu identifizieren. Der klinische Verlauf und die Rezidivrate der Patienten wurden 5 Jahre nach der initialen Prostatektomie retrospektiv bestimmt. Rezidivkategorien wurden erstellt und mit den bestimmten spektralen Intensitäten korreliert, um metabolomische Marker für das Auftreten von Prostatakrebsrezidiven zu identifizieren. Ergebnisse Die Immunfärbungen mit P504S und CK903 zeigten exzellente Qualität und Auswertbarkeit nach vorheriger 1H HRMAS MRS. Beide Marker eigneten sich zur Durchführung von quantitativer Immunhistochemie an spektroskopierten Gewebeproben. Im Gegensatz dazu war die Qualität der Immunfärbungen mit p63 nach Spektroskopie vermindert. Quantitative Immunfärbungen unter Einsatz der Immunmarker P504S und CK903 stellten eine praktikable diagnostische Methode dar, um zwischen malignen und benignem Prostatagewebe zu unterscheiden. Der Anteil von bösartig verändertem Prostatagewebe, bestimmt durch QIAP, korrelierte signifikant mit den Ergebnissen der quantitativen Analyse der Immunfärbungen durch den Pathologen (p < 0.001), sowie mit der quantitativen Auswertung der konventionellen histopathologischen Färbung (p = 0.001). Ebenso ließ sich die Bestimmung des Anteils von benignem Gewebe mit QIAP zu den Ergebnissen der pathologischen Analyse korrelieren (p < 0.001 und p = 0.0183). Für zwei metabolomische Regionen konnte ein signifikante Korrelation zwischen relativen spektralen Intensitäten, bestimmt mit 1H HRMAS NMR Spektroskopie, und dem Anteil von malignem Epithelium in derselben Gewebeprobe, ermittelt durch QIAP, festgestellt werden: 3.22 ppm (p = 0.015) und 2.68 ppm (p = 0.0144). Die zu diesen Regionen korrespondierenden Metaboliten, Phosphocholin und Zitrat, konnten als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Die retrospektiven Analyse der klinischen Daten der Patienten fünf Jahre nach Prostatektomie ergab eine Überlebensrate von 97.8%. Elf dieser Patienten (24.4%) erlitten ein Rezidiv ihrer Erkrankung. Die bestimmten Rezidivkategorien korrelierten signifikant mit zwei metabolomischen Regionen (2.33 – 2.3 ppm, p = 0.0403 und 1.28 ppm, p = 0.0144), welche zu den Metaboliten Phosphokreatin und Lipiden korrespondierten. Schlussfolgerung Die vorliegende Studie präsentiert einen diagnostischen Ansatz zur objektiven und quantitativen Bestimmung metabolomischer Marker von Prostatakrebs unter Verwendung von 1H HRMAS MRS und Immunhistochemie. P504S und CK903 eignen sich als Immunmarker für quantitative Immunfärbungen nach vorheriger Durchführung von 1H HRMAS MRS. Die Metaboliten Phosphocholin und Zitrat konnten in der vorliegenden Patientenkohorte als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Eine mögliche in vivo Anwendung der gefundenen metabolomischen Marker könnte als hochsensitives, objektives und nicht- invasives diagnostisches Werkzeug der Prostatakrebsdiagnostik dienen. Der vorliegende untersucherunabhängige, automatisierte und quantitative diagnostischer Ansatz hat das Potential, zwischen hochmalignen und weniger aggressiven Krebsfällen zu unterscheiden und somit unnötige Risiken und Komplikationen für Prostatakrebspatienten zu reduzieren. Weitere Untersuchungen sind notwendig, um die identifizierten metabolomischen Marker zu verifizieren und eine klinische Anwendung zu etablieren.:Table of Contents Glossary 1 Introduction 1. 1 Prostate Cancer 1. 2 Detection of Prostate Cancer – State of the Art 1. 2. 1 Prostate- Specific Antigen Test and Digital Rectal Examination 1.2.2 Radiographic Methods in PCa Detection 1.2.3 Transrectal Core Biopsies and Histopathological Analysis 1.2.4 Histopathological Grading of Prostate Cancer: GLEASON Score 1.3 Challenges and Need for New Approaches in PCa Diagnostic Management 2 Scientific Background I: Nuclear Magnetic Resonance,1H HRMAS NMR Spectroscopy and Metabolomic Profiles 2.1 Nuclear Magnetic Resonance 2.1.1 Spin Precession 2.1.2 Magnetic Resonance 2.1.3 Chemical Shift and J- coupling 2.2 Nuclear Magnetic Resonance 2.2.1 Magic Angle Spinning and 1H HRMAS NMR Spectroscopy 2.2.2 MAS Spinning Rates and Spinning Side Bands 2. 3 Metabolomics, Metabolite Profiles and Clinical Utility 3 Scientific Background II: Immunohistochemistry of Prostate Cancer 4 Aims of the Study 5 Material and Methods 5.1 Prostate Tissue Samples and Patient Demographics 5.2 1H HRMAS NMR Spectroscopy 5.2.1 Sample Preparation 5.2.2 Spectroscopy Scan 5.2.3 Data Processing 5.3 Immunohistochemistry 5.3.1 Immunohistochemistry Material and Equipment 5.3.2. Immunohistochemistry Protocol 5. 3. 3 Prostate Immunomarker Stability after 1H HRMAS NMR Spectroscopy 5.3.4 Qualitative IHC Analysis 5. 3.5 Quantitative IHC Analysis 5.3.5.1 Quantitative IHC Slide Review 5.3.5.2 Computer-Automated Quantitative IHC Analysis 5.3 Quantitative Histopathology 5. 4 Identification of Prostate Cancer Metabolomic Markers 5. 5 Patient Outcomes and Recurrence Categories 5.6 Statistical Analysis 6 Results 6. 1 Patient demographics 6. 2 Spectroscopy Results 6. 3 Immunohistochemistry 6. 3. 1 Evaluation of Prostate Immunomarker Stability after 1H HRMAS MRS 6. 3. 2 Qualitative Immunohistochemistry 6. 4 Quantitative Immunohistochemistry 6. 4. 1 Quantitative IHC Slide Review 6. 4. 2 Computer-Automated Quantitative IHC Evaluation using QIAP 6. 5 Quantitative Histopathology 6. 6 Identification of Prostate Cancer Metabolomic Markers using QIAP 6. 7 Patient Outcomes and Recurrence 7 Discussion 8 Summary / Abstract 9 Zusammenfassung 10 References 11 Erklärung über die eigenständige Abfassung der Arbeit 12 Danksagung 13 Lebenslauf und Publikationsverzeichnis Appendix A.1 Immunostaining protocols A.2 Spectral Intensities Measured by 1H HRMAS MRS in 51 Samples A.3 Graphs for Correlations of Spectral Intensities and CaE% determined by QIAP in 34 Additional Regions of Interest

<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>

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A study was undertaken of plants used for treatment of diabetic symptoms by traditional healers of the Eeyou Istchee Cree (Canada), Lukomir Highlanders (Bosnia & Herzegovina), and Q’eqchi’ Maya (Belize). All antidiabetic plants were ranked by syndromic importance value (SIV) based on 15 symptoms, all of which were recognized by the Cree and Maya and 8 by the Highlanders. The Cree used only 18 species, the Highlanders 41, and the Maya 150, numbers which reflect the diversity of flora in their region. Vaccinium (Ericaceae) was one of the few genera in all three regions and the only consensus genus between the Cree and Highlander study sites. The Q’eqchi’ Maya ethnobotany did not present any cross-cultural consensus genera with Cree or Highlander medicinal plants, perhaps due to major biogeographic differences. In ethnopharmacological studies, Vaccinium species and Q’eqchi’ antidiabetic plants were tested in an assay relevant to diabetes, the advanced glycation endproduct (AGE) inhibition assay. Boreal and tropical Vaccinium species were potent inhibitors of AGEs and demonstrated concentration dependent inhibition, with a half maximal inhibitory concentration (IC50) range of 5.93–100 µg/mL. Phenolic content ranged from 80.3 to 201 µg/mL in boreal samples and from 1470 to 2170 µg/mL in tropical samples. Tropical species have a greater phenolic content and AGE inhibition. Seven Q’eqchi’ antidiabetic plant species were tested and all plant extracts showed AGE-inhibition. The IC50s ranged from 40.8 to 733 µg/mL, and the most active was Tynanthus guatemalensis Donn.. Tynanthus guatemalensis IC50 was about fives times greater (less active) than the mean ± SE IC50 reported for six tropical Vaccinium species of Vaccinium (8.77 ± 0.79 μg/mL). The highest consensus and most active Maya antidiabetic plant, Tynanthus guatemalensis Donn. Sm. was discovered to be an important plant recorded in archeological artifacts from the Late Classic Maya period (~750 CE). Ancient Maya used a cross shaped sign (k’an glyph) as a decorative element on Late Classic polychrome vessels and murals. The sign was believed to be the xylem template for a plant used as a flavouring in cacao drinks. However, the plant was incorrectly identified in the literature as Pimenta dioica (L.) Merr. (common name: Allspice) based on a common name and aromatic plant quality – not from a botanical voucher specimen. Pimenta dioica wood does not have a cross shape visible in the xylem but a unique character visible after a cross section of T. guatemalensis, is the xylem's cross shape organization. Wood of T. guatemalensis' also has an "allspice" aroma. Tynanthus guatemalensis is most likely the true botanical template behind the ancient Maya k’an glyph and this finding would show the continuity of use of this medicinal plant from ancient to modern times. Vaccinium was selected for an in depth phytochemical analysis using modern metabolomic methods. Nuclear magnetic resonance (1H NMR) was used to evaluate leaf extract spectra to provide information on (1) the taxonomic identity and (2) quantities of bioactive metabolites across multiple sites. Spectra clearly differentiated leaf samples of V. angustifolium, V. boreale, V. corymbosum, V. macrocarpon, V. myrtilloides, V. myrtillus, V. ovalifolium, and V. uliginosum according to generic, subgeneric, specific, phenotypic circumscriptions. Quantification of chlorogenic acid and hyperoside were replicated with a method that is highly reproducible across multiple sites with different NMR equipment. This methodology provides an important new approach to taxonomy and quality control for plants and natural health products.

A study was undertaken of plants used for treatment of diabetic symptoms by traditional healers of the Eeyou Istchee Cree (Canada), Lukomir Highlanders (Bosnia & Herzegovina), and Q’eqchi’ Maya (Belize). All antidiabetic plants were ranked by syndromic importance value (SIV) based on 15 symptoms, all of which were recognized by the Cree and Maya and 8 by the Highlanders. The Cree used only 18 species, the Highlanders 41, and the Maya 150, numbers which reflect the diversity of flora in their region. Vaccinium (Ericaceae) was one of the few genera in all three regions and the only consensus genus between the Cree and Highlander study sites. The Q’eqchi’ Maya ethnobotany did not present any cross-cultural consensus genera with Cree or Highlander medicinal plants, perhaps due to major biogeographic differences. In ethnopharmacological studies, Vaccinium species and Q’eqchi’ antidiabetic plants were tested in an assay relevant to diabetes, the advanced glycation endproduct (AGE) inhibition assay. Boreal and tropical Vaccinium species were potent inhibitors of AGEs and demonstrated concentration dependent inhibition, with a half maximal inhibitory concentration (IC50) range of 5.93–100 µg/mL. Phenolic content ranged from 80.3 to 201 µg/mL in boreal samples and from 1470 to 2170 µg/mL in tropical samples. Tropical species have a greater phenolic content and AGE inhibition. Seven Q’eqchi’ antidiabetic plant species were tested and all plant extracts showed AGE-inhibition. The IC50s ranged from 40.8 to 733 µg/mL, and the most active was Tynanthus guatemalensis Donn.. Tynanthus guatemalensis IC50 was about fives times greater (less active) than the mean ± SE IC50 reported for six tropical Vaccinium species of Vaccinium (8.77 ± 0.79 μg/mL). The highest consensus and most active Maya antidiabetic plant, Tynanthus guatemalensis Donn. Sm. was discovered to be an important plant recorded in archeological artifacts from the Late Classic Maya period (~750 CE). Ancient Maya used a cross shaped sign (k’an glyph) as a decorative element on Late Classic polychrome vessels and murals. The sign was believed to be the xylem template for a plant used as a flavouring in cacao drinks. However, the plant was incorrectly identified in the literature as Pimenta dioica (L.) Merr. (common name: Allspice) based on a common name and aromatic plant quality – not from a botanical voucher specimen. Pimenta dioica wood does not have a cross shape visible in the xylem but a unique character visible after a cross section of T. guatemalensis, is the xylem's cross shape organization. Wood of T. guatemalensis' also has an "allspice" aroma. Tynanthus guatemalensis is most likely the true botanical template behind the ancient Maya k’an glyph and this finding would show the continuity of use of this medicinal plant from ancient to modern times. Vaccinium was selected for an in depth phytochemical analysis using modern metabolomic methods. Nuclear magnetic resonance (1H NMR) was used to evaluate leaf extract spectra to provide information on (1) the taxonomic identity and (2) quantities of bioactive metabolites across multiple sites. Spectra clearly differentiated leaf samples of V. angustifolium, V. boreale, V. corymbosum, V. macrocarpon, V. myrtilloides, V. myrtillus, V. ovalifolium, and V. uliginosum according to generic, subgeneric, specific, phenotypic circumscriptions. Quantification of chlorogenic acid and hyperoside were replicated with a method that is highly reproducible across multiple sites with different NMR equipment. This methodology provides an important new approach to taxonomy and quality control for plants and natural health products.

Les pollutions par les xénobiotiques, en particulier les pesticides, et les métaux lourds issus des activités agricoles présentent une grande complexité de composition chimique et de dynamique spatio-temporelle. La présence de bandes enherbées entre les parcelles cultivées et les cours d’eau permet une limitation de la diffusion de ces pollutions résiduelles vers les milieux naturels. Le compartiment végétal de ces bandes enherbées peut jouer de multiples rôles dans ce contexte de protection environnementale. L’étude comparative réalisée in situ et en conditions contrôlées de laboratoire a permis de mettre en évidence le rôle biologique du compartiment végétal avec son implication directe dans les processus in planta d’absorption, de stockage et/ou de dégradation au moins partielle. Un tel rôle phytoremédiateur est dépendant de la capacité des plantes à se maintenir sur ces milieux pollués, qui diffère selon l’espèce considérée et structure ainsi les communautés végétales des bandes enherbées. L’étude intégrative en conditions contrôlées des réponses des plantes aux interactions avec les xénobiotiques à faibles doses, à différentes échelles de complexité du fonctionnement végétal, a permis de montrer les effets de ces stress chimiques chez l’espèce modèle Arabidopsis thaliana et chez l’espèce prairiale Lolium perenne. Les xénobiotiques et les métaux lourds à des doses subtoxiques ont induit d’importants bouleversements métabolomiques et moléculaires chez ces espèces, avec des effets cryptiques de ces polluants et de leurs produits de dégradation. L’analyse en conditions de multi-pollution, qui reflètent de manière réaliste les pollutions péri-agricoles, a montré la complexité et la difficulté de prédiction des interactions entre les effets des contaminants en mélange. Ces mécanismes de réponses diffèrent selon l’espèce et le polluant et laissent supposer des divergences en termes de perception et/ou de transport des polluants, ou de coordination des réponses moléculaires et métaboliques. Arabidopsis a ainsi présenté une coordination de ses réponses orientée vers une augmentation des métabolites de stress, et une diminution des métabolites carbonés (sucres solubles), en parallèle de modifications de l’expression de gènes impliqués dans les défenses antioxydantes, les défenses contre les stress xénobiotiques, ou dans la dynamique des phytohormones. Le stress chimique a entraîné chez Lolium des modifications majeures du métabolisme azoté, ainsi qu’un remaniement des processus de photorespiration. L’analyse transcriptomique de cette espèce a de plus montré que la majorité des gènes identifiés sont impliqués dans des voies de transduction de signal, montrant ainsi la complexité des mécanismes de réponse et les couplages qui existent entre les signaux métaboliques, en particulier liés aux sucres, les voies de signalisation associées aux phytohormones, les signaux de stress et la photosynthèse. / Environmental pollutions by xenobiotics, especially by pesticides and heavy metals derived from agricultural activities, show an important complexity of chemical composition and of spatiotemporal dynamic. Vegetative filter strips between cultivated fields and streams limit the diffusion of these residual pollutions to natural environments. However, the exact biological role of plant in these buffer strips is poorly understood in this context of environmental and ecological protection. A comparative study carried out in situ and in controlled conditions highlighted the role of plant compartment in the processes of absorption, storage and/or partial degradation of pollutants in planta. Such capability of phytoremediation depends on the maintenance of a vegetal cover in area subjected to recurring flow of pesticides, it varies according to species and leads to the structuration of vegetative filter strip communities. An integrative study in controlled conditions of plant responses to low doses of pollutants allowed to analyze at different levels of complexity the impacts of chemical stresses on the model species Arabidopsis thaliana and the grassland species Lolium perenne. Low and sublethal doses of xenobiotics, associated degradation products and heavy metals induced cryptic perturbations at metabolic and molecular levels. Multi-pollution analyses, which reflect realistic conditions of environmental exposure, highlighted complex interactive effects between pollutants in mixture and the difficulty to predict them. The mechanisms of response to these chemical stresses differ according to the species and the pollutant, and suggest differences in term of perception and/or transport of pollutants, or of coordination of molecular and metabolic responses. Arabidopsis presented a coordination of its responses toward an increase of stress metabolites, a decrease of carbon metabolites (soluble carbohydrates), in parallel with modifications of gene expressions implicated on antioxidant defences, defence against xenobiotic stresses, or phytohormone dynamic. Chemical stress leads to major modifications of nitrogen metabolism in Lolium, and perturbations of processes of photorespiration. De novo transcriptomic analysis of Lolium therefore showed that a majority of identified genes are related to signal transduction pathways, highlighting the complexity of response mechanisms and the links between metabolic signals, especially linked to carbohydrate, hormonal signaling pathways, stress signals and photosynthesis. Subtoxic chemical stress induced cryptic re-engineering of plant processes that may explain the development of tolerance for some species and their persistence in area affected by residual pollution.

Les techniques de criblage actuelles (in vitro et in silico) sont dépendantes des efforts menés en biologie médicinale pour identifier des cibles biologiques pertinentes ; cibles difficiles à définir pour les maladies génétiques dites "perte de fonction". De plus, les composés issus de ces cribles s'avèrent souvent inefficaces et/ou toxiques une fois confrontés à la complexité physiologique d'un organisme entier. Pour contourner ce problème, nous proposons d'utiliser le nématode C. elegans, notamment pour des maladies répondant aux critères suivants : i) physiopathologie complexe et/ou mal comprise excluant le développement à court terme de médicaments sur une base rationnelle, ii) peu d’espoir de thérapie génique/cellulaire à court terme, iii) conservation chez C. elegans du gène relié à la maladie humaine et induisant un phénotype exploitable une fois inactivé. Nous démontrons ici que ce petit nématode permet de tester, à moindre coût, un grand nombre de composés chimiques tout en conservant la complexité physiologique d'un animal entier. De plus, la souplesse génétique de cet animal permet d'apporter rapidement des informations sur le mode d'action des composés identifiés. Ainsi, en plus du but initial visant à identifier des molécules bioactives à intérêt thérapeutique, cette approche peut permettre de dégager de nouvelles cibles moléculaires utiles pour l'industrie chimique, et cruciales pour la recherche de traitements contre les maladies perte de fonction. Finalement, nous présentons comment mettre en place une telle stratégie, notamment pour la myopathie de Duchenne, l'amyotrophie spinale et le syndrome de Schwartz-Jampel. Enfin, nous présentons les résultats obtenus lors des différentes campagnes de criblage, les validations des molécules les plus prometteuses et les travaux effectués pour tenter de comprendre leur mode d'action chez le nématode. / Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limits its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans is gaining momentum as screening chemical tool. This tiny worm combines genetic amenability, low cost, and culture conditions that are compatible with large-scale screens. Its main advantage is to allow high-throughput screening in a whole-animal context. Moreover, its use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. Here, we introduce this approach with the Duchenne Muscular Dystrophy, the Spinal Muscular Dystrophy and the Schwartz-Jampel syndrome. We present the methodology used with each model to screen up to 7,000 compounds and the results of these screening campaigns. We further present the validation of our best hits and try to understand their mechanism of action.

Ce travail de thèse a cherché à déterminer si la contamination résiduelle à long terme des sols agricoles par les pesticides induit le développement de mécanismes d’adaptation aux pesticides chez les vers de terre. Il a aussi visé à identifier les coûts potentiels de l’adaptation de l’échelle de l’individu à celle de la population, et les conséquences pour l’écosystème sol. Une contamination résiduelle du sol par les pesticides est mesurée et comparée dans trois champs cultivés en agriculture conventionnelle (classés en fonction de l’historique cultural comme « haut », « moyen » et « bas » niveaux d’intrants), un champ cultivé en agriculture biologique et une prairie permanente biologique, tous dans ce type de management agricole depuis plus de 20 ans. En utilisant une méthode d’extraction des pesticides en milieu aqueux (représentant la fraction « biodisponible » des pesticides), 6, 8 et 4 résidus de pesticides sont détectés dans les champs à « haut », « moyen », et « bas » niveaux d’applications, respectivement, et un seul pesticide dans le sol du champ biologique (un résidu d’atrazine potentiellement vieux de plus de 20 ans). Les deux espèces endogées Allolobophora chlorotica et Aporrectodea caliginosa , communnes dans les sols des 5 champs,- mis à part A. chlorotica qui est absente du champ cultivé en agriculture biologique-, ont servi de modèles biologiques d’étude. Les stratégies d’adaptation aux pesticides sont étudiées en comparant les réponses de ces populations de vers de terre sur le terrain et après des expositions aux pesticides en laboratoire. Les réponses mesurées s’étendent de l’échelle moléculaire (enzymes de biotransformation et du stress oxidatif), biochimique (ressources énergétiques), métabolique (taux de respiration, métabolomique) à l’échelle de l’individu (biomasse, longueur) et de la population (traits de vie des cocons et des juvéniles), et aux possibles conséquences pour l’écosystème sol en termes de bioturbation (creusement et ingestion de sol) et de dissipation des pesticides comme service ecosystémique. Une capacité de détoxification augmentée et un plus grand potentiel anti-oxidant sont observés le long du gradient de contamination du sol et en laboratoire après exposition des vers de terre des champs conventionnel (population « pré-exposée ») et biologique (« naïve) à des pesticides. Des demandes énergétiques et des réarrangements métaboliques différents sont observés dans les deux populations, et sont plus prononcés chez la population pré-exposée. Une adaptation physiologique est démontrée chez les animaux pré-exposés, qui est associée à une ’augmentation de la bioturbation, et en cascade à une dissipation du pesticide dans le sol. Les conséquences au niveau de la population sont étudiées en termes de traits d’histoire de vie des deux populations pré-exposées et naïves. Le management en conventionnel incluant l’utilisation de pesticides semble diminuer le poids des adultes au champ, et implique potentiellement la réallocation des ressources énergétiques, des mécanismes reproductifs vers les processus métaboliques. Ceci aboutit à une diminution de la fécondité et du pourcentage d’éclosion et pourrait être un facteur participant à la diminution des populations de vers de terre dans les champs cultivés avec utilisation de produits phytosanitaires. / This work investigated if long-term residual contamination of agricultural soils leads to adaptation of earthworm populations to pesticides. It also aimed at identifying the costs of adaptation from the individual to the population level, and the consequences for the ecosystem. Residual contamination by pesticides was assessed and compared in three fields under conventional management (classified after evaluation of pesticide applications as ''high-'', ''medium-'', and ''low-''pesticide input), one field under organic agriculture requirements and one organic permanent pasture, all in this type of management for more than 20 years. Using a water extraction method, as indicative of the amount of bioavailable pesticides 6, 8 and 4 residues of pesticides were recovered in the ''high-'', ''medium-'', and ''low-input'' fields, respectively, and almost no pesticides were detected in the soil of the organic field except for low levels of residual (possibly 20 years old) atrazine. The endogeic species Aporrectodea caliginosa and Allolobophora chlorotica were found in common to the five fields, -except A. chlorotica which was absent from the organic field-, and were used as biological models. Adaptation strategies were investigated by comparing the populations of these earthworms between the different fields according to several endpoints in field and laboratory assessments. The endpoints measured ranged from the molecular (biotransformation and anti-oxidant enzymes), biochemical (main energy resources), and metabolic (respiration rate, metabolomics) levels, to individual (weight, length) and population-related parameters (cocoon and juvenile life traits), and to the possible consequences for the ecosystem in terms of bioturbation (earthworm burrowing behaviour) and pesticide disappearance as an crucial ecosystem service. Enhanced detoxification and anti-oxidant potential was demonstrated along the gradient of contamination in the fields, and in particular comparing the response to an experimental pesticide exposure between the pre-exposed, thus possibly adapted earthworms from the ''high-input'' field-, and the naïve population from the organic field. Distinct energetic demands and metabolic rearrangements were observed between the populations, more pronounced in the pre-exposed earthworms. Physiological adaptation was demonstrated in pre-exposed animals, and this was associated with an increase in burrowing behaviour and pesticide disappearance in the soil. Population-level consequences were assessed in life traits of the two populations. The conventional farming including the use of pesticides decreased the weight of adult worms in the field and resulted in reallocation of energy resources, possibly from reproductive to metabolic function. This led to lower fecundity and hatching success and could partly explain lower earthworm densities in pesticide-impacted soils. / I dette arbejde blev der forsket i om langsigtet residualforurening af landbrugsjord fører til pesticidadaptation hos regnorme. Forskningen sigtede også efter at identificere adaptations-omkostningerne fra individ- til populationsniveau, og konsekvenserne for økosystemet. Pesticiders residualforurening blev vurderet og sammenlignet i; tre konventionelt styrede marker (klassificeret efter evaluering af pesticid-anvendelser som ”højt-”, ”medium-”, og ”lav-input”), en mark styret ud fra økologiske betingelser, og et økologisk permanent græsningsareal, alle havde været styret på denne måde i mere end 20 år. Ved hjælp af en vand-ekstraktionsmetode blev 6,8 og 4 pesticidresiduums udvundet i henholdsvis ”høj-”, ”medium-”, og ”lav-input” markerne, og næsten ingen pesticider blev detekteret i jorden fra den økologiske mark bortset fra lave niveauer af resterende (højst sandsynligt 20 år gammelt) atrazin. Regnorms-populationerne Aporrectodea caliginosa og Allolobophora chlorotica blev fundet i alle fem marker, bortset fra A. chlorotica som ikke var til stede i den økologiske mark. Adaptationsstrategier blev undersøgt ved at sammenligne populationerne af disse regnorme de forskellige marker imellem ifølge flere parametre i felt- og laboratoriebedømmelserne. De målte parametre rangerede fra molekylære- (biotransformation og antioxiderende enzymer), biokemiske- (primære energiressourcer), og metaboliske- (respirationsrate, metabolomics) niveauer, til individuelle- (vægt, længde) og populations-relaterede parametre (puppe og juvenile livskarakterer), og til de mulige konsekvenser for økosystemet med hensyn til bioturbation (regnormes grave-adfærd) og nedgang i koncentrationen af ekstraherbare pesticider. Forbedret afgiftnings- og antioxiderende potentiale blev demonstreret langs gradienten af pesticid forurening i felten. Forbedret afgiftning i den pre-eksponerede population var særdeles tydeliggjort ved eksponeringen til pesticider i laboratoriet sammenliget med den økologiske population. Der blev observeret distinkte energibehov og metabolisk omgruppering populationerne imellem, dette var mere udtalt hos de pre-eksponerede regnorme. Der blev detekteret fysiologiske adaptationer hos de pre-eksponerede dyr, og dette hang sammen med en kompensatorisk øgning i grave-adfærd og nedgang i koncentrationen af ekstraherbare pesticider i jorden. Konsekvenser, på populationsniveau, blev bedømt ud fra de to populationers livskarakterer. Brug af pesticider resulterede i vægtnedgang hos de voksne orme i felten og omfordeling af energiressourcer, formodentlig fra reproduktiv til metabolisk funktion. Dette førte til lavere frugtbarhed og udklæknings-succes og kunne til dels forklare de lavere regnorme-densiteter i den pesticid-påvirkede jord.

The aim of this project has been to investigate whether there are correlations to be found between gut microbes and serum metabolites, which could be involved in the development of epilepsy. To do so, metabolomics data containing metabolites and metagenomics data containing bacteria have been integrated and used in a pipeline utilizing the software package DIABLO in R Studio. DIABLO stands for Data Integration Analysis for Biomarker discovery using Latent cOmponents and utilizes multi-block pls-da to integrate multiple omics data sets to find potential biomarkers. The results in this project are mainly divided into two groups, the first group being from taking samples at an early time point, where subjects have not yet developed symptoms of epilepsy and the second group being from taking samples at a late time point, where the subjects have developed epilepsy. To find biomarkers in the data used for the integration, two subgroups are of highest interest, namely subgroup PTE, which is the group that develops epilepsy symptoms after an induced trauma to the brain, as well as subgroup TBI which do not develop epilepsy symptoms after an induced trauma to the brain. Results from the early time point suggests that bacteria such as those from Phelethenecus, Christenselellales, Ventrimonas, Ruminococcaceae and Acetatifactor, as well as metabolites such as LPC 17:0, Indole and Indole-3-carboxyaldehyde might be of interest in finding biomarkers previous to the development of epilepsy after induced brain trauma.  Results from the late time point suggests that bacteria such as those from Muribaculaceae and Avidehalobacter, as well as metabolites such as Dioctyl sulfosuccinate, Canrenone, LPC 18:0, Uric acid, Arjunolic acid and Pseudouridine might be of interest in finding underlying mechanisms behind the existing condition of epilepsy. The hope is that findings in this paper might aid in future development of knowledge behind this disease as well as its underlying mechanisms.