Tesis por compendio / [EN] Cannabis sativa L. (Cannabaceae) is an angiosperm, allogamous and dicotyledonous species that includes short and neutral-day varieties with dioecious specimens (males and females), and monoecious plants. Among its many applications, its industrial and medicinal uses stand out. Despite the fact that cannabis has been used by humans since ancient times and the growing interest that the C. sativa therapeutic properties have aroused in researchers around the world, the psychoactivity of some of its varieties, derived from its ¿9-tetrahydrocannabinol (THC) content, has motivated the prohibition of its cultivation for almost sixty years. The strict control to which cannabis has been subjected has prevented professionals from all over the world from carrying out genetic breeding programs for this species, which has resulted in the absence of uniform varieties. In this Doctoral Thesis, different biotechnological tools for cannabis genetic improvement have been developed. In the first place, given the lack of reproducibility of some cannabis plant in vitro regeneration protocols and the great influence that the genotype exerts on their effectiveness, plant in vitro regeneration competence of different explants was evaluated. As a result, an hormone-free protocol from C. sativa hypocotyls that presents high regeneration rates (ranging from 32.26% to 71.15%) in all the genotypes evaluated, also presenting a 17.94% of spontaneous rooting rate of regenerants has been developed. At the same time, the polysomatic pattern of different cannabis explants has been studied, and it has been possible to regenerate, from them, a significant percentage of mixoploid specimens (17.65% from cotyledons and 13.33% from hypocotyls) that, as described in the existing literature, could show a greater capacity for cannabinoid synthesis. On the other hand, given the absence of scientific publications in this regard, and the potential that this technique presents to alleviate the intrinsic variability of this species, the most in-depth study to date on the male floral biology of C. sativa has been developed. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% have been found. Furthermore, all stages of development of the microgametophyte have been correlated with an easily measurable floral morphological marker such as the bud length, identifying bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains in all the phenotypes evaluated. In this way, and although the starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis, it has been possible to address the induction of microspore embryogenesis in this species, obtaining for the first time microspore-derived multicellular structures after one week long cold-shock bud pretreatment. Finally, as a prerequisite for the genetic editing of C. sativa by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, and taking advantage of the in vitro plant regeneration protocol which resulted from this Doctoral Thesis, it has been possible to develop for the first time a protocol for the production of stably transformed cannabis plants, which represents a historical milestone in the genetic improvement of the species. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyls achieved 23.1% and 5.0% of regeneration and transformation rates respectively. As a whole, the present Doctoral Thesis provides a range of biotechnological tools that will allow the development of a new generation of high-yield cannabis varieties with uniform traits, resistant to multiple biotic and abiotic stresses, and therefore being suitable for both industrial and medicinal use. / [ES] Cannabis sativa L. (Cannabaceae) es una especie angiosperma, alógama y dicotiledónea compuesta por variedades de día corto y día neutro que presentan ejemplares dioicos (machos y hembras), y plantas monoicas. Entre sus múltiples aplicaciones destacan tanto su uso industrial como su uso medicinal. A pesar de que el cannabis ha sido empleado por el ser humano desde tiempos ancestrales, la psicoactividad que presentan algunas de sus variedades, derivada de su contenido en ¿ 9 -tetrahidrocannabinol (THC), ha motivado la prohibición de su cultivo durante casi sesenta años. La estricta fiscalización a la que ha sido sometido el cannabis, ha impedido llevar a cabo programas de mejora genética de esta especie, lo que se ha traducido en la ausencia de variedades uniformes. En esta Tesis Doctoral se han desarrollado diferentes herramientas biotecnológicas para la mejora genética del cannabis. En primer lugar, dada la falta de reproducibilidad de algunos protocolos de cultivo in vitro de cannabis y la gran influencia que el genotipo ejerce en la efectividad de los mismos, se evaluó la capacidad de regeneración in vitro de diferentes explantes. Como resultado, se ha desarrollado un protocolo libre de hormonas a partir de hipocótilos de C. sativa que presenta altas tasas de regeneración (las cuales oscilan del 32,26% al 71,15%) en todos los genotipos evaluados, presentando además un 17,94% de tasa de enraizado espontáneo de los regenerantes. A su vez, se ha estudiado el patrón polisomático de diferentes explantes de cannabis y se ha conseguido regenerar, a partir de los mismos, un porcentaje significativo de ejemplares mixoploides (17,65% procedentes de cotiledones y 13,33% de hipocotilos) que, tal y como describe la bibliografía existente, podrían mostrar una mayor capacidad de síntesis de cannabinoides. Por otro lado, dada la ausencia de publicaciones científicas al respecto y el potencial que esta técnica presenta para paliar la variabilidad intrínseca de esta especie, se ha desarrollado el estudio más profundo hasta la fecha relativo a la biología floral masculina de C. sativa. Se han descrito hasta 476.903 microsporas y granos de polen por flor masculina, con tasas de viabilidad in vivo de las microsporas del 53,71 al 70,88%. Además, se han correlacionado todas las etapas de desarrollo del microgametofito con la longitud de la yema, identificando intervalos de longitud de yema que contienen mayoritariamente microsporas vacuoladas y granos de polen joven bicelular en todos los fenotipos evaluados. De este modo, y aunque la presencia de almidón en las microsporas y granos de polen de C. sativa sigue un patrón similar al observado en especies recalcitrantes a la androgénesis, ha sido posible abordar la inducción de la embriogénesis de microsporas en esta especie, consiguiendo producir por primera vez estructuras multicelulares derivadas de las microsporas tras aplicar sobre las yemas un pretratamiento de frío de una semana de duración. Finalmente, como requisito previo para la edición genética de C. sativa mediante los sistemas CRISPR/Cas, y haciendo uso del protocolo de regeneración in vitro de plantas surgido de la presente Tesis Doctoral, se ha conseguido desarrollar por primera vez un protocolo para producir plantas de cannabis transformadas genéticamente de forma estable, lo que supone un hito histórico en la mejora genética de la especie. Después del cocultivo con A. tumefaciens y el posterior cultivo en medio de regeneración selectiva con antibióticos, los hipocótilos lograron respectivamente un 23,1% y un 5,0% de tasas de regeneración y transformación. En su conjunto, la presente Tesis Doctoral proporciona un abanico de herramientas biotecnológicas que permitirán el desarrollo de una nueva generación de variedades de cannabis de alto rendimiento, que presenten caracteres homogéneos, resistentes a múltiples estreses tanto bióticos como abióticos, y siendo así aptas tanto para un uso industrial como medicinal. / [CAT] Cannabis sativa L. (Cannabaceae) és una espècie angiosperma, alógama i dicotiledònia composta per varietats de dia curt i dia neutre que presenten exemplars dioics (mascles i femelles), i plantes monoiques. Entre les seues múltiples aplicacions destaquen tant el seu ús industrial com el seu ús medicinal. Tot i que el cànnabis ha sigut emprat per l'ésser humà des de temps ancestrals, la psicoactivitat que presenten algunes de les seues varietats, derivada del seu contingut en ¿9-tetrahidrocannabinol (THC), ha motivat la prohibició del seu cultiu durant gairebé seixanta anys. L'estricta fiscalització a la qual ha sigut sotmés el cànnabis, ha impedit que professionals de tot el món puguen dur a terme programes de millora genètica d'aquesta espècie, la qual cosa s'ha traduït en l'absència de varietats uniformes. En aquesta Tesi Doctoral s'han desenvolupat diferents eines biotecnològiques per a la millora genètica del cànnabis. En primer lloc, donada la falta de reproducibilitat d'alguns protocols de cultiu in vitro de cànnabis i la gran influència que el genotip exerceix en l'efectivitat d'aquests, es va avaluar la capacitat de regeneració in vitro de diferents explants. Com a resultat, s'ha desenvolupat un protocol lliure d'hormones a partir de hipocòtils de C. sativa que presenta altes taxes de regeneració (les quals oscil·len del 32,26% al 71,15%) en tots els genotips avaluats, presentant a més un 17,94% de taxa d'arrelat espontani dels regenerants. Al mateix temps, s'ha estudiat el patró polisomàtic de diferents explants de cànnabis i s'ha aconseguit regenerar, a partir d'aquests, un percentatge significatiu d'exemplars mixoploids (17,65% procedents de cotilèdons i 13,33% de hipocòtils) que, tal com descriu la bibliografia existent, podrien mostrar una major capacitat de síntesi de cannabinoids. D'altra banda, donada l'absència de publicacions científiques sobre aquest tema i el potencial que aquesta tècnica presenta per a pal·liar la variabilitat intrínseca d'aquesta espècie, s'ha desenvolupat l'estudi més profund fins hui relatiu a la biologia floral masculina de C. sativa. S'han descrit fins a 476.903 microspores i grans de pol·len per flor masculina, amb taxes de viabilitat in vivo de les microspores del 53,71 al 70,88%. A més, s'han correlacionat totes les etapes de desenvolupament del microgametòfit amb la longitud de la gemma, identificant intervals de longitud de gemma que contenen majoritàriament microspores vacuolades i grans de pol·len jove bi-cel·lular en tots els fenotips avaluats. D'aquesta manera, i encara que la presència de midó en les microspores i grans de pol·len de C. sativa segueix un patró similar a l'observat en espècies recalcitrants a la androgènesi, ha sigut possible abordar la inducció de la embriogènesi de microspores en aquesta espècie, aconseguint produir per primera vegada estructures multicel·lulars derivades de les microspores després d'aplicar sobre les gemmes un pretractament de fred d'una setmana de duració. Finalment, com a requisit previ per a l'edició genètica de C. sativa mitjançant els sistemes CRISPR/Cas, i fent ús del protocol de regeneració in vitro de plantes sorgit de la present Tesi Doctoral, s'ha aconseguit desenvolupar per primera vegada un protocol per a produir plantes de cànnabis transformades genèticament de manera estable, la qual cosa suposa una fita històrica en la millora genètica de l'espècie. Després del cocultiu amb A. tumefaciens i el posterior cultiu en medi de regeneració selectiva amb antibiòtics, els hipocòtils van aconseguir respectivament un 23,1% i un 5,0% de taxes de regeneració i transformació. En el seu conjunt, la present Tesi Doctoral proporciona un ventall d'eines biotecnològiques que permetran el desenvolupament d'una nova generació de varietats de cànnabis d'alt rendiment, que presenten caràcters homogenis, resistents a múltiples estressos tant biòtics com abiòtics, i sent així aptes tant per a un ús industrial com medicinal. / Galán Ávila, A. (2021). Development of biotechnological tools for the genetic improvement of Cannabis sativa L [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/176013 / Compendio

Cannabinoid (CB) receptors have been found in most vertebrates that have been studied. The location of various CB receptors in the body and brain are known, but their physiological functions are not fully understood. The effects CBs have on the cardiovascular system have been of growing interest in recent years. Increasing reports from emergency departments and law enforcement agencies detail acute cardiovascular and psychological effects from synthetic CB intoxication, such as JWH-018. This major health concern is substantiated by governmental agencies like the CDC and NIDA. This pilot study investigates the acute toxic effects of the synthetic CB, JWH-018, on the cardiovascular and neuroendocrine systems in Ictalurus punctatus (channel catfish). Research in organisms besides the traditional mammal models can provide new insights into CB function and physiology. Ictalurus punctatus lend multiple benefits as a model organism that permits researchers to investigate in vivo effects of both cardiovascular and neuroendocrine systems without much influence from traditional sampling methods, and further more provide ample size and tissue to perform specific cardiovascular experiments. Multiple methods were used to assess cardiovascular function and sympathetic nervous system activation. Two different doses, low (500 µg/kg) and high 1,500 µg/kg, of JWH-018 were evaluated in the study. Delivery of JWH-018, via dorsal aorta cannulation, was administered to channel catfish in order to measure cardiovascular functions and sample blood. Plasma levels of the hypothalamus-pituitary-adrenal/interrenal (HPA/I) biomarkers; ACTH, cortisol, epinephrine, and norepinephrine, were measured using ELISAs. Myocardial and neural tissue was collected after the exposures for rt-PCR analysis on β2 adrenergic and glucocorticoid receptor density change. Acute exposure of JWH-018 in undisturbed channel catfish yielded several findings: (1) High dose of JWH-018 was responsible for cardio depressor effects in catfish with a tendency to produce tachycardia, (2) rt-PCR results showed a 2.7 fold increase of glucocorticoid receptor mRNA density in catfish cardiomyocytes when exposed to each dose of JWH-018, (3) Catfish plasma ACTH levels were increased with high doses of JWH-018, while plasma cortisol was increased by low doses. Channel catfish is an excellent animal model to examine the effects of synthetic cannabinoids and cardiovascular function. Acute exposures to high levels of JWH-018 appear to produce cardiovascular dysfunction providing evidence that substantiates emergency department reports, in addition yields novel information about the interaction of CBs exposure and the increase of glucocorticoid receptors levels on cardiomyocytes. The channel catfish is a new animal model that can aid in further investigations of CB exposure and multiple physiological functions for health and toxicology studies. With relatively easy adjustments from this pilot study, the effects on CBs can be monitored on Ictalurus punctatus with confident results concerning human health.

Ce projet de maitrise implique le développement et l’optimisation de deux méthodes utilisant la chromatographie liquide à haute performance couplée à la spectrométrie de masse en tandem (HPLC-MS/MS). L'objectif du premier projet était de séparer le plus rapidement possible, simultanément, 71 médicaments traitant la dysfonction érectile (ED) et 11 ingrédients naturels parfois retrouvés avec ces médicaments dans les échantillons suspectés d’être adultérés ou contrefaits. L'objectif du deuxième projet était de développer une méthode de dépistage permettant l'analyse rapide simultanée de 24 cannabinoïdes synthétiques et naturels pour une grande variété d'échantillons tels que les mélanges à base de plantes, des bâtons d'encens, de sérums et de cannabis. Dans les deux projets, la séparation a été réalisée en moins de 10 min et cela en utilisant une colonne C18 à noyau solide 100 x 2,1 mm avec des particules de 2,6 µm de diamètre couplée à un système MS avec trappe ionique orbitale fonctionnant en électronébulisation positive. En raison du nombre élevé de composés dans les deux méthodes et de l’émergence de nouveaux analogues sur le marché qui pourraient être présents dans les échantillons futurs, une méthode de dépistage LC-MS/MS ciblée/non-ciblée a été développée. Pour les deux projets, les limites de détection étaient sous les ng/mL et la variation de la précision et de l’exactitude étaient inférieures de 10,5%. Le taux de recouvrement à partir des échantillons réels variait entre 92 à 111%. L’innovation des méthodes LC-MS/MS développées au cours des projets est que le spectre de masse obtenu en mode balayage lors de l'acquisition, fournit une masse exacte pour tous les composés détectés et permet l'identification des composés initialement non-ciblés, comme des nouveaux analogues. Cette innovation amène une dimension supplémentaire aux méthodes traditionnellement utilisées, en permettant une analyse à haute résolution sur la masse de composés non-ciblés. / This master’s project involved the development and optimization of two rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. The objective of the first project was to simultaneously separate, as rapidly as possible, 71 erectile dysfunction (ED) treatment drugs and 11 natural ingredients sometimes found alongside ED drugs present in suspected adulterated or counterfeit samples. The objective of the second project was to develop a screening method allowing rapid, simultaneous analysis of 24 synthetic and natural cannabinoids for a wide variety of samples such as herbal smoking mixtures, incense sticks, serums and Cannabis plant material. In both projects, the separations were achieved in ≤ 10 min using 2.6 µm fused-core C18 particles packed into a 100 x 2.1 mm column coupled to an LTQ Orbitrap XL mass spectrometer operated in positive electrospray mode. Because of the very high number of compounds in both methods and the knowledge that future analogues are always immerging on the market that could thus be present in samples, a targeted/untargeted LC-MS/MS screening method was developed. For both projects, detection limits were in the sub ng/mL range and intra- and inter-assay precisions were below 10.5%. Recovery from real samples ranged from 92 to 111%. The innovation of the developed LC-MS/MS methods is that the full scan event in the MS acquisition provides accurate masses for all detected species and thus allows post-analysis identification of initially untargeted compounds, i.e., the immerging analogues. This innovation adds an additional dimension to traditional MS/MS methods, allowing high mass resolution of untargeted compounds.

Objective: This research aimed at reviewing the information provided by the online drug misuse communities with reference to the available evidence-based literature on the novel psychoactive substances. Methodology: Among hundreds of novel psychoactive substances, four groups (phenethylamines, tryptamines, piperazines and miscellaneous) were selected for the study. Various website drug fora were identified by Google and Yahoo search engines using a set of specific key words. The methods consisted of extracting and analysing qualitative data from the identified website fora. This was also supplemented by critical reviewing the existing evidence-based literature search for each of the selected psychoactive compounds. Results: The combined search results identified 84 unique website fora from which qualitative data were extracted for thirty novel psychoactive substances and organised into technical folders. This data extracted from online communities has thrown some light on factors such as the mode of purchase, subjective experiences, reasons for use, combinations, legislation, mechanisms of action in the CNS, side effects, toxicity and its management. This would enable the clinicians to be obtain full history when assessing and would inform better treatment choices. Conclusions: A range of novel psychoactive substances have been made recently available across the globe. The sale is easily achieved through the Internet. New legislations are made to control some recreational substances whilst newer substances appear. Furthermore, the distributors sell the backlog of products even after controlling of the substance has occured and hence are liable to potentiating criminal investigations. It is here suggested as well that the 'genuinity' of each onlince susbtance is questionable. Evidence-based literature is scant for the vast majority of these substances. Accidental overdoses are common occurences and some of the potential life-threatening clinical situations include sympathomimetic toxidrome and serotonin syndrome. Benzodiazepines appear to help with agitation and neuropsychiatric manifestations. Better levels of international cooperation and rapid share of available information may be needed to tackle the emerging problem of the novel psychoactive substances.

The use of hair and oral fluid for drugs of abuse testing has increased over the last years. For this reason, the assurance that results provided using these matrices are reliable and error-free is needed. The objective of this thesis is to develop tools to assess the quality of results provided by laboratories analysing drugs of abuse in hair and in oral fluid and to evaluate the effect on the quality of results of different actions carried out. For this reason, intercomparison exercises have been organized and some studies have been performed to develop appropriate quality control materials. Regarding the analysis of drugs of abuse in hair, nine different intercomparison exercises have been organized. The evaluation of qualitative and quantitative results reported by laboratories together with the study of the methodology used, has led to know the quality of the results, to identify the sources of error and to know the corrective actions that should be developed. Concerning the quality control material, these exercises have enabled to know the influence on the results of the type of hair used to perform the analysis. Concerning the analysis of drugs of abuse in oral fluid, a method has been developed to identify and quantify 6-monoacetyl morphine, morphine, codeine, cocaine and benzoylecgonine in oral fluid samples. On the other hand, stability studies of the main drugs of abuse in oral fluid have been done to establish the optimal preparation, transport and storage conditions, and finally, two intercomparison exercises have been conducted to know the performance of analytical laboratories when analysing drugs of abuse in oral fluid and to know the stability of some drugs of abuse in two commercial collection devices. / La creixent utilització del cabell i del fluid oral per a l’anàlisi de drogues d’abús, ha portat a la necessitat d’assegurar que els resultats obtinguts utilitzant aquestes matrius són fiables i lliures d’error. L’objectiu d’aquesta tesi és desenvolupar eines que permetin avaluar la qualitat dels resultats dels laboratoris que analitzen drogues d’abús en cabell i en fluid oral i avaluar l’efecte de diferents accions sobre la qualitat final dels resultats. Per això s’han organitzat exercicis interlaboratori i s’han realitzat estudis per desenvolupar material d’assaig adequat. Pel que fa a l’anàlisi de drogues d’abús en cabell, s’han realitzat nou exercicis interlaboratori. A través de l’avaluació dels resultats qualitatius i quantitatius informats pels laboratoris i mitjançant l’estudi de la metodologia emprada, ha estat possible conèixer la qualitat dels resultats obtinguts, detectar les fonts d’error i conèixer quines mesures correctives caldria desenvolupar. Pel que fa al material d’assaig, aquests exercicis han permès conèixer la influència que té, en els resultats, el tipus de cabell del qual es parteix per realitzar l’anàlisi. Pel que fa a l’anàlisi de drogues d’abús en fluid oral, s’ha desenvolupat i validat un mètode analític que ha permès identificar i quantificar 6-monoacetil morfina, morfina, codeïna, cocaïna i benzoïlecgonina en mostres de fluid oral. Per altra banda, s’han realitzat estudis d’estabilitat de les principals drogues en fluid oral que han permès establir les condicions òptimes de preparació, transport i conservació del material d’assaig i, per últim, s’han realitzat dos exercicis interlaboratori que han permès conèixer la qualitat dels resultats analítics obtinguts pels laboratoris i també l’estabilitat de les drogues en dos dispositius de recollida comercials.