By using cryo-electron microscopy, we analyzed the morphology and structure of long double-stranded DNA chains condensed upon addition of varying amounts of the tetravalent polycation spermine (polyamine). Experiments have been performed i) with chains diluted in the bulk and ii) with individual chains confined in a virus capsid.Bulk experiments have been done with lambda DNA (48.5 kbp) at low concentration (0.03 mM Ph) and in low salt conditions (10 mM Tris HCl, 1 mM EDTA, pH 7.6). We explored a wide range of spermine concentration, from the onset of precipitation (0.05 mM sp) up to above the resolubilization limit (400 mM sp). Sixteen min after mixing spermine and DNA, samples have been trapped in thin films and vitrified in liquid ethane to keep ionic conditions unchanged, and imaged at low temperature with low doses of electrons (cryoTEM). DNA chains mostly form large aggregates of toroids in which DNA chains are hexagonally packed with interhelical spacings of 2.93, 2.88, and 2.95 nm at 0.05, 1 and 100 mM spermine, respectively, in agreement with previous X-ray data. At higher spermine concentration (200 mM), hexagonal toroids are replaced by cholesteric bundles with a larger interhelical spacing (3.32 nm). We conclude that the shape and the structure of the liquid crystalline sp-DNA condensates are linked to the DNA interhelix spacing and determined by the ionic conditions i.e. by the cohesive energy between DNA strands. Outside of the precipitation domain (400 mM spermine), DNA chains form a soluble network of thin fibers (4-6 nm in diameter) that let us reconsider the state of these DNA chains in excess of spermine. We also designed experiments to visualize condensates formed 6-60 sec after mixing Lambda DNA with 0.05 mM spermine, under identical buffer conditions. Among multiple original shapes (not found after 16 min), the presence of stretched and helical elongated fibers seen only 9sec after addition of spermine let us propose that DNA chains are immediately stretched upon addition of spermine, relax into helical structures and finally form small toroids (containing in some cases less than one Lambda chain) that further grow and aggregate. We also analyzed the dimensions and structural details of the complete collection of toroids, and reveal the existence of geometric constraints that remain to be clarified. Since it was only exceptionally possible to prevent the aggregation of DNA in dilute solution, we used another approach to observe the collapse of single DNA chains. We handled a population of T5 viruses containing a fraction of their initial genome (12-54 kbp long). The Na-DNA chain, initially confined in the small volume of the capsid (80nm in diameter) is collapsed by the addition of spermine. Compared to the first set of experiments, we explored a higher DNA concentration range (0.45 mM Phosphates in the whole sample) and the spermine concentration was varied from 0.05 to 0.5 mM (which corresponds to much lower +/- charge ratios). Experiments are thus performed close to the precipitation line, in the coexistence region, between the region where all chains are in a coil conformation, and the region where all chains are collapsed into toroids. We describe the existence of intermediate states between the coil and the toroidal globule that were not reported yet. In these "hairy toroids", part of the DNA chain is condensed in the toroid and the other part stays uncondensed outside of it. The interhelical spacing was also measured; it is larger in these partly-condensed toroids than in the fully organized toroids formed at higher spermine concentration.These two series of experiments show the interest of cryoEM to analyze the structural polymorphism and local structure of spermine-DNA aggregates. We also demonstrated how the confinement interferes with DNA condensation and the interest to investigate such effects that are important in the biological context.

Skeletal muscle atrophy is a debilitating condition that commonly occurs as a secondary consequence of many acute and chronic medical conditions, including muscle disuse, heart and renal failure, starvation, cancer, HIV/AIDS, and aging. Though it leads to weakness, falls, and fractures, and reduces independence and quality of life for millions of Americans annually, no effective pharmacologic therapies for muscle atrophy exist. This is largely due to a poor understanding of the pathogenesis of skeletal muscle atrophy at a molecular level. In this thesis, I describe my studies into the molecular pathogenesis of skeletal muscle atrophy. Using mouse models, I showed that the gene encoding the pro-atrophy nuclear protein Gadd45a is regulated by distinct pathways after muscle denervation and fasting, and also identified a novel protein regulating skeletal muscle fiber size. First, we demonstrated that denervation-induced muscle atrophy, unlike atrophy mediated by fasting, does not require the bZIP transcription factor ATF4. However, the lysine deacetylase HDAC4 is sufficient to induce Gadd45a mRNA and necessary for Gadd45a mRNA induction after denervation, but not after fasting. Taken together, these data show that Gadd45a is a central convergence point for muscle atrophy caused by several stimuli, and also demonstrate that distinct pathways mediate Gadd45a induction in different models of skeletal muscle atrophy. Second, we identified spermine oxidase as a critical regulator of muscle fiber size. We observed that spermine oxidase mRNA and spermine oxidase protein were reduced by several distinct causes of muscle atrophy (i.e. immobilization, denervation, fasting, and aging). Furthermore, spermine oxidase overexpression increased muscle fiber size, while spermine oxidase knockdown caused muscle fiber atrophy. Restoring spermine oxidase expression significantly attenuated muscle atrophy after limb immobilization, denervation, and fasting. Finally, we identified p21 as a key upstream regulator of spermine oxidase expression, and spermine oxidase as a required mediator of p21-mediated skeletal muscle fiber atrophy. Collectively, these findings greatly advance our understanding of the molecular pathogenesis of skeletal muscle atrophy. These data demonstrate that Gadd45a is a convergence point for multiple pro-atrophy pathways and identify spermine oxidase as a novel therapeutic target for the treatment of skeletal muscle atrophy. These discoveries suggest several important new areas for future research, and further our understanding of this common, debilitating condition.

Abstract not available

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

La synthèse et la vectorisation d’agents anticancéreux constituent des axes de recherche majeurs du LCSN. De nombreux composés naturels possèdent des propriétés anticancéreuses, mais ils sont abandonnés en raison de leur manque de sélectivité vis-à-vis des cellules cancéreuses ou de leur faible biodisponibilité. Ainsi, un grand intérêt est actuellement porté sur le développement de médicaments spécifiquement vectorisés vers les cellules cancéreuses. Les vecteurs utilisés dans ce travail sont des dérivés de polyamines et des nano objets de type β-cyclodextrines / nanocristaux de cellulose (β-CD/CNCx). Les polyamines vont permettre un ciblage actif des cellules cancéreuses grâce au système de transport de polyamine (PTS) surexprimé dans ces cellules. Les nano objets vont cibler spécifiquement les tumeurs via un ciblage passif dû à l’effet EPR. Les principes actifs employés dans cette étude sont des flavonoïdes, et plus particulièrement des chalcones. En effet, les flavonoïdes, qui constituent une large famille de composés phénoliques naturels, sont connus pour leurs nombreux effets biologiques comme les activités antioxydantes, anti-inflammatoires et anti-prolifératives.L’intérêt du LCSN à la fois pour les chalcones et les agents anticancéreux nous a conduits à concevoir de nouveaux composés antiprolifératifs vectorisés. Ce travail présente dans un premier temps la synthèse de chalcones et l’obtention de dérivés couplés aux différents vecteurs décrits précédemment (motifs polyaminés,β-CD/CNCx) ; un travail sur la synthèse d’une bis-chalcone via le couplage de Suzuki est également exposé.L’ensemble des molécules obtenues est caractérisé par des analyses RMN 1H, 13C et HRMS. Dans une seconde partie, nous présentons l’ensemble des évaluations biologiques des composés précédemment obtenus. Ces évaluations sont réalisées par un test de viabilité cellulaire (test MTT) sur quatre lignées cancéreuses : deux colorectales (HT-29 et HCT-116) et deux prostatiques (PC-3 et DU-145). / Synthesis and vectorization of anticancer agents are major research themes of LCSN. Many natural compoundspossess anti-cancer properties, but they are dropped because of their lack of selectivity to cancer cells or theirlow bioavailability. Thus, great interest is currently focused on the development of drugs specifically vectorizedto cancer cells. The vectors used in this work are polyamine derivatives and nano-objects type β-cyclodextrin /cellulose nanocrystals (β-CD/CNCx). Polyamines allow active targeting of cancer cells through the polyaminetransport system (PTS) overexpressed in these cells. Nano-objects specifically target tumors using a passivetargeting due to the EPR effect. Drugs used in this study are flavonoids, especially chalcones. Indeed,flavonoids, which constitute a large family of natural phenolic compounds, are known for their numerousbiological effects such as antioxidant, anti-inflammatory and anti-proliferative activities. The interest of LCSNfor both chalcones and anticancer agents led us to design new vectorized anti-proliferative compounds. Firstly,this work shows the synthesis of chalcones and their derivatives coupled to various above-described vectors(polyamines units, β-CD/CNCx); a work on the synthesis of a bis-chalcone through the Suzuki coupling reactionis also exposed. All molecules obtained are characterized by 1H NMR, 13C NMR and HRMS analysis. In thesecond part of this work, we present all biological evaluations of compounds previously obtained. Theseassessments are performed through a cell viability test (MTT test) on four cancer cell lines: two colorectal (HT-29 and HCT-116) and two prostate (PC-3 and DU-145) cell lines.

Les oligonucleotides sont utilisés pour de nombreuses applications dans le domaine du diagnostic et ils peuvent également être utilisés comme traitement pour de nombreuses maladies. Les oligonucléotides sont des polyanions qui viennent s'hybrider sur leurs séquences complémentaires elles aussi anioniques. Les répulsions électrostatiques impliquent que l'addition de charges positives sur les oligonucléotides serait bénéfique pour diminuer les répulsions et améliorer l'hybridation. Dans le but de diminuer ces répulsions électrostatiques, des conjugués oligonucléotide-oligocation sur lesquels sont greffées des unités spermines ont été développés. Les conjugués oligonucléotideoligocation sont synthétisés sur un synthétiseur automatique d'oligonucléotide en utilisant la chimie des phosphoramidites. Les" Zip Nucleic Acid "ou ZNAs sont des oligonucléotides portant une queue cationique de quelques unités de spermine et sont de charge globale négative. Les modifications apportées permettent d'améliorer l'hybridation en accélérant la reconnaissance de la séquence cible et en augmentant la température de fusion linéairement avec le nombre de spermines greffées sans altérer la spécificité. Les ZNAs se révèlent être efficaces utilisés comme amorces ou sondes en PCR et ils apparaissent comme de nouveaux outils intéressants pour la biologie moléculaire. Les petits ARNintérferents (si RNA) induisant l'extinction d'un gène par la voie d' ARN interférence ont suscités un grand engouement ces dernières années, cependant leur très faible pénétration cellulaire est un frein majeur à leur utilisation. C'est pour cela que les conjugués oligonucléotide- oligospermine ont un réel intérêt pour le domaine de la thérapie in vivo. Les duplexes SIRNAPLUS cationiques sont des siRNAs ciblantspécifiquement un ARN messager. Ils sont constitués d'un brin sens ARN-oligospermine de charge globale positive hybridé à un brin antisens. Les résultats ont montré que lesSIRNAPLUS pouvaient entrer seuls dans les cellules sans agent de transfert pour induire l'extinction d'un gène cible et les premières expériences montrent qu'ils sont actifs in vivo. Mes travaux de thèse ont porté sur le développement des conjugués oligonucléotide oligospermine et démontrent des applications potentielles dans le domaine du diagnostic et de la thérapie.

The polyamines are a group of natural compounds which have been found in almost all living tissues. N1- and N8- acetylspermidine have been known for a considerable time as normal constituents in human urine, but their physiological role is unknown. Recent studies have indicated the presence of enzymes in the tissues capable of converting spermidine into N1-acetylspermidine and N8-acetylspermidine and other enzyme activities which catalyze deacetylation and interconversion reactions. One approach for determining physiologic activity of an endogenous compound is to observe their pharmacologic and toxicologic effects. In the present study, the LD50 for N1-acetylspermidine, N8-acetylspermidine, spermidine, spermine and putrescine, were determined by intraperitoneal injection in mice. The LD50 for N1-acetylspermidine •2HCl and N8-acetylspermidine •2HCl were 1150 and 820 mg/kg, respectively, and the respective LD50 for spermidine •3HCl, spermine n#8226;4HCl and putrescine n#8226;2HCl were 870, 370, and 1400 mg/kg. The major difference between the toxicity of N1-acetylspermidine and N8acetylspermidine and that of spermidine, spermine and putrescine appeared to be the latency period for the time of death. While N1- and N8-acetylspermidine have very rapid effects in which animals died in the first ten minutes after the injection with very few delayed deaths, the effects of spermidine, spermine and putrescine occurred over a wider range of time intervals with some deaths occurring as late as ten days after treatment The other signs of toxicity with N1acetylspermidine, N8-acetylspermidine, spermidine, spermine, and putrescine were qualitatively quite similar as each compound produced hypothermia, sedation, muscle incoordination, decreased motor activity, decreased respiration, and clonic convulsions.

Snyder-Robinson syndrome (SRS) is an X-linked neurodegenerative disorder affecting males. The disease appears early in childhood with symptoms like bone abnormalities, reduced muscle mass, and mobility issues. It results from disrupted polyamine biosynthesis, which is due to a mutation in spermine synthase (Sms). This causes an abnormally elevated Spermidine: Spermine ratios. There is no approved SRS treatment, rather only management of symptoms. A genetic mouse model for the SmsG56S mutation found in some SRS patients was characterized, and a novel spermine Prodrug treatment was tested. The hypothesis is that male SmsG56S mutant mice exhibit polyamine dysregulation and SRS traits, while Prodrug intervention may rebalance abnormal ratios and facilitate a normalized phenotype. Mice were phenotyped in pure C57BL/6J, mixed C3H, and backcrossed C57BL/6J x C3H backgrounds. Lethality of the mutation, especially in C57BL/6J mice, was an obstacle. Viable mutant mice exhibited reduced body weight, typically smaller size, and lower bone densities compared to age-matched wild-type mice. Prodrug treatment was performed using different dosing strategies and in all backgrounds. Upon histological examination, testes and bone exhibited subtle defects in affected mutant male mice, while no detectable differences were found in skeletal muscle, liver, or kidney compared to wild-type mice. Acute prodrug treatment using oral gavage 5 times per week over 2 weeks was found to rebalance the Spermidine: Spermine ratio. Repeated efforts to dose Prodrug over a longer 6-8 week duration in mice required lower intraperitoneal doses but outcomes may have been moderated by the mice background. Overall, the SmsG56S phenotype in the testes and bone are consistent with other mutant Sms models, although C57BL/6J mice seemed to be more sensitive to the SmsG56S mutation. Further testing of Prodrug is needed, including in younger mice and for a longer duration of treatment, to evaluate Prodrug effectiveness in improving traits in SmsG56S mice.

Asthma is a chronic respiratory disease with a high prevalence in Western countries, including Canada, and increased exacerbations have been associated with ambient air pollution. The maintenance of airways tone is critically dependent on the endogenous bronchodilator, nitric oxide (NO). The nitric oxide synthase (NOS) isoenzymes produce NO from the amino acid, L-arginine, and competition for substrate with the arginase isoenzymes can limit NO production. Imbalances between these pathways have been implicated in the airways hyperresponsiveness (AHR) of asthma. The overall objective of this work was to determine whether arginase and downstream polyamine metabolites are functionally involved in airways responsiveness in animal models of asthma and the adverse responses of allergic animals to air pollution. To this purpose, the expression profiles of proteins involved in L-arginine metabolism were determined in lung tissues from human asthmatics and murine models of ovalbumin (OVA)-induced airways inflammation. Expression of arginase 1 was increased in human asthma and animal models. Competitive inhibition of arginase attenuated AHR in vivo. The roles of the downstream metabolites of arginase, the polyamines (putrescine, spermidine and spermine) were examined by administering them via inhalation to anaesthetized mice. It was demonstrated that spermine increases methacholine responsiveness in normal and allergic mice. Additionally, inhibition of polyamine synthesis improved AHR in a murine model. Thus, arginase and downstream polyamine metabolites contribute to AHR in asthma. Finally, the potential role of arginase in the exacerbation of asthma by air pollution was investigated. For this purpose, murine sub-acute and chronic murine models of allergic airways inflammation were employed, which exhibit inflammatory cell influx and remodeling/AHR, respectively, to determine the role of arginase in the response to concentrated ambient fine particles plus ozone. Allergic mice that were exposed to air pollution exhibited increased arginase activity and expression, compared to filtered air-exposed controls. Furthermore, inhibition of arginase attenuated the air pollution-induced AHR. Thus, the studies of the arginase pathway and downstream metabolites described in this thesis indicate that arginase inhibition may be a therapeutic target in asthma and may also protect susceptible populations against the adverse health effects of air pollution.