Currently, opioids are extensively used in clinical practices in order to treat pain in patients. However, prolonged administration of opioids are not feasible due to the development of side effects especially tolerance, constipation, addiction and dependence. Our drug design is mainly aimed to reduce opioid induce side effects such as development of tolerance. The first strategy examined involves design and synthesis of peptide based single molecules that have a mu agonist and delta agonist pharmacophore in combination with a COX2 inhibitory pharmacophore. A new molecule, 3-17 having good delta agonist activity, partial COX2 inhibitory activity and weak mu agonist activity was produced. Moreover, Investigation of the bioactivities of the synthesized ligands including 3-17 in terms of their ligand receptor interactions were probed using NMR conformational analysis along with docking analysis to the respective homology modeled mu and delta opioid receptors as well as the COX2 enzyme. As a further continuation of this work, instead of peptide based mu agonist and delta agonist type pharmacophore, the highly mu selective fentanyl pharmacophore was used in combination with a pyrazole based and a pyrazolone based COX pharmacophore. Based on the SAR study and docking analysis of synthesized ligands to the homology modeled mu opioid receptor, an ideal tolerant position without significant loss of mu opioid agonist activity for fentanyl were found. The second strategy involves design and synthesis of a peptide based single molecule that has a mu agonist and a delta antagonist pharmacophore in combination with a NK1 antagonist pharmacophore. A novel molecule (4-2) containing delta antagonist activity, weak mu agonist activity and NK1 antagonist activity was identified. Its homology modeled mu opioid receptor bound conformation was compared with that of reference ligands. Docking analysis of modified 4-2 to the homology modeled mu opioid receptor revealed that it can be further modified to obtain better mu agonist activity. 4-2 showed antinociception for 45 min period of time after injection in tail flick assay. In addition to studies that were directed to avoid tolerance development due to opioid administration, peptide based potential analgesics such as biphalin was modified by introducing more peptidomimetic character in order to enhance its blood brain barrier permeability and proteolytic stability. The novel molecule (6-7) was produced in this study and its antinociception lasted for 30 min period of time after injection in the tail flick assay.

Entomopathogenic fungi, including Beauveria bassiana are being developed as an alternative to chemical insecticides. Their effectiveness can be enhanced through understanding of the mechanisms of response to environmental stresses and conditions. An aspect of repair of ultraviolet radiation induced DNA damage and response to high temperature were studied here. A region of the photolyase gene (phr-1), encoding cyclobutane pyrimidine dimer photolyase (CPD-PHR), an enzyme pivotal to DNA repair, was cloned, sequenced and identified for species of the genera Beauveria, Isaria, Lecanicillium, Metarhizium and Tolypocladium. The DNA and deduced amino acid sequences were analysed using several in silico methods and annotated for functionality. The data suggested that the DNA encoded a protein with conserved residues known in CPD-PHR function, which had structural homology with other CPD-PHRs and molecular phylogeny that was generally consistent amongst this group of fungi. These results are the first for a phr-1 from the genera Isaria, Lecanicillium and Tolypocladium. In bacteria and yeasts, tolerance to environmental stress was shown to be aided through an inducible phenomenon that involves extracellular sensory component (ESC) proteins in Escherichia coli, which have yet to be purified or sequenced. The presence of an ESC-like factor (ELF) was examined in cell-free filtrate (CFF) from B. bassiana cultures. It was revealed that the tolerance of conidiospores and blastospores (BS) to ultraviolet radiation or heat could be increased by preheated CFF, respectively, but not after pretreating the CFF with trypsin. Several novel polyacrylamide-based in situ and binding bioassays were developed to screen for and characterize ELF candidate (EC) proteins. Two were detected (EC1 and EC2) and EC1 was found to interact with BS, while bioassays with purified ECs showed that EC1 could increase heat tolerance in BS. De novo peptide sequencing revealed that the ECs were the same protein, but differed by EC1 being glycosylated. An expressed sequence tag from B. bassiana, encoding six peptides that were also found in the ECs, was identified in the public data base. This sequence information was exploited to amplify the remaining coding regions of the suspected ELF gene (elf) using polymerase chain reactions. Through this a 741 nucleotide open reading frame was cloned and sequenced. Structure-function analyses of the amino acid sequence encoded by the open reading frame revealed features that were consistent with the ECs, such as eight shared peptides, its nascent derived size (26 kDa), potential glycosylation sites and secretion signal peptide. In addition, other features such as the high proportion of cysteine residues and internal amino acid repeats will be discussed. The elf gene was inserted into an expression vector and introduced into the methylotrophic yeast Pichia pastoris for its controlled over-expression. Heterologously expressed ELF conferred elevated tolerance to heat in BS to similar levels produced by ELF synthesized by B. bassiana. Several functional and molecular features of the ELF system have certain commonalities with many agonist-receptor systems involved in signal transduction, but remain to be detailed. This is the first report of the cloning and functional analyses of elf and ELF, respectively, from any organism.

Les modifications de structure et de diversité des communautés biologiques au sein d’un écosystème soumis à une perturbation, se traduisent généralement par la raréfaction, la disparition d’espèces sensibles et/ou l’apparition de nouvelles espèces tolérantes ou par la prolifération d’autres espèces tolérantes déjà présentes mais à une faible densité. Dans le cas d’une perturbation d’origine toxique, ceci a pour conséquence une diminution de la sensibilité globale de la communauté par rapport à la (aux) substance(s) responsable(s) de cette modification de structure et de diversité. L’évaluation de la tolérance vis-à-vis d’un toxique peut donc nous permettre de révéler a posteriori l’exposition d’une communauté biologique à ce toxique, en mettant en évidence le lien entre pression et impact sur le compartiment biotique d’un écosystème. Malgré de nombreux travaux en ce domaine, il reste cependant de nombreuses lacunes scientifiques dans la compréhension de cette tolérance induite par les pollutions (PICT). Le modèle d’étude retenu est le biofilm aquatique (ou périphyton), dont les spécificités biologiques et écologiques en font un outil d’étude très intéressant. Ce travail a permis de montrer que l’intégration du concept PICT comme un outil complémentaire dans les systèmes d'évaluation environnementale donnerait plus de pertinence écologique et de spécificité écotoxicologique à la batterie actuelle des bioindicateurs utilisés. Par ailleurs, le PICT est aussi une approche conceptuelle, à l’échelle des communautés, très riche et qui confirme l’intérêt d’aborder l’écotoxicologie avec le regard de l’écologue plus holistique que celui du toxicologue. En effet, les mesures de tolérance-induite qui prennent en compte la diversité fonctionnelle du biofilm, ainsi que les analyses taxonomiques associées, nous ont permis une meilleure compréhension de la résistance et de la résilience de cet écosystème suite à des perturbations d’origine chimique. Nos travaux nous ont aussi permis d’aborder le concept des seuils de résistance et de résilience écologiques, et de mettre en évidence le fait qu’une acquisition de tolérance à un stress donné, pourrait se traduire par le déplacement des communautés d’un état initial vers un état « alternatif » stable, même après le retrait du stress. Ces seuils écologiques ainsi que cet état alternatif stable signifient que la disparition des espèces les plus sensibles (comme l'un des processus expliquant le PICT) n'affecte donc pas les fonctions de la communauté dans son ensemble au début et ce seulement jusqu’à un certain seuil de résistance. Le PICT pourrait ainsi se traduire par une réduction de la diversité ou avec des modifications dans la composition spécifique, sans pour autant qu’il y ait un effet négatif sur le fonctionnement de la communauté. Cependant, la capacité des communautés à devenir tolérantes à une perturbation peut avoir des conséquences négatives sur les capacités de résilience et de résistance des écosystèmes. Nous avons donc abordé dans nos travaux le concept de « co-tolérance négative entre espèces » et de coût de la tolérance. / Changes in structure and diversity of biological communities within ecosystems subjected to disturbances are generally synonymous of the scarcity, loss and/or the emergence of new tolerant species or by the proliferation of other species already present but at low density. This leads to a decrease in the overall sensitivity of the community toward the substance(s) responsible for this change of community structure and diversity. The evaluation (and if possible the quantification) of tolerance towards a toxicant may therefore enable us to reveal a posteriori the exposure of a biological community to this toxicant (its exposure history), and demonstrate the specific link between pressure and impact on the biotic compartment of an ecosystem and, more generally, on the ecosystem. Despite numerous studies in this area, there are still many gaps in scientific understanding of the pollution induced-tolerance. The biological model that we used is the lotic biofilm (or periphyton) whose biological and ecological characteristics make it a very interesting tool for study. This work has shown firstly that the integration of the acquired tolerance concept as a complementary tool in the environmental assessment systems would allow more ecological relevance and ecotoxicological specificity to the current set of used bio-indicators. Furthermore, the PICT is also a conceptual approach at the community level, which confirms the interest to address ecotoxicology from the viewpoint of the ecologist that is more holistic than the toxicologist one. Indeed, measures of pollution-induced tolerance, by taking into account the functional diversity of biofilms, and the associated taxonomic analysis, allowed to have a better understanding of resistance and resilience of the ecosystem submitted to chemical perturbations. Our works enabled us to tackle the concept of ecological thresholds of resistance and resilience, and to highlight the fact that enhanced tolerance to a given stress, could result in the displacement of communities from an initial state to an « alternative » stable state, even after the stress removal. These ecological thresholds and the alternative stable state mean that the disappearance of the most sensitive species (as a process explaining the PICT) does not affect the functions of the community until reaching a threshold of resistance. Thus the PICT could correspond to a reduction in diversity or changes in species composition, without having a negative effect on the functioning of the community. However, the ability of communities to be tolerant toward disturbance can have negative consequences on the resilience and resistance of ecosystems. Consequently, we addressed in our work the concept of "negative co-tolerance between species" and costs of tolerance.