Homologous genetic recombination, because of its fundamental roles in the maintenance of genome stability and evolution, is an essential cellular function common to all organisms. This process also plays important roles in the repair of damaged DNA molecules, generation of genetic diversity and proper segregation of chromosomes. The genetic exchange is a highly orchestrated process that entails a plethora of control mechanisms and a large number of proteins, of which RecA and SSB are two proteins that have been chosen for further investigation(s) in the present study. In addition, we have also investigated the interaction between SSB and UvrD1, which plays an important role in DNA repair pathways, especially nucleotide excision repair (NER) and mismatch repair as well as DNA replication and recombination. Chapter 1 reviews the literature regarding various aspects of homologous recombination, with an emphasis on the biochemical and the biophysical aspects of RecA and SSB proteins. In addition, it provides an overview of the study of DNA repair and recombination in mycobacteria. RecA protein is ubiquitous and well conserved among bacterial species. Many archaeal species possess two RecA homologues (RadA and RadB) and eukarya possess multiple homologues of RecA including, Rad51, Rad51B, Rad51C, Rad51D, DMC1, XRCC2, or XRCC3. RecA or its homologues function as polymers, consisting of hundreds of monomers that cooperatively polymerize on single-stranded DNA to form a nucleoprotein filament. E. coli RecA protein participates in Trans Lesion Synthesis (TLS) of DNA and forms the minimal mutasome in association with DNA polymerase V (UmuD’2C). The fundamental mechanism underlying HR, i.e. DNA strand exchange, is one of the most fascinating examples of molecular recognition and exchange between biological macromolecules. Since the isolation of E. coli recA gene and the subsequent purification of its gene product and also from other organisms, RecA protein has been studied extensively for more than three decades. E. coli RecA protein has pivotal roles in DNA recombination and repair, and binding to DNA in the presence of ATP, is a fundamental property of RecA protein resulting in the formation of a nucleoprotein filament. This is the slow step of the HR process, and is considerably faster on ssDNA than on duplex DNA. Binding of RecA to dsDNA is slower at physiological pH, is accelerated at acidic pH, and the lag in binding at the higher pH values is due to slow nucleation. The ATP and the DNA binding functions of RecA display allosteric interaction such that ATP- binding leads to an increase in affinity to ssDNA-binding and vice-versa. X-ray structures of E. coli RecA complexed with nucleotide cofactors have implicated a highly conserved Gln196 in Mycobacterium smegmatis RecA in the coupling of ATP and the DNA binding domains. The carboxyamide group of Gln196 makes an H-bond with the γ-phosphate group of ATP and the side chain of this residue is observed to move by approximately 2Å towards the ATP, relative to the other residues involved in ATP binding. In addition, a highly conserved Arg198 has also been postulated to interact with the γ-phosphate group of bound ATP and position it for a nucleophilic attack by a conserved residue-Glu96 leading to ATP hydrolyses. To elucidate the role of Gln196 and Arg198 in the allosteric modulation of RecA functions, we generated MsRecA variant proteins, where in Gln196 was substituted with alanine, asparagine or glutamate; Arg198 was mutated to a lysine. The biochemical characterization of MsRecA and its variant proteins with the objective of defining the allosteric interaction between the ATP- and the DNA-binding sites has been described with in Chapter 2. We observed that while the mutant MsRecA proteins were proficient in ATP-binding they were deficient in ATP hydrolyses. We assayed for the ability of these proteins to bind ssDNA using either nitrocellulose filter binding or Surface Plasmon Resonance (SPR). While we did not detect any ssDNA-binding by the mutant MsRecA proteins in the filter binding assay, we observed only ten-fold reduction in the affinity for ssDNA as compared to wild type MsRecA protein in MsRecAQ196A, Q196N and R198K in the SPR assay. MsRecA Q196E did not show any binding to ssDNA, in both nitrocellulose filter-binding as well as SPR assays. We assayed for the ability of the mutant RecA proteins for their ability to promote DNA-pairing as well as DNA strand exchange. While we observed limited pairing promoted by the mutant proteins relative to the wild-type MsRecA, we observed a complete abrogation of strand exchange in the case of mutant proteins. In addition, we assayed for the co-protease function of MsRecA, by monitoring the cleavage of MtLexA. We observed that only the wild-type MsRecA protein was able to cleave MtLexA, while none of the mutant RecA proteins were able to do so. In order to understand the differences observed between the wild -type and the mutant MsRecA proteins, we analyzed the conformational state of MsRecA and its variant proteins by circular dichroism spectroscopy upon ATP-binding. We observed that while MsRecA and MsRecAQ196N displayed a reduction in the absorbance at 220 nm upon ATP binding, we did not observe any such structural transitions in the other mutant MsRecA proteins that we tested. Based on our observations and the crystal structure of E. coli RecA bound to ssDNA, in Chapter 2, we propose a dual role for the Gln196 and Arg198 in modulating RecA activities. In the presynaptic filament Gln196 and Arg198 sense the presence of the nucleotide in the nucleotide binding pocket and initiate a series of conformation changes that culminate in the transition to an active RecA nucleoprotein filament. In the active RecA nucleoprotein filament these residues are repositioned such that they now form a part of the protomer-protomer interface. As such they perform two vital functions; they stabilize the protomer-protomer interface by participating in the formation of hydrogen bonds that span the interface as well transmit the wave of ATP hydrolysis across the interface leading to a coordinated hydrolyses of ATP essential for the heteroduplex extension phase of strand exchange reaction. The members of the super family of single stranded DNA binding proteins (SSB) play an important role in all aspects of DNA metabolism including DNA replication, repair, transcription and recombination. Prokaryotic SSBs bind ssDNA with high affinity and generally with positive cooperativity. Several lines of evidence suggest that prokaryotic SSBs are modularly organized into three distinct domains: the N-terminal DNA binding domain and acidic C-terminal domain are linked by a flexible spacer. Studies from our laboratory have revealed that M. smegmatis SSB plays a concerted role in recombination-like activities promoted by the cognate RecA. The C- terminal of SSB is known to be involved in its ability to interact with other proteins. We have previously reported that the C-terminal domain of M. smegmatis SSB, which is not essential for interaction with DNA, is the site for the binding of cognate RecA. The data in Chapter 3 describes the characterization of the SSB C-terminus with the objective of delineating the elements responsible for mediating protein-protein interaction, as well as to define the mechanism by which SSB is able to modulate the activities of RecA. To map the RecA interaction domain of SSB we created deletion mutants in MsSSB lacking 5, 10, 15 or 20 residues from the C-terminal. The truncated SSB proteins were expressed with a His- tag at the N- terminus and purified to homogeneity using a Ni-NTA affinity matrix. We observed unlike MsSSB, MsSSB∆C5 and MsSSB∆C10, MsSSB∆C15 and MsSSB∆C20 were unable to support three-strand exchange catalyzed by MsRecA. Based on the observation that interaction with SSB is essential for MsRecA to catalyze the strand Exchange reaction, we postulate that the RecA interacting domain of SSB is situated between the 15th and the 20th residue from the C-terminal. Further, the C-terminal of MsSSB modulates the transitions between DNA binding modes. Unlike the case with EcSSB where deletion of the last 8 residues from the C-terminal stabilizes the (SSB)35 mode of ssDNA binding, we observe that in case of MsSSB the deletion of C-terminal seems to destabilize the (SSB)35. In addition, the transition from the low density binding mode to a high density mode involves the formation of several intermediates when the C-terminal residues are deleted. With the objective of understanding the functions to the C-terminal of SSB independent of its DNA-binding domain in modulating RecA functions, we employed a peptide corresponding to the 35 residues from the C-terminal of the MsSSB. We observed that the C-terminal region alone is capable of interacting with RecA. In addition we also observed that the C-terminal domain of SSB stimulates RecA functions independent of its DNA binding domain. To address the question, whether the stimulatory effect of the C-terminal domain of SSB in the absence of its DNA-binding domain is restricted to RecA or is a generalized phenomenon associated with all SSB interacting proteins; we tested the effect of C-terminal domain of SSB on UvrD which is known to interact with SSB. UvrD participates in several pathways of DNA metabolism, which include the nucleotide excision repair (NER) and mismatch repair pathway, replication and recombination. Genetic evidence suggests that UvrD and SSB interact in vivo. We tested the effect of mycobacterial SSB on M. tuberculosis UvrD1 (MtUvrD1) functions in vitro. We observe that MtUvrd1 physically interacts with SSB. Further, presence of SSB has an inhibitory effect on the helicase activity of MtUvrD1 and that this effect is dependent on the C-terminal region as the deletion of residues from the C-terminal of SSB abrogates the inhibitory effect of SSB. However, unlike RecA, the C-terminal region of SSB alone had no effect on the helicase activity of UvrD1. We also observed that MsSSB has opposing effects on the ATPase activity of MtUvrD1. In the presence of low concentrations of SSB the ATPase activity is enhanced, while we observed an inhibition when the concentration of MsSSB is high. The precise mechanistic details of how SSB is able to act as an accessory protein to RecA, in context of homologous recombination and stimulates its biochemical activities have been a subject of debate. Whereas research from some groups has shown that the stimulatory effect SSB is mediated through its ability to melt DNA secondary structure, thereby allowing RecA to overcome the kinetic barrier imposed by the presence of secondary structure in ssDNA, others postulate that SSB plays a direct role in the stabilization of RecA nucleoprotein filament and prevents its dissociation. Chapter 3 discusses the experimental evidence in favor of the aforesaid models and based on the results of our experiments; we propose that the accessory functions of SSB may be mediated by a mechanism that involves elements of both models. While interaction with SSB can bring about a conformational change in RecA that is reflected in the enhanced levels of strand exchange and co-protease activity, the helix destabilizing function of SSB is essential during heteroduplex extension and to sequester the displaced strand such that it does not participate in any further pairing reactions. The novel finding that we present in Chapter 3 is that the interaction of SSB C-terminal alone has a stimulatory effect upon RecA activities. Furthermore, we observed that M. tuberculosis UvrD1 is a weak interaction partner of SSB. The physical and functional interactions between MsSSB with RecA on the one hand, and MsSSB and UvrD1 on the other highlight different types of cross-talk between the components of HR and DNA repair pathways. In contrast to the results of earlier studies, our results indicate that protein-protein interactions alone between SSB and RecA may modulate the RecA mediated processes of presynapsis, homologous pairing and strand exchange between homologous DNA molecules as well as modulate its co-protease activity. In addition, our studies indicate that a direct protein-protein interaction is responsible for the modulation of UvrD1 activities by SSB.

La stabilité génomique des organelles de plantes suscite un grand intérêt dans le domaine de la biologie végétale. En effet, plusieurs études récentes suggèrent que ce type d’instabilité génomique pourrait mener à l’isolation de traits intéressants en l’agronomie. Plusieurs protéines sont d’ailleurs déjà été identifiés comme étant impliqués dans le maintien de la stabilité de ces génomes, tels que MSH1, la famille des POLI, OSB1, les protéines Whirly et les Recombinases A (RECA). Le génome nucléaire d’Arabidopsis thaliana encode trois protéines s’apparentant à la Recombinase A bactérienne et qui sont ciblées à la mitochondrie et/ou au chloroplaste, soit RECA1, RECA2 et RECA3. Globalement, ces gènes partagent une similarité de séquence de 61% avec leur homologue bactérien chez Escherichia coli. Chez les bactéries ces protéines jouent un rôle essentiel dans la recombinaison homologue et sont impliquées dans la réparation de l’ADN. Chez Arabidopsis, il a été démontré que RECA2 et RECA3 sont nécessaires au maintien de l’intégrité du génome mitochondriale. Toutefois leur contribution à la stabilité du génome chloroplastique ainsi que le rôle de RECA1 restent obscures. Le but de ce projet est donc de déterminer la contribution éventuelle des protéines RECA d’Arabidopsis dans la réparation de l’ADN chloroplastique et plus précisément le rôle du gène RECA1. Nous énonçons l’hypothèse que les RECA de plantes se comportent effectivement comme leurs orthologues bactériens en étant impliqués dans la recombinaison homologue. Dans le cadre de ce projet, nous avons tenté d’isoler des lignées mutantes pour chacun des gènes RECA d’Arabidopsis. En somme, nous avons pu obtenir des lignées convenables pour notre étude que dans le cas du gène RECA1. Ces lignées ont été utilisées pour évaluer la contribution de ce gène à la stabilité du génome du chloroplaste. Ensuite, pour étudier la relation épistatique des gènes RECA1, WHY1 et WHY3, un croisement des différentes lignées mutantes pour ces gènes a été réalisé. Nous avons ensuite étudié la sensibilité de toutes ces lignées mutantes à la ciprofloxacine, un agent causant des bris double brin exclusivement dans les organelles de plantes. Finalement, iii nous avons testé la présence de réarrangements dans le génome du chloroplaste en condition normal ou en présence de stress génotoxique. Nos résultats démontrent que les protéines Whirly et RECA1 sont impliquées dans deux voies de réparation de l’ADN différentes et que les Whirly sont suffisantes pour s’occuper des bris d’ADN double brin en l’absence de RECA1. Nous démontrons également que l’absence de Whirly et RECA1 entraine une forte augmentation de la quantité de réarrangements dans le génome du chloroplaste. De plus nous proposons que la polymérase POLIB est impliquée dans la même voie de réparation que RECA1. Finalement nous proposons un modèle pour expliquer nos résultats et impliquons RECA1 dans un mécanisme de réparation d’ADN et aussi un rôle potentiel dans la réplication. / The stability of plant organelles genomes elicits a great interest in the domain of plant biology. In fact, numerous studies suggest that genomic instability can lead to the isolation of interesting traits in the field of agronomy. Some factors such as MSH1, the POLI family, OSB1, the Whirly proteins and the Recombinase A (RECA), have already been identified has being implicated in the maintenance of genome stability. The nuclear genome of Arabidopsis thaliana encodes three proteins, RECA1, RECA2 and RECA3, that shares a high resemblance with bacterial Recombinase A. They are targeted to the mitochondria and/or to the chloroplast. Globally, these genes share a similarity of sequence of 61% with their bacterial homologue in Escherichia coli. In bacteria these proteins play an essential part in homologous recombination and are implicated in DNA repair. In Arabidopsis, RECA2 and RECA3 have been shown as being essential to maintain the integrity of the mitochondrial genome but their contribution to the stability of the chloroplast as well as the role of RECA1 remains obscure. The goal of this project is to establish the eventual contribution of the Arabidopsis RECA proteins in the repair of chloroplast DNA and more precisely the role of the RECA1 gene. We propose the hypothesis that plants RECA act in the same fashion as their bacterial orthologues by being implicated in homologous recombination. Within the framework of this project, we have attempted to isolate mutant lines for each RECA gene of Arabidopsis. In the end, we were able to obtain appropriate lines for our study only for the RECA1 gene. These lines were then used to evaluate the contribution of the gene to chloroplast genome stability. Afterwards, in order to study the epistatic relationship between the RECA1, WHY1 and WHY3 genes, a cross between different mutant lines of these genes was realised. We then studied the sensitivity of all of those mutant lines to ciprofloxacine, an agent causing double stranded breaks exclusively in plant organelles. Finally, we evaluated the presence of rearrangements in the chloroplast genome under normal conditions and under the presence of a genotoxic stress. Our v results show that the Whirly and RECA1 proteins are implicated in two separate pathways of DNA reparation and that the Whirly proteins are sufficient to take in charge DNA double strand breaks generated by the absence of RECA1. We also demonstrate that the absence of Whirly and RECA1 causes an increasein the quantity of rearrangements in the chloroplast genome. Furthermore, we propose that the polymerase POLIB is implicated in the same repair pathway as RECA1. Finally we propose a model to explain our results and implicate RECA1 in a DNA repair mechanism and propose a role for RECA1 in DNA replication.

No description available.

Homologous recombination (HR) is conserved across all three domains of life and is associated with a number of key biological processes. Over the years, numerous genetic, biochemical and structural studies have uncovered important mechanistic details and established a role for HR in DNA damage repair, control of DNA replication fidelity and suppression of various types of cancer. Much of our current understanding of the mechanistic aspects of HR is gained from the study of Escherichia coli paradigm. E. coli RecA is the founding member of a nearly ubiquitous family of multifunctional proteins and is substantially conserved among eubacterial species. During HR, RecA protein promotes homologous pairing followed by strand exchange reaction leading to heteroduplex formation. In addition to HR, RecA is a central component of SOS response, recombinational DNA repair and rescue of collapsed replications forks. Moreover, recent work has suggested that DNA recombination/repair mechanisms might contribute to genome evolution and consequently to the generation of multidrug-resistant strains of the pathogen. The disease caused by Mycobacterium tuberculosis, endemic in certain regions of the world, is a leading cause of disability and death. A thorough knowledge of the function and interaction of specific HR proteins/enzymes involved in the maintenance of genome integrity is essential in order to elucidate the impact of genome perturbation effects on M. tuberculosis. Toward this end, modulation of RecA protein activity, a central component of HR, represents a potential novel target for design of new drugs because of its involvement in various processes of DNA metabolism. Additionally, small molecule modulators of RecA activity may offer novel insights into the regulation and its role in cellular physiology and pathology. Traditionally, antibiotics have been used to treat infections caused by bacteria. Despite their importance, the development of new antibiotics against M. tuberculosis has considerably decreased over the past several years due to disappointing results in clinical trials. These failures may be due the fact that they suffer from low potency or low cell permeability. Therefore, one of the aims of studies described in this thesis was to test the effect of suramin, a known inhibitor of E. coli RecA, on various biochemical activities of mycobacterial RecA proteins and determine its mechanism of action. Furthermore, the most crucial step in the HR pathway and rescue of collapsed DNA replication forks is the resolution of Holliday junctions and other branched intermediates. Because Holliday junction resolvases are essential for the resolution of different types of DNA recombination/repair intermediates, therefore, we considered it worthwhile to study the genomic expression and biochemical properties of HJRs in M. tuberculosis. Suramin is a commonly used antitrypanosomal and antifiliarial drug, and a novel experimental agent for the treatment of several cancers. A forward chemical screen assay identified several small molecule inhibitors of E. coli RecA. In this screen, suramin (also called germanin), a polysulfonated naphthylurea, and suramin-like agents were found to inhibit EcRecA catalyzed ATPase and DNA strand exchange activity. However, the mechanism underlying such inhibitory action of suramin and whether it can exert antibacterial activity under in vivo conditions remains largely unknown. In an attempt to delineate the range of suramin action, we reasoned that it might be useful to test its effect on mycobacterium RecA proteins. We found that suramin is a potent inhibitor of all known biochemical activities of mycobacterial RecA proteins with IC50 values in the low μM range. The mechanism of action involves, in part, its ability to disassemble the nucleoprotein filaments of RecA-ssDNA. To validate the above results and to obtain quantitative data, a pull-down assay was developed to assess the effect of suramin on RecA–ssDNA filaments. The data indicated that suramin was able to dissociate >80% of RecA bound to ssDNA. Altogether, these results indicated the effectiveness of suramin in the disassembly of RecA nucleoprotein filament. Next, we sought to test whether suramin binds to RecA by using a CD spectropolarimeter. Significant spectral changes were observed upon addition of increasing concentrations of suramin, indicating alterations in the secondary structure of RecA protein. Additional evidence revealed that suramin impaired RecA catalyzed proteolytic cleavage of LexA repressor and blocked ciprofloxacin-inducible recA gene expression and SOS response. More importantly, suramin potentiated the cidal action of ciprofloxacin and reduced the growth of Mycobacterium smegmatis recA+ strain but not its isogenic recA∆ mutant, consistent with the idea that it acts directly on RecA protein. This approach, which appears as an appealing concept, opens up new possibilities to chemically disrupt the pathways controlled by RecA and treat drug-sensitive as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies. The annotated genome sequence of M. tuberculosis revealed the presence of putative homologues of E. coli DNA recombination/repair genes. However, it is unknown whether these putative genes have the ability to encode catalytically active proteins or participate in biochemical reactions intrinsic to the process of HR or DNA repair. Studies in the second half of the thesis originated from an in silico analysis for genes that encode functional equivalents of E. coli RuvC HJ resolvase(s) in M. tuberculosis. The central intermediate formed during mitotic and meiotic recombination is a four-way DNA junction, also known as the Holliday junction (HJ), and its efficient resolution is essential for proper segregation of chromosomes. The resolution of HJ is mediated by a group of structure specific endonucleases known as the Holliday junction resolvases (HJR) which have been identified in a wide variety of organisms based on their shared biochemical characteristics. Bioinformatics analyses of the evolutionary relationships among HJ resolvases suggests that HJR function has arisen independently from four distinct structural folds, namely RNase H, endonuclease VII-colicin E, endonuclease and RusA. Furthermore, similar analyses of HJRs identified another family within the RNaseH fold, along with previously characterized RuvC family of junction resolvases. This new family of putative HJRs is typified by E. coli Yqgf protein. The yqgf gene is highly conserved among bacterial genomes. Nuclear magnetic resonance structural studies have disclosed notable structural similarities between E. coli RuvC and YqgF proteins. Utilizing homology-based molecular modelling, YqgF is predicted to function as a nuclease in various aspects of nucleic acid metabolism. Sequence analysis of M. tuberculosis genome has revealed the presence of two putative HJ resolvases, ruvC (Rv2594c) and ruvX (Rv2554c, yqgF homolog). Previous studies have demonstrated that M. tuberculosis ruvC is induced following DNA damage and ruvX is expressed during active growth phase of M. tuberculosis. More importantly, the absence of ruvC increased the potency of moxifloxacin in M. smegmatis. Although, these results imply that the ruv genes play crucial roles in DNA recombination and repair in M. tuberculosis, the biochemical properties of their gene products have not been characterized. In this study, we have isolated M. tuberculosis ruvC and yqgF genes and purified their encoded proteins, M. tuberculosis RuvC (MtRuvC) and M. tuberculosis RuvX (MtRuvX), respectively, to near homogeneity. Protein-DNA interaction assays conducted with purified MtRuvC and MtRuvX revealed that both can bind HJ, albeit with different affinities. However, in contrast to MtRuvC, MtRuvX showed robust HJ resolvase activity. The endonuclease activity of MtRuvX was completely dependent on Mg2+and Mn2+ partially substituted for Mg2+. Additional experiments showed that RuvX exhibits >2-fold higher binding affinity for HJ over other recombination/ replication intermediates. As demonstrated for other HJRs, MtRuvX failed to cleave static HJ and linear duplex DNA. The cleavage sites were mapped within the homologous core of a branch-migratable HJ. To identify catalytic residues in RuvX, we conducted mutational analysis of an acidic amino acid residue guided by the bioinformatics data. The product of MtRuvXD28N retained full HJ-binding activity, but showed extremely reduced HJ-specific endonuclease activity. Further biochemical characterization revealed that MtRuvX exists as a homodimer in solution. Notably, we found that disulfide-bond mediated intermolecular homodimerization is crucial for the ability of MtRuvX to cleave Holliday junctions, implicating that stable junction binding is necessary to promote branch migration and to create cleavable sites. Analysis of qPCR data suggested that the pattern of yqgF gene expression was similar to those of ruvC and recA genes following DNA damage. Together, these data indicate that ruvX expression is induced by DNA-damaging agents and that RuvX might be functionally involved in recombinational DNA repair in M. tuberculosis. These findings are all consistent with the idea that RuvX might be the bona fide HJ resolvase in M. tuberculosis analogous to that of E. coli RuvC. More importantly, we provide the first detailed characterization of RuvX and present important insights into the mechanism of HJ resolution, which could be directly linked to the regulation of different DNA metabolic processes, including HR, DNA replication and DNA repair. Overall, this study opens a new avenue in the understanding of HR in this human pathogen, together with elucidation of the function of some of the uncharacterized genes may represent a novel set of recombination enzymes.

O avanço das ações de desenvolvimento vem colocando desafios à gestão de recursos; as diferentes particularidades nos tipos de recursos disponíveis para uso pelas organizações (na área de administração de empresas), seja por localização geográfica, clima, relevo e/ou outras especificidades locais, como por exemplo, recursos naturais, fogem aos padrões impostos pelas abordagens teóricas estratégicas dominantes, atualmente, nas ciências sociais. No caso do megabioma Amazônico, devido à grandeza patrimonial e suas especificidades locais, há recursos naturais que são únicos e inimitáveis, seja de maneira individual seja pelo conjunto do megabioma, apesar do contexto de pobreza da região. Na medida em que se pressiona para o uso desses recursos, seja pelos indivíduos, seja pelas organizações, podem-se gerar diferentes tipos de conflitos, até porque o uso dos recursos naturais e os conflitos socioambientais são cenários de repercussões para a sociedade, devido às mudanças que promovem em termos ecológicos e das propostas de desenvolvimento social. Assim existem recursos naturais, principalmente, os renováveis disponíveis para uso, que exige novos conhecimentos teóricos e práticos, tanto para acadêmicos como para os gestores atuais. Nesse contexto, são necessárias as preocupações dos cidadãos, instituições e governos, necessitando, no entanto, de um conhecimento adequado à natureza, suas potencialidades e dimensões, no tocante ao uso no presente e garantia para as gerações futuras dos recursos necessários e sua sobrevivência com qualidade de vida, ou seja, a sustentabilidade nesse sentido vem ao encontro de um manejo ambientalmente correto (sustentável) socialmente justo e economicamente rentável, considerando como pilares do Desenvolvimento Sustentável as dimensões Sociais, ambiental e Econômica, dimensões que nortearam a presente pesquisa. Nesse quesito de conhecer como esse processo poderia ocorrer, na Amazônia, foi tirado proveito da teoria Path Dependence que possibilita organizar de forma metodológica e teórica a trajetória e os conhecimentos necessários para que se obtenha uma produção sustentável, com satisfação de mercado aliados a oportunidade de negócio para as comunidades locais. Sendo assim, nesse contexto que envolve recursos, preocupações com a sustentabilidade as inovações que orientaram a presente pesquisa são coladas com a realidade local, com intuito de atender tanto a sustentabilidade quanto resolver impasses que tem impedido progressos substanciais no cruzamento da pobreza, sustentabilidade e meio ambiente. As preocupações, relacionadas à redução da pobreza é um fato que tem sido motivo de preocupação, inclusive no megabioma amazônico, apesar desse ser considerado rico e potencialmente rentável; tem transcendido os limites individuais, empresariais e nacionais, passando a ser uma preocupação global. Nos anos 2000, se consolidou a ideia de que as empresas têm um papel importante na redução da pobreza e a partir disso surge a teoria Base da Pirâmide (BoP) visando possibilitar o consumo para os pobres (BoP 1.0); no entanto por mais que essa teoria tenha avançado possibilitando maior participação dos pobres os envolvendo como co-criadores (BoP 2.0) esta ainda continua os considerando como sujeito passivo ou meros consumidores/colaboradores, visto que a renda por agregação de valor aos produtos (co-criadores) fica com a empresa, ou seja, a teoria não tem sido aproveitada para a produção a partir dos recursos naturais, com vistas a geração de renda, a melhoria na qualidade de vida e organização sustentável, principalmente numa região como a Amazônica. Assim, considerando as oportunidades existentes no megabioma amazônico, tem-se o entendimento que as oportunidades da BoP, podem ir além do consumo (BoP 1.0) ou co-criação de valor (BoP 2.0) e, com foco nos produtores da BoP (produtores de PFNMs), de forma organizada, podem ser desenvolvidas alternativas de melhoria de qualidade de vida, baseado em recursos naturais renováveis sustentáveis, emergindo a questão principal da presente pesquisa: A exploração dos recursos naturais amazônicos, de forma sustentável, pelos produtores BoP, caracteriza-se como uma nova abordagem da BoP, indo além do que é preconizado pela BoP 1.0 e BoP 2.0? Para se chegar a isso, a presente pesquisa está sendo norteada pelos temas: Sustentabilidade e Desenvolvimento sustentável, Recursos Naturais; Visão Baseada em Recursos Internos (VBRI); Inovação e Inovação orientada para a sustentabilidade; Base da Pirâmide (BoP), e PFNMs (produtos florestais não madeiráveis), nesse caso são os produtos naturais renováveis amazônicos. O objetivo é o de propor e aplicar um framework de sustentabilidade da BoP, para organizações que atuem com recursos naturais renováveis, para os produtores (PFNMs) da BoP, do megabioma amazônico. A pesquisa é exploratória e descritiva, de natureza qualitativa. Foram coletados dados secundários e primários. Os dados primários utilizou como método de coleta de dados a entrevista semiaberta, visita e observação in loco. Para tratamento dos dados, foi utilizado a técnica de análise de conteúdo. O objeto de estudo foi a organização RECA – Reflorestamento Econômico Consorciado Adensado, que é uma Associação de pequenos agrossilvicultores, localizada em Nova Califórnia (RO). O projeto RECA possui mais de 2.700 hectares de Sistema Agroflorestal - SAFs implantados, utilizando vários tipos e esquemas de plantios com menos de 5% de áreas de monoculturas. Os resultados obtidos foram ricos e os mais relevantes foram: para que os produtores da BoP, de recursos naturais renováveis, de forma sustentável, possam ser sujeitos e tenham a governança das suas atividades necessita-se de uma nova visão da BoP (BoP 3.0); um novo tipo de negócio social do tipo associação-cooperativa foi criada; o uso dos tipos de recursos mobilizáveis e mobilizadores permitem uma análise multinível sustentável dos recursos naturais renováveis; numa BoP para produtores da BoP a ordem de importância das dimensões de sustentabilideade é social, ambiental e econômica, respectivamente; foi identificado um sistema de produção resultado de uma articulação do uso de recursos coletivos (da organização) e individuais (de cada produtor); necessita-se de um olhar para a inovação orientada para a sustentabilidade que seja interativa e não-linear; as relações deixam de ser dos produtores da ToP (topo da pirâmide) para a BoP e passam a ser dos produtores da BoP, tanto para os consumidores da ToP como os próprios consumidores da BoP. / The progress of the development actions have been placing challenges on the resource management, the various details of the types of resources available for use by organizations (in the area of business administration), or by geographical location, climate, topography and / or other local specificities, such as natural resources, they are escaping the standards imposed by the dominant strategic theoretical approaches currently in social sciences. In the case of megabioma Amazon, because of the patrimonial greatness and their specific locations, there are natural resources that are unique and inimitable, either individually or by the whole megabioma, despite the context of poverty in the region. To the extent that you are forced to use these resources, either by individuals, or by organizations, one can generate different types of conflicts, because the use of natural resources and environmental conflicts are repercussion scenarios for society due to promoting changes in the ecological and social development proposals. In this context, it takes the concerns of citizens, institutions and governments, requiring, however, an adequate knowledge of nature, its potential and dimensions, regarding the use of this guarantee and for future generations of the resources needed and their survival with quality of life, i.e., sustainability in this direction is in line with an environmentally sound management (sustainable) socially, just and economically viable, considering them as pillars of Sustainable Social dimensions, Environmental and Economic dimensions that guided this research. In this aspect of knowing how this process could occur in the Amazon, the Path Dependence theory was taken advantage of, enabling to organize in a methodological and theoretical trajectory way and the knowledge needed to obtain sustainable production, satisfaction with market allies and the business opportunities for local communities. So in this context, that involves resources, concerns about the sustainability innovations that guided this research are bonded with the local reality, in order to meet both sustainability as much as resolve impasses that have prevented substantial progress at the intersection of poverty, sustainability and environment. The concerns, related to the reduction of poverty is a fact that has been of concern, including Amazon megabioma, despite this being considered rich and potentially profitable, it has transcended the individual limits, corporate and national, becoming a global concern. In the 2000s, it has consolidated the idea that businesses have an important role in poverty reduction and from this arises the theory Base of the Pyramid (BoP), in order to enable the consumption for the poor (BoP 1.0), however as much as this theory has advanced, enabling greater participation, by involving the poor as co-creators (BoP 2.0), this is still considering the taxpayer or as mere consumers / employees, since the income for adding value to the products (co-creators) is with the company, ie, the theory has not been used for the production of the natural resources, with a view to generating income, improving the quality of life and sustainable organization, especially in a region like the Amazon. Thus, considering the opportunities in megabioma Amazon, it has been the understanding that the BoP opportunities, can go beyond consumption (BoP 1.0) or co-creation of value (BoP 2.0), and focusing on the BoP producers (producers NTFP), in an organized manner, alternatives can be developed to improve quality of life, based on sustainable renewable natural resources, emerging from the main question of this research: Is the Amazonian exploitation of natural resources in a sustainable manner, by BoP producers, characterizes as a new approach to the BoP, going beyond what is recommended by the BoP 1.0 and BoP 2.0? To achieve this, this research is guided by themes: Sustainability and Sustainable Development, Natural Resources, Internal Resource Based View (VBRI); Innovation and Innovation geared towards sustainability, Base of the Pyramid (BoP) and NTFPs (non-timber forest products), in which case the products are natural renewable Amazonia resources. The goal is to propose and apply a framework of sustainability of BoP for organizations that operate with natural renewable resources for producers (NTFPs) of BoP, the megabioma Amazon. The research is exploratory and descriptive; of qualitative nature. We collected primary and secondary data. The primary data used as a method of data collection to semi-open interview, visits and on-site observation. For data processing, we used the technique of content analysis. The object of study was the organization RECA - Reforestation Economic Dense Joint Venture, which is an association of small agro foresters, localized in New California (RO). The project RECA has over 2,700 acres of Agro Forestry System - SAF deployed using various types and planting schemes with less than 5% of areas of monoculture. The results were rich and the most relevant were: for the BoP producers of renewable natural resources sustainably, and they may be subject to the governance, what their activities need, is a new vision of BoP (BoP 3.0) , a new type of social business-type cooperative association was created, the use of the types of resources deployable and mobilizes allowing a multilevel analysis of sustainable natural renewable resources for BoP and for a producers of BoP, in order of importance of the social dimensions of sustainability is, environmental and economic, respectively; identified a production system result of a joint use of collective resources (the organization) and individual (each producer); it needs a look at innovation geared towards sustainability that is interactive and non-linear; relations cease to be producers of ToP (top of the pyramid) to the BoP and become producers of BoP, both for consumers of ToP as for the consumer of BoP.

O avanço das ações de desenvolvimento vem colocando desafios à gestão de recursos; as diferentes particularidades nos tipos de recursos disponíveis para uso pelas organizações (na área de administração de empresas), seja por localização geográfica, clima, relevo e/ou outras especificidades locais, como por exemplo, recursos naturais, fogem aos padrões impostos pelas abordagens teóricas estratégicas dominantes, atualmente, nas ciências sociais. No caso do megabioma Amazônico, devido à grandeza patrimonial e suas especificidades locais, há recursos naturais que são únicos e inimitáveis, seja de maneira individual seja pelo conjunto do megabioma, apesar do contexto de pobreza da região. Na medida em que se pressiona para o uso desses recursos, seja pelos indivíduos, seja pelas organizações, podem-se gerar diferentes tipos de conflitos, até porque o uso dos recursos naturais e os conflitos socioambientais são cenários de repercussões para a sociedade, devido às mudanças que promovem em termos ecológicos e das propostas de desenvolvimento social. Assim existem recursos naturais, principalmente, os renováveis disponíveis para uso, que exige novos conhecimentos teóricos e práticos, tanto para acadêmicos como para os gestores atuais. Nesse contexto, são necessárias as preocupações dos cidadãos, instituições e governos, necessitando, no entanto, de um conhecimento adequado à natureza, suas potencialidades e dimensões, no tocante ao uso no presente e garantia para as gerações futuras dos recursos necessários e sua sobrevivência com qualidade de vida, ou seja, a sustentabilidade nesse sentido vem ao encontro de um manejo ambientalmente correto (sustentável) socialmente justo e economicamente rentável, considerando como pilares do Desenvolvimento Sustentável as dimensões Sociais, ambiental e Econômica, dimensões que nortearam a presente pesquisa. Nesse quesito de conhecer como esse processo poderia ocorrer, na Amazônia, foi tirado proveito da teoria Path Dependence que possibilita organizar de forma metodológica e teórica a trajetória e os conhecimentos necessários para que se obtenha uma produção sustentável, com satisfação de mercado aliados a oportunidade de negócio para as comunidades locais. Sendo assim, nesse contexto que envolve recursos, preocupações com a sustentabilidade as inovações que orientaram a presente pesquisa são coladas com a realidade local, com intuito de atender tanto a sustentabilidade quanto resolver impasses que tem impedido progressos substanciais no cruzamento da pobreza, sustentabilidade e meio ambiente. As preocupações, relacionadas à redução da pobreza é um fato que tem sido motivo de preocupação, inclusive no megabioma amazônico, apesar desse ser considerado rico e potencialmente rentável; tem transcendido os limites individuais, empresariais e nacionais, passando a ser uma preocupação global. Nos anos 2000, se consolidou a ideia de que as empresas têm um papel importante na redução da pobreza e a partir disso surge a teoria Base da Pirâmide (BoP) visando possibilitar o consumo para os pobres (BoP 1.0); no entanto por mais que essa teoria tenha avançado possibilitando maior participação dos pobres os envolvendo como co-criadores (BoP 2.0) esta ainda continua os considerando como sujeito passivo ou meros consumidores/colaboradores, visto que a renda por agregação de valor aos produtos (co-criadores) fica com a empresa, ou seja, a teoria não tem sido aproveitada para a produção a partir dos recursos naturais, com vistas a geração de renda, a melhoria na qualidade de vida e organização sustentável, principalmente numa região como a Amazônica. Assim, considerando as oportunidades existentes no megabioma amazônico, tem-se o entendimento que as oportunidades da BoP, podem ir além do consumo (BoP 1.0) ou co-criação de valor (BoP 2.0) e, com foco nos produtores da BoP (produtores de PFNMs), de forma organizada, podem ser desenvolvidas alternativas de melhoria de qualidade de vida, baseado em recursos naturais renováveis sustentáveis, emergindo a questão principal da presente pesquisa: A exploração dos recursos naturais amazônicos, de forma sustentável, pelos produtores BoP, caracteriza-se como uma nova abordagem da BoP, indo além do que é preconizado pela BoP 1.0 e BoP 2.0? Para se chegar a isso, a presente pesquisa está sendo norteada pelos temas: Sustentabilidade e Desenvolvimento sustentável, Recursos Naturais; Visão Baseada em Recursos Internos (VBRI); Inovação e Inovação orientada para a sustentabilidade; Base da Pirâmide (BoP), e PFNMs (produtos florestais não madeiráveis), nesse caso são os produtos naturais renováveis amazônicos. O objetivo é o de propor e aplicar um framework de sustentabilidade da BoP, para organizações que atuem com recursos naturais renováveis, para os produtores (PFNMs) da BoP, do megabioma amazônico. A pesquisa é exploratória e descritiva, de natureza qualitativa. Foram coletados dados secundários e primários. Os dados primários utilizou como método de coleta de dados a entrevista semiaberta, visita e observação in loco. Para tratamento dos dados, foi utilizado a técnica de análise de conteúdo. O objeto de estudo foi a organização RECA – Reflorestamento Econômico Consorciado Adensado, que é uma Associação de pequenos agrossilvicultores, localizada em Nova Califórnia (RO). O projeto RECA possui mais de 2.700 hectares de Sistema Agroflorestal - SAFs implantados, utilizando vários tipos e esquemas de plantios com menos de 5% de áreas de monoculturas. Os resultados obtidos foram ricos e os mais relevantes foram: para que os produtores da BoP, de recursos naturais renováveis, de forma sustentável, possam ser sujeitos e tenham a governança das suas atividades necessita-se de uma nova visão da BoP (BoP 3.0); um novo tipo de negócio social do tipo associação-cooperativa foi criada; o uso dos tipos de recursos mobilizáveis e mobilizadores permitem uma análise multinível sustentável dos recursos naturais renováveis; numa BoP para produtores da BoP a ordem de importância das dimensões de sustentabilideade é social, ambiental e econômica, respectivamente; foi identificado um sistema de produção resultado de uma articulação do uso de recursos coletivos (da organização) e individuais (de cada produtor); necessita-se de um olhar para a inovação orientada para a sustentabilidade que seja interativa e não-linear; as relações deixam de ser dos produtores da ToP (topo da pirâmide) para a BoP e passam a ser dos produtores da BoP, tanto para os consumidores da ToP como os próprios consumidores da BoP. / The progress of the development actions have been placing challenges on the resource management, the various details of the types of resources available for use by organizations (in the area of business administration), or by geographical location, climate, topography and / or other local specificities, such as natural resources, they are escaping the standards imposed by the dominant strategic theoretical approaches currently in social sciences. In the case of megabioma Amazon, because of the patrimonial greatness and their specific locations, there are natural resources that are unique and inimitable, either individually or by the whole megabioma, despite the context of poverty in the region. To the extent that you are forced to use these resources, either by individuals, or by organizations, one can generate different types of conflicts, because the use of natural resources and environmental conflicts are repercussion scenarios for society due to promoting changes in the ecological and social development proposals. In this context, it takes the concerns of citizens, institutions and governments, requiring, however, an adequate knowledge of nature, its potential and dimensions, regarding the use of this guarantee and for future generations of the resources needed and their survival with quality of life, i.e., sustainability in this direction is in line with an environmentally sound management (sustainable) socially, just and economically viable, considering them as pillars of Sustainable Social dimensions, Environmental and Economic dimensions that guided this research. In this aspect of knowing how this process could occur in the Amazon, the Path Dependence theory was taken advantage of, enabling to organize in a methodological and theoretical trajectory way and the knowledge needed to obtain sustainable production, satisfaction with market allies and the business opportunities for local communities. So in this context, that involves resources, concerns about the sustainability innovations that guided this research are bonded with the local reality, in order to meet both sustainability as much as resolve impasses that have prevented substantial progress at the intersection of poverty, sustainability and environment. The concerns, related to the reduction of poverty is a fact that has been of concern, including Amazon megabioma, despite this being considered rich and potentially profitable, it has transcended the individual limits, corporate and national, becoming a global concern. In the 2000s, it has consolidated the idea that businesses have an important role in poverty reduction and from this arises the theory Base of the Pyramid (BoP), in order to enable the consumption for the poor (BoP 1.0), however as much as this theory has advanced, enabling greater participation, by involving the poor as co-creators (BoP 2.0), this is still considering the taxpayer or as mere consumers / employees, since the income for adding value to the products (co-creators) is with the company, ie, the theory has not been used for the production of the natural resources, with a view to generating income, improving the quality of life and sustainable organization, especially in a region like the Amazon. Thus, considering the opportunities in megabioma Amazon, it has been the understanding that the BoP opportunities, can go beyond consumption (BoP 1.0) or co-creation of value (BoP 2.0), and focusing on the BoP producers (producers NTFP), in an organized manner, alternatives can be developed to improve quality of life, based on sustainable renewable natural resources, emerging from the main question of this research: Is the Amazonian exploitation of natural resources in a sustainable manner, by BoP producers, characterizes as a new approach to the BoP, going beyond what is recommended by the BoP 1.0 and BoP 2.0? To achieve this, this research is guided by themes: Sustainability and Sustainable Development, Natural Resources, Internal Resource Based View (VBRI); Innovation and Innovation geared towards sustainability, Base of the Pyramid (BoP) and NTFPs (non-timber forest products), in which case the products are natural renewable Amazonia resources. The goal is to propose and apply a framework of sustainability of BoP for organizations that operate with natural renewable resources for producers (NTFPs) of BoP, the megabioma Amazon. The research is exploratory and descriptive; of qualitative nature. We collected primary and secondary data. The primary data used as a method of data collection to semi-open interview, visits and on-site observation. For data processing, we used the technique of content analysis. The object of study was the organization RECA - Reforestation Economic Dense Joint Venture, which is an association of small agro foresters, localized in New California (RO). The project RECA has over 2,700 acres of Agro Forestry System - SAF deployed using various types and planting schemes with less than 5% of areas of monoculture. The results were rich and the most relevant were: for the BoP producers of renewable natural resources sustainably, and they may be subject to the governance, what their activities need, is a new vision of BoP (BoP 3.0) , a new type of social business-type cooperative association was created, the use of the types of resources deployable and mobilizes allowing a multilevel analysis of sustainable natural renewable resources for BoP and for a producers of BoP, in order of importance of the social dimensions of sustainability is, environmental and economic, respectively; identified a production system result of a joint use of collective resources (the organization) and individual (each producer); it needs a look at innovation geared towards sustainability that is interactive and non-linear; relations cease to be producers of ToP (top of the pyramid) to the BoP and become producers of BoP, both for consumers of ToP as for the consumer of BoP.

O avanço das ações de desenvolvimento vem colocando desafios à gestão de recursos; as diferentes particularidades nos tipos de recursos disponíveis para uso pelas organizações (na área de administração de empresas), seja por localização geográfica, clima, relevo e/ou outras especificidades locais, como por exemplo, recursos naturais, fogem aos padrões impostos pelas abordagens teóricas estratégicas dominantes, atualmente, nas ciências sociais. No caso do megabioma Amazônico, devido à grandeza patrimonial e suas especificidades locais, há recursos naturais que são únicos e inimitáveis, seja de maneira individual seja pelo conjunto do megabioma, apesar do contexto de pobreza da região. Na medida em que se pressiona para o uso desses recursos, seja pelos indivíduos, seja pelas organizações, podem-se gerar diferentes tipos de conflitos, até porque o uso dos recursos naturais e os conflitos socioambientais são cenários de repercussões para a sociedade, devido às mudanças que promovem em termos ecológicos e das propostas de desenvolvimento social. Assim existem recursos naturais, principalmente, os renováveis disponíveis para uso, que exige novos conhecimentos teóricos e práticos, tanto para acadêmicos como para os gestores atuais. Nesse contexto, são necessárias as preocupações dos cidadãos, instituições e governos, necessitando, no entanto, de um conhecimento adequado à natureza, suas potencialidades e dimensões, no tocante ao uso no presente e garantia para as gerações futuras dos recursos necessários e sua sobrevivência com qualidade de vida, ou seja, a sustentabilidade nesse sentido vem ao encontro de um manejo ambientalmente correto (sustentável) socialmente justo e economicamente rentável, considerando como pilares do Desenvolvimento Sustentável as dimensões Sociais, ambiental e Econômica, dimensões que nortearam a presente pesquisa. Nesse quesito de conhecer como esse processo poderia ocorrer, na Amazônia, foi tirado proveito da teoria Path Dependence que possibilita organizar de forma metodológica e teórica a trajetória e os conhecimentos necessários para que se obtenha uma produção sustentável, com satisfação de mercado aliados a oportunidade de negócio para as comunidades locais. Sendo assim, nesse contexto que envolve recursos, preocupações com a sustentabilidade as inovações que orientaram a presente pesquisa são coladas com a realidade local, com intuito de atender tanto a sustentabilidade quanto resolver impasses que tem impedido progressos substanciais no cruzamento da pobreza, sustentabilidade e meio ambiente. As preocupações, relacionadas à redução da pobreza é um fato que tem sido motivo de preocupação, inclusive no megabioma amazônico, apesar desse ser considerado rico e potencialmente rentável; tem transcendido os limites individuais, empresariais e nacionais, passando a ser uma preocupação global. Nos anos 2000, se consolidou a ideia de que as empresas têm um papel importante na redução da pobreza e a partir disso surge a teoria Base da Pirâmide (BoP) visando possibilitar o consumo para os pobres (BoP 1.0); no entanto por mais que essa teoria tenha avançado possibilitando maior participação dos pobres os envolvendo como co-criadores (BoP 2.0) esta ainda continua os considerando como sujeito passivo ou meros consumidores/colaboradores, visto que a renda por agregação de valor aos produtos (co-criadores) fica com a empresa, ou seja, a teoria não tem sido aproveitada para a produção a partir dos recursos naturais, com vistas a geração de renda, a melhoria na qualidade de vida e organização sustentável, principalmente numa região como a Amazônica. Assim, considerando as oportunidades existentes no megabioma amazônico, tem-se o entendimento que as oportunidades da BoP, podem ir além do consumo (BoP 1.0) ou co-criação de valor (BoP 2.0) e, com foco nos produtores da BoP (produtores de PFNMs), de forma organizada, podem ser desenvolvidas alternativas de melhoria de qualidade de vida, baseado em recursos naturais renováveis sustentáveis, emergindo a questão principal da presente pesquisa: A exploração dos recursos naturais amazônicos, de forma sustentável, pelos produtores BoP, caracteriza-se como uma nova abordagem da BoP, indo além do que é preconizado pela BoP 1.0 e BoP 2.0? Para se chegar a isso, a presente pesquisa está sendo norteada pelos temas: Sustentabilidade e Desenvolvimento sustentável, Recursos Naturais; Visão Baseada em Recursos Internos (VBRI); Inovação e Inovação orientada para a sustentabilidade; Base da Pirâmide (BoP), e PFNMs (produtos florestais não madeiráveis), nesse caso são os produtos naturais renováveis amazônicos. O objetivo é o de propor e aplicar um framework de sustentabilidade da BoP, para organizações que atuem com recursos naturais renováveis, para os produtores (PFNMs) da BoP, do megabioma amazônico. A pesquisa é exploratória e descritiva, de natureza qualitativa. Foram coletados dados secundários e primários. Os dados primários utilizou como método de coleta de dados a entrevista semiaberta, visita e observação in loco. Para tratamento dos dados, foi utilizado a técnica de análise de conteúdo. O objeto de estudo foi a organização RECA – Reflorestamento Econômico Consorciado Adensado, que é uma Associação de pequenos agrossilvicultores, localizada em Nova Califórnia (RO). O projeto RECA possui mais de 2.700 hectares de Sistema Agroflorestal - SAFs implantados, utilizando vários tipos e esquemas de plantios com menos de 5% de áreas de monoculturas. Os resultados obtidos foram ricos e os mais relevantes foram: para que os produtores da BoP, de recursos naturais renováveis, de forma sustentável, possam ser sujeitos e tenham a governança das suas atividades necessita-se de uma nova visão da BoP (BoP 3.0); um novo tipo de negócio social do tipo associação-cooperativa foi criada; o uso dos tipos de recursos mobilizáveis e mobilizadores permitem uma análise multinível sustentável dos recursos naturais renováveis; numa BoP para produtores da BoP a ordem de importância das dimensões de sustentabilideade é social, ambiental e econômica, respectivamente; foi identificado um sistema de produção resultado de uma articulação do uso de recursos coletivos (da organização) e individuais (de cada produtor); necessita-se de um olhar para a inovação orientada para a sustentabilidade que seja interativa e não-linear; as relações deixam de ser dos produtores da ToP (topo da pirâmide) para a BoP e passam a ser dos produtores da BoP, tanto para os consumidores da ToP como os próprios consumidores da BoP. / The progress of the development actions have been placing challenges on the resource management, the various details of the types of resources available for use by organizations (in the area of business administration), or by geographical location, climate, topography and / or other local specificities, such as natural resources, they are escaping the standards imposed by the dominant strategic theoretical approaches currently in social sciences. In the case of megabioma Amazon, because of the patrimonial greatness and their specific locations, there are natural resources that are unique and inimitable, either individually or by the whole megabioma, despite the context of poverty in the region. To the extent that you are forced to use these resources, either by individuals, or by organizations, one can generate different types of conflicts, because the use of natural resources and environmental conflicts are repercussion scenarios for society due to promoting changes in the ecological and social development proposals. In this context, it takes the concerns of citizens, institutions and governments, requiring, however, an adequate knowledge of nature, its potential and dimensions, regarding the use of this guarantee and for future generations of the resources needed and their survival with quality of life, i.e., sustainability in this direction is in line with an environmentally sound management (sustainable) socially, just and economically viable, considering them as pillars of Sustainable Social dimensions, Environmental and Economic dimensions that guided this research. In this aspect of knowing how this process could occur in the Amazon, the Path Dependence theory was taken advantage of, enabling to organize in a methodological and theoretical trajectory way and the knowledge needed to obtain sustainable production, satisfaction with market allies and the business opportunities for local communities. So in this context, that involves resources, concerns about the sustainability innovations that guided this research are bonded with the local reality, in order to meet both sustainability as much as resolve impasses that have prevented substantial progress at the intersection of poverty, sustainability and environment. The concerns, related to the reduction of poverty is a fact that has been of concern, including Amazon megabioma, despite this being considered rich and potentially profitable, it has transcended the individual limits, corporate and national, becoming a global concern. In the 2000s, it has consolidated the idea that businesses have an important role in poverty reduction and from this arises the theory Base of the Pyramid (BoP), in order to enable the consumption for the poor (BoP 1.0), however as much as this theory has advanced, enabling greater participation, by involving the poor as co-creators (BoP 2.0), this is still considering the taxpayer or as mere consumers / employees, since the income for adding value to the products (co-creators) is with the company, ie, the theory has not been used for the production of the natural resources, with a view to generating income, improving the quality of life and sustainable organization, especially in a region like the Amazon. Thus, considering the opportunities in megabioma Amazon, it has been the understanding that the BoP opportunities, can go beyond consumption (BoP 1.0) or co-creation of value (BoP 2.0), and focusing on the BoP producers (producers NTFP), in an organized manner, alternatives can be developed to improve quality of life, based on sustainable renewable natural resources, emerging from the main question of this research: Is the Amazonian exploitation of natural resources in a sustainable manner, by BoP producers, characterizes as a new approach to the BoP, going beyond what is recommended by the BoP 1.0 and BoP 2.0? To achieve this, this research is guided by themes: Sustainability and Sustainable Development, Natural Resources, Internal Resource Based View (VBRI); Innovation and Innovation geared towards sustainability, Base of the Pyramid (BoP) and NTFPs (non-timber forest products), in which case the products are natural renewable Amazonia resources. The goal is to propose and apply a framework of sustainability of BoP for organizations that operate with natural renewable resources for producers (NTFPs) of BoP, the megabioma Amazon. The research is exploratory and descriptive; of qualitative nature. We collected primary and secondary data. The primary data used as a method of data collection to semi-open interview, visits and on-site observation. For data processing, we used the technique of content analysis. The object of study was the organization RECA - Reforestation Economic Dense Joint Venture, which is an association of small agro foresters, localized in New California (RO). The project RECA has over 2,700 acres of Agro Forestry System - SAF deployed using various types and planting schemes with less than 5% of areas of monoculture. The results were rich and the most relevant were: for the BoP producers of renewable natural resources sustainably, and they may be subject to the governance, what their activities need, is a new vision of BoP (BoP 3.0) , a new type of social business-type cooperative association was created, the use of the types of resources deployable and mobilizes allowing a multilevel analysis of sustainable natural renewable resources for BoP and for a producers of BoP, in order of importance of the social dimensions of sustainability is, environmental and economic, respectively; identified a production system result of a joint use of collective resources (the organization) and individual (each producer); it needs a look at innovation geared towards sustainability that is interactive and non-linear; relations cease to be producers of ToP (top of the pyramid) to the BoP and become producers of BoP, both for consumers of ToP as for the consumer of BoP.

Bacillus subtilis est la bactérie modèle des bactéries Gram-positif à bas pourcentage en GC et possède un intérêt marqué en biotechnologie. Par ailleurs, la phosphorylation des protéines est un mécanisme de régulation essentiel chez les bactéries qui reste encore largement à explorer. B. subtilis possède plusieurs ser/thr kinases potentielles (PrkA, YbdM, YabT et PrkC, qui a été déjà largement caractérisée), mais très peu de substrats de ces kinases ont été mis en évidence. Récemment, des études phosphoprotéomiques ont permis d’identifier de nombreux peptides phosphorylés sur des sérines ou des thréonines chez B. subtilis, incluant: a) deux régulateurs globaux de la phase de transition, DegS et AbrB et b) RecA, qui joue un rôle essentiel dans la réparation des cassures double-brin de l’ADN et la recombinaison. Des tests de phosphorylation in vitro nous ont permis d’identifier les ser/thr kinases capables de phosphoryler DegS, RecA et AbrB. La phosphorylation de DegS sur son résidu sérine 76 par la kinase YbdM influence, in vitro et in vivo, son activité kinase vis à vis de son substrat DegU. L’expression chez B. subtilis d’un allèle codant la protéine DegS-S76D (la sérine étant remplacée par un aspartate phosphomimétique) perturbe l’ensemble des processi cellulaires régulés par le système à deux composants DegS/DegU. Ces résultats suggèrent un lien entre la phosphorylation de DegS sur sa sérine 78 et le niveau de phosphorylation de son substrat DegU, cette modification post-traductionnelle représentant un degré supplémentaire de régulation pour ce système à deux composants. Au cours du démarrage de la sporulation, B. subtilis exprime une ser/thr kinase atypique, YabT, qui localise au septum et est activée grâce à la liaison de séquences ADN non spécifiques. YabT activée phosphoryle RecA sur sa sérine 2, ce qui induit la formation de foci RecA. Dans une souche exprimant une protéine RecA non phosphorylable (RecA-S2A) ou inactivée pour yabT, la formation de spores en présence de lésions de l’ADN est diminuée. Ces résultats suggèrent une homologie fonctionnelle au cours du développement entre la phosphorylation de RecA chez B. subtilis et la phosphorylation de son homologue eukaryote Rad51, qui permet leur recrutement sur des lésions de l’ADN. Nous proposons donc que la phosphorylation de RecA serve de signal pour promouvoir la formation de foci au cours de la sporulation. In vitro, le régulateur transcriptionnel AbrB est phosphorylé par les kinases YabT, YbdM et PrkC, L’utilisation de protéines mutées AbrB-S86A (non phosphorylable) et AbrB-S86D (forme phosphomimétique) nous a permis de montrer que la phosphorylation d’AbrB diminue son affinité pour l’ADN cible. L’expression chez B. subtilis des protéines AbrB-S86A et –S86D perturbe des phénomènes mis en place au cours de la phase stationnaire comme la production d’exoprotéases, la compétence et la sporulation via la dérégulation des gènes et opérons AbrB-dépendants correspondants. Nous proposons donc que la phosphorylation d’AbrB par les Hanks-kinases constitue un mécanisme de contrôle supplémentaire nécessaire à l’inactivation de ce régulateur transcriptionnel, qui peut être activateur ou répresseur, pendant la phase de transition. / Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

La tesis estudia la evolución del parque de generación de energía eléctrica durante el franquismo (1940-1975). En este período comenzó la formación de un sistema unificado de explotación, y durante su desarrollo se pasó de 1.731Mw a 25.467Mw. El sistema que se denominó “autorregulación”, permitió la coexistencia de las empresas eléctricas privadas, agrupadas alrededor de UNESA y el INI en su vertiente eléctrica. Sin embargo, las decisiones y la política eléctrica, y más tarde energética, estuvo directamente influida por el gobierno. Se analiza la importancia de las fuentes (hidráulica, carbón, fuel oil, gas natura, nuclear) de energía primaria en la generación de energía eléctrica. Asimismo, se estudia, aunque no de manera exhaustiva el impacto del medio ambiente y sus consecuencias y efectos a largo plazo. Todo ello se desarrolla en el contexto histórico pertinente bajo una vertiente técnico-económica. / The thesis studies the evolution of the power generation system of electricity, during the Franco’s Regime (1940-1975). In this period, it began the construction of a unified system of exploitation, where the power installed was moved from 1.731Mw to 25.467Mw. The system was called "self regulation", allowing the coexistence of private electric companies, grouped around UNESA and the INI in its electrical aspect. However, the decisions and the power policy, and later on the energy, was directly influenced by the government. It discusses the importance of the primary energy sources (hydro, coal, fuel oil, natural gas, nuclear) in electric power generation. However, it is not intended to illustrate a comprenhensive explanation of the environmental impact and its consequences and effects long term. To sum up, all this takes place in the relevant historical context in a technical and economical perspective.

Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.