Micropollutant bioremoval in wastewater treatment systems: from microbial population structure to function

Dissertation presented to obtain the Ph.D degree in Biology

The role of water in determining structure and function of macromolecules and macromolecular assemblies /

The interaction of biological molecules with water is an important determinant of structural properties both in molecular assemblies, and in conformation of individual macromolecules. By observing the effects of manipulating the activity of water (which can be accomplished by limiting its concentration or by adding additional solutes, "osmotic stress"), one can learn something about intrinsic physical properties of biological molecules as well as measure an energetic contribution of closely associated water molecules to overall equilibria in biological reactions. Here two such studies are reported. The first of these examines several species of lysolipid which, while present in relatively low concentrations in biomembranes, have been shown to affect many cellular processes involving membrane-protein or membrane-membrane interactions. Monolayer elastic constants were determined by combining X-ray diffraction and the osmotic stress technique. Spontaneous radii of curvature of lysophosphatidylcholines were determined to be positive and in the range +30A to +70A, while lysophosphatidylethanolamines proved to be essentially flat. Neither lysolipid significantly affected the bending modulus of the monolayer in which it was incorporated. The second study examines the role of water in theprocess of polymerization of actin into filaments. Water activity was manipulated by adding osmolytes and the effect on the equilibrium dissociation constant (measured as the criticalmonomer concentration) was determined. As water activity was decreased, the critical concentration was reduced for Ca-actin but not for Mg-actin, suggesting that 10-12 fewer water molecules are associated with Ca-actin in the polymerized state. Thisunexpectedly small amount of water is discussed in the context of the common structural motif of a nucleotide binding cleft.

The structure of arabidopsis NPR1 : its function as a salicylic acid receptor and a metal-binding protein

The Arabidopsis NPRI protein regulates systemic acquired resistance dependent on salicylic acid. Analyses by plant two-hybrid analysis in vivo and pull-down assays in vitro showed that the BTB/POZ domain of NPRI at the N-terminus serves as an autoinhibitory domain to negate the function of the transactivation domain at the C-terminus through direct binding of these two domains. I t was also shown that the binding of the BTB/POZ domain to the C-terminus of NPRI was abolished by SA treatment, suggesting that SA could interfere directly with this binding. By gel filtration, it was demonstrated that SA affects the conformation of full-length NPRl , confirming the role of NPRI as an SA receptor. Gel filtration analysis also indicated that NPRI could be converted from an oligomer to a dimer with SA treatment. Furthermore, one N-terminal deletion ~513 has been shown to act as a metal-binding protein and its two Cys-521 and Cys-529 are important for binding to Ni 2 + by pull-down assays.

The effects of oxidative stress on the retinal structure and function

Des études antérieures ont démontré que le métabolisme de la rétine, son apport sanguin et sa consommation de l'oxygène sont plus élevés dans le noir (Riva C.E. et al. 1983, Wang L. et al. 1996, Tam B.M. and Moritz O.L. 2007). Les stimuli physiologiques jouent supposément un rôle important dans le développement des différents systèmes nerveux (Arthur W. Spira, David Parkinson 1991). La privation de la rétine de son stimulus physiologique, la lumière, est un moyen valable de démontrer la validité de ce concept. D'autres études ont affirmé que les injections de dichlorure de paraquat dans la cavité vitréenne causent une sévère rétinopathie (Rétinopathie induite par paraquat: RIP). Cette rétinopathie est provoquée par les dérivés réactifs de l'oxygène (DRO) générés par le paraquat (Cingolani C. et al. 2006, Lu L. et al. 2006). Le but de notre premier projet (''Dark rearing project'') était de déterminer si les conséquences nocives de l'hyperoxie postnatale chez les rats albinos SD pourraient être amoindries en élevant une portée de rats au noir. Nos résultats suggèrent qu'une augmentation du métabolisme de la rétine causée par la déprivation de lumière chez les ratons, pourrait protéger ou masquer certains effets néfastes de l'hyperoxie postnatale. Le but de notre deuxième étude (''Paraquat project'') était d'examiner les possibles points de similitude entre RIP et d'autres modèles de rétinopathies oxydatives étudiés présentement par notre équipe, à savoir: Rétinopathie induite par l'oxygène (RIO) et Rétinopathie induite par la lumière (RIL). Nos résultats suggèrent que l'injection de dichlorure de paraquat dans la cavité vitréenne cause des changements sévères de la fonction de la rétine, tandis que sa structure semble intacte. La sévérité de ces changements dépend inversement de la maturité de la rétine au moment de l'injection.

The study of RNA tertiary interactions in tRNA structure and function

Le rôle des deux paires de bases universelles inverse Hoogsteen U : A ( RHUAs ) présentent chez les ARNt standards , une dans la boucle T et l'autre dans le noyau de la forme en L , a été étudiée. Pour chacun des RHUAs , un criblage génétique spécialisé in vivo chez les bactéries , le système suppresseur ambre ( pour l'étude de la RHUA dans la boucle T ) et le système d'ARNt de la sélénocystéine ( tRNASec ) ( pour l'étude de la RHUA dans le noyau ) , ont été utilisé pour générer des variants fonctionnels à partir de multiples librairies combinatoires . Ces variants ont ensuite été séquencé et soumis à une analyse systématique qui comprend la modélisation informatique et un type d'analyse phylogénétique. Les résultats du système suppresseur ambre ont montré un ensemble de variants fonctionnels qui ne nécessitent pas le motif RHUA dans la boucle T et qui ont remplacé la méthode standard de l'interaction entre les boucles D et T avec une double hélice interboucle , ILDH . D'autres études ont abouti à la détermination d'un modèle In silico de l'alternative à la norme standard de la boucle T, sous le nom de type III . Les résultats du système tRNASec ont révélé que pour cette ARNt exceptionnel, l'absence de RHUA ( dans le noyau ) assure une flexibilité accrue qui est spécifiquement nécessaire pour la fonction de tRNASec . Ainsi, les ARNt standards , à la différence de tRNASec , avec la présence universelle de RHUA dans le noyau , a été naturellement sélectionnée pour être rigide . Pris ensemble, la RHUA joue un rôle essentiel dans la stabilisation des interactions tertiaires.

Germin and germin-like proteins: evolution, structure, and function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

Mapping CD55 function: the structure of two pathogen-binding domains at 1.7 A

Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells. It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection. We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement. We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

The gynoecium of male Anchietea pyrifolia (Violaceae): Preserved structure with a new function

Structure of inflorescences and flowers and flowering behaviour are reported for the woody liana Anchietea pyrifolia (Violaceae) from Brazil. The specimen studied is grown for some decades now in the greenhouses of Halle Botanical Garden and turned out unisexually male, which adds a further example of dioecism to the family Violaceae, in which this type of sex distribution is rarely encountered. The flowers are exceptional also for the strongly asymmetric anterior petal, which represents a rare case of a species with enantiomorphic flowers pollinated by Lepidoptera. They have a fully developed gynoecium with a complicated architecture comparable to the pistil of bisexual Violaceae flowers, though without ovules. The style head is capable to release viscose liquid on tactile stimulation or pressure, which is known to act as pollen-gathering mechanism in bisexual Violaceae species with usually dry pollen and buzz-pollination. This function has switched in male A. pyrifolia to a mechanism for efficient pollen release mediated by insect pollinators from its short-lived flowers. (C) 2009 Elsevier GmbH. All rights reserved.

Percolation in a network with long-range connections: Implications for cytoskeletal structure and function

Cell shape, signaling, and integrity depend on cytoskeletal organization. In this study we describe the cytoskeleton as a simple network of filamentary proteins (links) anchored by complex protein structures (nodes). The structure of this network is regulated by a distance-dependent probability of link formation as P = p/d(s), where p regulates the network density and s controls how fast the probability for link formation decays with node distance (d). It was previously shown that the regulation of the link lengths is crucial for the mechanical behavior of the cells. Here we examined the ability of the two-dimensional network to percolate (i.e. to have end-to-end connectivity), and found that the percolation threshold depends strongly on s. The system undergoes a transition around s = 2. The percolation threshold of networks with s < 2 decreases with increasing system size L, while the percolation threshold for networks with s > 2 converges to a finite value. We speculate that s < 2 may represent a condition in which cells can accommodate deformation while still preserving their mechanical integrity. Additionally, we measured the length distribution of F-actin filaments from publicly available images of a variety of cell types. In agreement with model predictions, cells originating from more deformable tissues show longer F-actin cytoskeletal filaments. (C) 2008 Elsevier B.V. All rights reserved.

Bulliform cells in Loudetiopsis chrysothrix (Nees) Conert and Tristachya leiostachya Nees (Poaceae): structure in relation to function

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)