Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein–DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

Conceptual Decolonization of Space: Worldview and Language in Anishinaabe Akiing

This dissertation examines the role of worldview and language in the cultural framework of American Indian people. In it I develop a theory of worldview which can be defined as an interrelated set of logics that orients a culture to space (land), time, the rest of life, and provides a prescription for understanding that life. Considering the strong links between language and worldview, it is methodologically necessary to focus on a particular language and culture to decolonize concepts of and relationships to land. In particular, this dissertation focuses on an Anishinaabe worldview as consisting of four components, which are; (1) an intimate relationship to a localized space; (2) a cyclical understanding of time; (3) living in a web of relatedness with all life, and (4) understanding the world around us in terms of balance. The methodological approach draws from Anishinaabemowin, the traditional Anishinaabe language, as a starting place for negotiating a linguistic-conceptual analysis of these logics to decolonize the understandings of land, time, relatedness and balance. This dissertation helps to demonstrate that the religious language as codified in the 1st Amendment to the United States Constitution as religious freedom is unable to carry the meaning of the fundamental relationships to land that are embedded in Anishinaabemowin and culture. I compare the above Anishinaabe worldview to that of the eurowestern culture in America, which is; (1) the domination of space; (2) a linear progression of time; (3) a hierarchical organization of life; and (4) understanding the world as a Manichean battle of good versus evil. This dissertation seeks to decolonize American Indian translational methodologies and undermine the assumptions of eurowestern cultural universality.

Tratamiento de lixiviados de residuos de origen urbano mediante MBR

Se ha utilizado una planta de tratamiento a escala laboratorio consiste en un biorreactor de membrana (MBR). Esta planta está compuesta por un reactor biológico de 25 L de capacidad. Se utilizó una membrana plana de micro filtración marca Kubota de polietileno clorado, tamaño de poro 0,1 μm y área de filtración 0.116 m2. Se utilizaron como condiciones de operación: tiempo de residencia hidráulico 3 días, caudal de permeado 0.35 L/h y LMH 3 L/m2h. Se ha podido comprobar que es posible adaptar una población microbiológica a las particulares características químicas del lixiviado procedente de la planta y tratar estos lixiviados en un reactor biológico de membrana sumergida operando en condiciones habituales de sólidos en suspensión en el reactor entre 8-12 g/L durante un periodo de 6 meses. El proceso utilizado permite reducir la materia orgánica (97% DBO5 y 40% DQO) presente en estas corrientes residuales, agotando prácticamente toda la materia biodegradable. Respecto a los contenidos de nutrientes, el tratamiento MBR ensayado permite reducir de 35-40% el nitrógeno total, 45-50% el nitrógeno amoniacal y un 65-70% el fósforo total. Los sólidos en suspensión se han reducido en el efluente tratado en más de un 99%.

Gasification and Pyrolysis of Posidonia oceanica in the Presence of Dolomite

Resumen del póster presentado en Symposium on Renewable Energy and Products from Biomass and Waste, CIUDEN (Cubillos de Sil, León, Spain), 12-13 May 2015

La conjetura de Poincaré

Con la ayuda de los grupos de homología – que él mismo había definido – Poincaré dio una clasificación completa de las superficies topológicas, y posteriormente intentó clasificar, con ayuda de estos grupos y también del grupo fundamental, las variedades topológicas de cualquier dimensión. Una de las preguntas que se planteó fue si toda variedad topológica 3-dimensional simplemente conexa era homeomorfa a la esfera S3. Esta pregunta – conocida como la conjetura de Poincaré – ha sido objeto de estudio durante casi 100 años, y ha impulsado de modo notable el desarrollo de la Topología Algebraica. La conjetura se extendió a dimensiones arbitrarias y se fue resolviendo en todas las dimensiones salvo en la dimensión 3 que era aquella en la que había sido originariamente planteada. Por fin en el año 2003, Perelman resolvió la famosa conjetura. Los objetivos de este trabajo son los siguientes: hacer un estudio histórico de la Conjetura de Poincaré y de la clasificación de las variedades topológicas, definir con precisión los conceptos que se usan en la clasificación de las variedades topológicas, presentar una selección de los principales resultados, y por último, construir ejemplos de variedades topológicas que justifiquen el desarrollo de esta teoría.

Construção tridimensional de atlas dos núcleos anteriores do tálamo a partir de cortes de um bloco de encéfalo

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

Prospective longitudinal voxel-based morphometry study of major depressive disorder in young individuals at high familial risk

BACKGROUND Previous neuroimaging studies indicate abnormalities in cortico-limbic circuitry in mood disorder. Here we employ prospective longitudinal voxel-based morphometry to examine the trajectory of these abnormalities during early stages of illness development. METHOD Unaffected individuals (16-25 years) at high and low familial risk of mood disorder underwent structural brain imaging on two occasions 2 years apart. Further clinical assessment was conducted 2 years after the second scan (time 3). Clinical outcome data at time 3 was used to categorize individuals: (i) healthy controls ('low risk', n = 48); (ii) high-risk individuals who remained well (HR well, n = 53); and (iii) high-risk individuals who developed a major depressive disorder (HR MDD, n = 30). Groups were compared using longitudinal voxel-based morphometry. We also examined whether progress to illness was associated with changes in other potential risk markers (personality traits, symptoms scores and baseline measures of childhood trauma), and whether any changes in brain structure could be indexed using these measures. RESULTS Significant decreases in right amygdala grey matter were found in HR MDD v. controls (p = 0.001) and v. HR well (p = 0.005). This structural change was not related to measures of childhood trauma, symptom severity or measures of sub-diagnostic anxiety, neuroticism or extraversion, although cross-sectionally these measures significantly differentiated the groups at baseline. CONCLUSIONS These longitudinal findings implicate structural amygdala changes in the neurobiology of mood disorder. They also provide a potential biomarker for risk stratification capturing additional information beyond clinically ascertained measures.

Physical and acoustic sediment properties of sediment cores from the eastern equatorial Atlantic

To reconstruct the deep-water circulation for the last 3.5 Ma from deep-sea sediments of the eastern equatorial Atlantic, sea floor morphology, sub-bottom reflectors and the echo character have been mapped on the basis of 3.5 kHz records and sediment cores. Physical properties of sediments and synthetic seismograms derived from them enable us to decipher reflector sequences in environments of pelagic, current-resuspended and turbidity sedimentation. The individual reflectors originate from carbonate dissolution, hiatus, coarse sand layers and interferences. Those which are related to carbonate dissolution and hiatus provide evidence of water-mass boundaries by their distribution. Five phases of different deep-water circulation can be seen in the record of th elast 3.5 Ma, and these are related to climate history: 1. Between 3.7 Ma and 2.2 Ma a strong deep-water circulation indicates a northward flow of bottom water below 4200 m (AABW = Antarctic-Bottom Water) and a southward flow of deep-water above 4200 m (NADW = North-Atlantic Deep Water). 2. Between 1.6 and 1.4 Ma a southward flow of bottom water below 4500 m and a diminished southward flow above 4500 m can be detected. This water-mass geometry can be interpreted by an expansion of the NADW-masses and a displacement of the AABW-masses during the same time. 3. Since 1.4 Ma a northward flow of a bottom-water current developed again. This current flow created a leeside sediment ridge in the southern part of the Kane Gap and furrows in the northern part of it. 4. Between 400,000 and 200,000 yrs B. P. the oceanic and atmospheric circulation increased. The strengthened oceanic circulation caused and increase in carbonate dissolution, which is documented by a traceable reflector from 2800 m to 4500 m water depth. At the same time an increase of the atmospheric circulation caused a drastic rise in the pelagic sediment accumulation (> 100 %) through an intensification of upwelling. This runs parallel with a higher oceanic productivity in the northern equatorial divergence zone and an enhanced supply of fluvial and probably eolian sediments from the Senegal and Guinea. 5. Before 10,000 yrs B. P. an erosive northward flowing bottom-water current prevailed below 4500 m water depth. After 10,000 yrs B.P. the bottom-water flow was sluggish and non erosive.

Observations of Joints Present in the Lawton Clay Member of the Vashon Stade, Exposed at a Coastal Bluff in Discovery Park, Seattle, Washington

The southwest-facing coastal bluff present at Discovery Park, Seattle, Washington, displays distinctive joints throughout the exposed Lawton Clay Member. Exhibiting a characteristic local stratigraphy of permeable advance outwash over the impermeable proglacial lacustrine clay, this bluff is located in an area of Seattle at high risk from landslides. This project addressed the relationship between the joints observed at this coastal bluff and the coherency of the bluff as a whole, through remote sensing and field measurements. Aerial drone photography taken of the bluff was processed through a photogrammetry software to produce a 3-dimensional Structure from Motion model, allowing for a digital manipulation and broad examination of the bluff not possible by foot. Stereonet plots produced from these measurements provided insight into patterns of varying joint strike along a horizontal transect of the observed bluff face. Taken together, these two visualizations provided a better picture of the possible chicken-and-egg interaction of the joints and bluff topography; they demonstrated the likelihood that the joint formation at the bluff was most likely to be primarily influenced by the local topography of the bluff over other sources of possible tensional stress in the immediate area.

On Selmer groups and factoring p-adic L-functions

Thesis (Ph.D.)--University of Washington, 2016-06

Structural Insights into Viral Membrane Fusion Machinery via Cryo-Electron Tomography: Influenza Virus and Human Parainfluenza Virus

Thesis (Ph.D.)--University of Washington, 2016-06

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.

Structure of Petunia hybrida Defensin 1, a Novel Plant Defensin with Five Disulfide Bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by H-1 NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.

Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone

Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., Peduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthelemy, M., Prigent, Y., Salomon, R. A., Farias, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.

Twists, knots, and rings in proteins: Structural definition of the cyclotide framework

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Mobius cyclotides) and knots (a knotted arrangement of the disulfide bonds).