Evaluation and comparison of mammalian subcellular localization prediction methods

Background: Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results: In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, and lysosome). The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion: No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE dataset and variable performance on individual subcellular localizations was observed. Proteins localized to the secretory pathway were the most difficult to predict, while nuclear and extracellular proteins were predicted with the highest sensitivity.

Sequence distributions in free-radical polymers

This thesis is concerned with demonstrating how the visual representation of the sequence distribution of individual monomer units, of a polymer, that would be observed upon polymerisation, may be utilised in designing and synthesizing polymers with relatively low cell adhesion characteristics, The initial part of this thesis is concerned with demonstrating the use of a computer simulation technique, in illustrating the sequence distribution that would be observed upon the polymerisation of a set of monomers. The power of the computer simulation technique has been demonstrated through the simulation of the sequence distributions of some generic contact lens materials. These generic contact lens materials were chosen simply because in the field of biomaterials their compositions are amongst the most systematically regulated and they present a wide range of compositions. The validity of the computer simulation technique has been assessed through the synthesis and analysis of linear free-radical polymers at different conversions. Two main parameters were examined, that of composition and the number-average sequence lengths of individual monomer units, at various conversions. The polymers were synthesized through the solution polymerisation process. The monomer composition was determined by elemental analysis and 13C nuclear magnetic analysis (NMR). Number-average sequence lengths were determined exclusively through 13C NMR. Although the computer simulation technique provides a visual representation of the monomer sequence distribution up to 100% conversion, these assessments were made on linear polymers at a reasonably high conversion (above 50%) but below 100% conversion of ease for analysis. The analyses proved that the computer simulation technique was reasonably accurate in predicting the sequence distribution of monomer units, upon polymerisation, in the polymer.An approach has been presented which allows one to manipulate the use of monomers, with their reactivity ratios, thereby enabling us to design polymers with controlled sequence distributions.Hydrogel membranes, with relatively controlled sequence distributions and polymerised to 100% conversion, were synthesized to represent prospective biomaterials. Cell adhesion studies were used as a biological probe to investigate the susceptibility of the surface of these membranes to cell adhesion. This was necessary in order to assess the surface biocompatibility or biotolerance of these prospective biomaterials.

Utilização de pulsos de radiofrequência fortemente modulados no processamento de informação quântica via ressonância magnética nuclear

Nesta dissertação apresentamos um trabalho de desenvolvimento e utilização de pulsos de radiofreqüência modulados simultaneamente em freqüência, amplitude e fase (pulsos fortemente modulados, SMP, do inglês Strongly Modulated Pulses) para criar estados iniciais e executar operações unitárias que servem como blocos básicos para processamento da informação quântica utilizando Ressonância Magnética Nuclear (RMN). As implementações experimentais foram realizas em um sistema de 3 q-bits constituído por spins nucleares de Césio 133 (spin nuclear 7/2) em uma amostra de cristal líquido em fase nemática. Os pulsos SMP´s foram construídos teoricamente utilizando um programa especialmente desenvolvido para esse fim, sendo o mesmo baseado no processo de otimização numérica Simplex Nelder-Mead. Através deste programa, os pulsos SMP foram otimizados de modo a executarem as operações lógicas desejadas com durações consideravelmente menores que aquelas realizadas usando o procedimento usual de RMN, ou seja, seqüências de pulsos e evoluções livres. Isso tem a vantagem de reduzir os efeitos de descoerência decorrentes da relaxação do sistema. Os conceitos teóricos envolvidos na criação dos SMPs são apresentados e as principais dificuldades (experimentais e teóricas) que podem surgir devido ao uso desses procedimentos são discutidas. Como exemplos de aplicação, foram produzidos os estados pseudo-puros usados como estados iniciais de operações lógicas em RMN, bem como operações lógicas que foram posteriormente aplicadas aos mesmos. Utilizando os SMP\'s também foi possível realizar experimentalmente os algoritmos quânticos de Grover e Deutsch-Jozsa para 3 q-bits. A fidelidade das implementações experimentais foi determinadas utilizando as matrizes densidade experimentais obtidas utilizando um método de tomografia da matriz densidade previamente desenvolvido.

Source localization of reaction-diffusion models for brain tumors

We propose a mathematically well-founded approach for locating the source (initial state) of density functions evolved within a nonlinear reaction-diffusion model. The reconstruction of the initial source is an ill-posed inverse problem since the solution is highly unstable with respect to measurement noise. To address this instability problem, we introduce a regularization procedure based on the nonlinear Landweber method for the stable determination of the source location. This amounts to solving a sequence of well-posed forward reaction-diffusion problems. The developed framework is general, and as a special instance we consider the problem of source localization of brain tumors. We show numerically that the source of the initial densities of tumor cells are reconstructed well on both imaging data consisting of simple and complex geometric structures.

RNA Localization and Translational Regulation on the Endoplasmic Reticulum

mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.

Spliceosomal small nuclear ribonucleoprotein particle trafficking and maturation in the nuclear compartment

We show for the first time that upon injection into the cytoplasm of the oocyte, fluorescein-labeled spliceosomal snRNAs, in the context of functional snRNPs, are targeted to elongating pre-mRNAs. This finding presents us with a novel assay with which to dissect the mechanism by which snRNPs are targeted to nascent pre-mRNA transcripts. Two critical advantages offered by this system are immediately evident. First, it allows us to investigate the mechanisms employed to recruit snRNPs as it actually transpires within the realm of the cell nucleus. Second, it allows a genome-wide analysis of snRNP recruitment to nascent transcripts, and, hence, the conclusions drawn from these studies do not depend on the sequence of any particular promoter or pre-mRNA. Indeed, it is with this assay that we have stumbled upon a most unanticipated discovery: Contrary to the current paradigm, the co-transcriptional recruitment of splicing snRNPs to nascent transcripts is not contingent on their role in splicing in vivo. Based on these and other data, we have constructed a two-step recruitment-loading model wherein snRNPs are first recruited to pre-mRNA transcripts and only then loaded directly onto cis-acting sequences on nascent pre-mRNA. While conducting studies on snRNP trafficking, a new discovery was made. We found that the lampbrush chromosomes could be visualized by light microscopy in vivo, and that these chromosomes have an architecture that is identical with those in formaldehyde treated nuclear spread preparations. Importantly, we now have the first system with which we can examine the dynamic interactions of macromolecules with specific RNA polymerase II transcriptional units in the live nucleus.

Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91-phox and p22-phox in Endothelial Cells

The production of reactive oxygen species (ROS) within endothelial cells may have several effects, including alterations in the activity of paracrine factors, gene expression, apoptosis, and cellular injury. Recent studies indicate that a phagocyte-type NAD(P)H oxidase is a major source of endothelial ROS. In contrast to the high-output phagocytic oxidase, the endothelial enzyme has much lower biochemical activity and a different substrate specificity (NADH.NADPH). In the present study, we (1) cloned and characterized the cDNA and predicted amino acid structures of the 2 major subunits of rat coronary microvascular endothelial cell NAD(P)H oxidase, gp91-phox and p22-phox; (2) undertook a detailed comparison with phagocytic NADPH oxidase sequences; and (3) studied the subcellular location of these subunits in endothelial cells. Although these studies revealed an overall high degree of homology (.90%) between the endothelial and phagocytic oxidase subunits, the endothelial gp91-phox sequence has potentially important differences in a putative NADPH-binding domain and in putative glycosylation sites. In addition, the subcellular location of the endothelial gp91-phox and p22-phox subunits is significantly different from that reported for the neutrophil oxidase, in that they are predominantly intracellular and collocated in the vicinity of the endoplasmic reticulum. This first detailed characterization of gp91-phox and p22-phox structure and location in endothelial cells provides new data that may account, in part, for the differences in function between the phagocytic and endothelial NAD(P)H oxidases.

Exploring the Function and Regulation of Arabidopsis EIN2 in Ethylene Signaling

Ethylene is an essential plant hormone involved in nearly all stages of plant growth and development. EIN2 (ETHYLENE INSENSITIVE2) is a master positive regulator in the ethylene signaling pathway, consisting of an N-terminal domain and a C-terminal domain. The EIN2 N-terminal domain localizes to the endoplasmic reticulum (ER) membrane and shows sequence similarity to Nramp metal ion transporters. The cytosolic C-terminal domain is unique to plants and signals downstream. There have been several major gaps in our knowledge of EIN2 function. It was unknown how the ethylene signal gets relayed from the known upstream component CTR1 (CONSTITUTIVE RESPONSE1) a Ser/Thr kinase at the ER, to EIN2. How the ethylene signal was transduced from EIN2 to the next downstream component transcription factor EIN3 (ETHYLENE INSENSITIVE3) in the nucleus was also unknown. The N-terminal domain of EIN2 shows homology to Nramp metal ion transporters and whether EIN2 can also function as a metal transporter has been a question plaguing the ethylene field for almost two decades. Here, EIN2 was found to interact with the CTR1 protein kinase, leading to the discovery that CTR1 phosphorylates the C-terminal domain of EIN2 in Arabidopsis thaliana. Using tags at the termini of EIN2, it was deduced that in the presence of ethylene, the EIN2 C-terminal domain is cleaved and translocates into the nucleus, where it could somehow activate downstream ethylene responses. The EIN2 C-terminal domain interacts with nuclear proteins, RTE3 and EER5, which are components of the TREX-2 mRNA export complex, although the role of these interactions remains unclear. The EIN2 N-terminal domain was found to be capable of divalent metal transport when expressed in E. coli and S. cerevisiae leading to the hypothesis that metal transport plays a role in ethylene signaling. This hypothesis was tested using a novel missense allele, ein2 G36E, substituting a highly conserved residue that is required for metal transport in Nramp proteins. This G36E substitution did not disrupt metal ion transport of EIN2, but the ethylene insensitive phenotype of this mutant indicates that the EIN2 N-terminal domain is important for positively regulating the C-terminal domain. The defect of the ein2 G36E mutant does not prevent proper expression or subcellular localization, but might affect protein modifications. The ein2 G36E allele is partially dominant, mostly likely displaying haploinsufficiency. Overexpression of the EIN2 N-terminal domain in the ein2 G36E mutant did not rescue ethylene insensitivity, suggesting the N-terminal domain functions in cis to regulate the C-terminal domain. These findings advance our knowledge of EIN2, which is critical to understanding ethylene signaling.

NEW DATA ON THE CHRONOLOGY OF THE VALE DO FORNO SEDIMENTARY SEQUENCE (LOWER TAGUS RIVER TERRACE STAIRCASE) AND ITS RELEVANCE AS FLUVIAL ARCHIVE OF THE MIDDLE PLEISTOCENE IN WESTERN IBERIA

NEW DATA ON THE CHRONOLOGY OF THE VALE DO FORNO SEDIMENTARY SEQUENCE (LOWER TAGUS RIVER TERRACE STAIRCASE) AND ITS RELEVANCE AS FLUVIAL ARCHIVE OF THE MIDDLE PLEISTOCENE IN WESTERN IBERIA Pedro P. Cunha 1, António A. Martins 2, Jan-Pieter Buylaert 3,4, Andrew S. Murray 4, Luis Raposo 5, Paolo Mozzi 6, Martin Stokes 7 1 MARE - Marine and Environmental Sciences Centre, Department of Earth Sciences, University of Coimbra, Portugal: pcunha@dct.uc.pt 2 MARE - Marine and Environmental Sciences Centre, Dep. Geociências, University of Évora, Portugal; aam@uevora.pt 3 Centre for Nuclear Technologies, Technical University of Denmark, Risø Campus, Denmark; jabu@dtu.dk 4 Nordic Laboratory for Luminescence Dating, Aarhus University, Risø DTU, Denmark; anmu@dtu.dk 5 Museu Nacional de Arqueologia, Lisboa, Portugal; 3raposos@sapo.pt 6 Department of Geosciences, University of Padova, Italy; paolo.mozzi@unipd.it 7 School of Geography, Earth and Environmental Sciences, University of Plymouth, UK; m.stokes@plymouth.ac.uk The stratigraphic units that record the evolution of the Tagus River in Portugal (study area between Vila Velha de Ródão and Porto Alto villages; Fig. 1) have different sedimentary characteristics and lithic industries (Cunha et al., 2012): - a culminant sedimentary unit (the ancestral Tagus, before the drainage network entrenchment) – SLD13 (+142 to 262 m above river bed – a.r.b.; with probable age ca. 3,6 to 1,8 Ma), without artefacts; - T1 terrace (+84 to 180 m; ca. 1000? to 900 ka), without artefacts; - T2 terrace (+57 to 150 m; top deposits with a probable age ca. 600 ka), without artefacts; - T3 terrace (+43 to 113 m; ca. 460 to 360? ka), without artefacts; - T4 terrace (+26 to 55 m; ca. 335 a 155 ka), Lower Paleolithic (Acheulian) at basal and middle levels but early Middle Paleolithic at top levels; - T5 terrace (+5 to 34 m; 135 to 73 ka), Middle Paleolithic (Mousterian; Levallois technique); - T6 terrace (+3 to 14 m; 62 to 32 ka), late Middle Paleolithic (late Mousterian); - Carregueira Sands (aeolian sands) and colluvium (+3 a ca. 100 m; 32 to 12 ka), Upper Paleolithic to Epipaleolithic; - alluvial plain (+0 to 8 m; ca. 12 ka to present), Mesolithic and more recent industries. The differences in elevation (a.r.b.) of the several terrace staircases results from differential uplift due to active faults. Longitudinal correlation with the terrace levels indicates that a graded profile ca. 200 km long was achieved during terrace formation periods and a strong control by sea base level was determinant for terrace formation. The Neogene sedimentary units constituted the main source of sediments for the fluvial terraces (Fig. 2). Geomorphological mapping, coupled with lithostratigraphy, sedimentology and luminescence dating (quartz-OSL and K-feldspar post-IRIR290) were used in this study focused on the T4 terrace, which comprises a Lower Gravels (LG) unit and an Upper Sand (US) unit. The thick, coarse and dominantly massive gravels of the LG unit indicate deposition by a coarse bed-load braided river, with strong sediment supply, high gradient and fluvial competence, during conditions of rapidly rising sea level. Luminescence dating only provided minimum ages but it is probable that the LG unit corresponds to the earlier part of the MIS9 (ca. 335 to 325 ka), immediately postdating the incision promoted by the very low sea level (reaching ca. -140 m) during MIS10 (362 to 337 ka), a period of relatively cold climate conditions with weak vegetation cover on slopes and low sea level. Fig. 1. Main Portuguese reaches in which the Tagus River can be divided (Lower Tagus Basin): I – from the Spanish border to Arneiro (a general E–W trend, mainly consisting of polygonal segments); II – from Arneiro to Gavião (NE–SW); III – from Gavião to Arripiado (E–W); IV – from Arripiado to Vila Franca de Xira (NNE-SSW); V – from Vila Franca de Xira to the Atlantic shoreline. The faults considered to be the limit of the referred fluvial sectors are: F1 – Ponsul-Arneiro fault (WSW-ENE); F2 – Gavião fault (NW-SE); F3 – Ortiga fault (NW-SE); F4 – Vila Nova da Barquinha fault (W-E); F5 – Arripiado-Chamusca fault (NNE-SSW). 1 – estuary; 2 – terraces; 3 – faults; 4 – Tagus main channel. The main Iberian drainage basins are also represented (inset). The lower and middle parts of the US unit, comprising an alternation of clayish silts with paleosols and minor sands to the east (flood-plain deposits) and sand deposits to the west (channel belt), have a probable age of ca. 325 to 200 ka. This points to formation during MIS9 to MIS7, under conditions of high to medium sea levels and warm to mild conditions. The upper part of the US unit, dominated by sand facies and with OSL ages of ca. 200 to 154 ka, correlates with the early part of the MIS6. During this period, progradation resulted from climate deterioration and relative depletion of vegetation that promoted enhanced sediment production in the catchment, coupled with initiation of sea-level lowering that increased the longitudinal slope. The Vale do Forno and Vale da Atela archaeological sites (Alpiarça, central Portugal) document the earliest human occupation in the Lower Tagus River, well established in geomorphological and environmental terms, within the Middle Pleistocene. The Lower Palaeolithic sites were found on the T4 terrace (+26 m, a.r.b.). The oldest artefacts previously found in the LG unit, display crude bifacial forms that can be attributed to the Acheulian, with a probable age of ca. 335 to 325 ka. The T4 US unit has archaeological sites stratigraphically documenting successive phases of an evolved Acheulian, that probably date ca. 325 to 300 ka. Notably, these Lower Palaeolithic artisans were able to produce tools with different sophistication levels, simply by applying different strategies: more elaborated reduction sequences in case of bifaces and simple reduction sequences to obtain cleavers. Fig. 2. . Simplified geologic map of the Lower Tagus Cenozoic basin, adapted from the Carta Geológica de Portugal, 1/500000, 1992). The study area (comprising the Vale do Forno and Vale de Atela sites) is located on the more upstream sector of the Lower Tagus River reach IV, between Arripiado and Chamusca villages. 1 – alluvium (Holocene); 2 – terraces (Pleistocene); 3 – sands, silts and gravels (Paleogene to Pliocene); 4 – Sintra Massif (Cretaceous); 5 – limestones, marls, silts and sandstones (Mesozoic); 6 – quartzites (Ordovician); 7 – basement (Proterozoic to Palaeozoic); 8 – main fault. The main Portuguese reaches of the Tagus River are identified (I to V). The VF3 site (Milharós), containing a Final Acheulian industry, with fine and elaborated bifaces) found in a stratigraphic level located between the T4 terrace deposits and a colluvium associated with Late Pleistocene aeolian sands (32 to 12 ka), has an age younger than ca. 154 ka but much older than 32 ka. In the study area, the sedimentary units of the T4 terrace seem to record the river response to sea-level changes and climatically-driven fluctuations in sediment supply. REFERENCES Cunha P. P., Almeida N. A. C., Aubry T., Martins A. A., Murray A. S., Buylaert J.-P., Sohbati R., Raposo L., Rocha L., 2012, Records of human occupation from Pleistocene river terrace and aeolian sediments in the Arneiro depression (Lower Tejo River, central eastern Portugal). Geomorphology, vol. 165-166, pp. 78-90.