Succinate:ubiquinone oxidoreductase from Paracoccus denitrificans and particulate methane monooxygenase from Methylococcus capsulatus (Bath): experimental and theoretical EPR studies of the metal cofactors

This thesis summarizes the application of conventional and modern electron paramagnetic resonance (EPR) techniques to establish proximity relationships between paramagnetic metal centers in metalloproteins and between metal centers and magnetic ligand nuclei in two important and timely membrane proteins: succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans and particulate methane monooxygenase (pMMO) from Methylococcus capsulatus. Such proximity relationships are thought to be critical to the biological function and the associated biochemistry mediated by the metal centers in these proteins. A mechanistic understanding of biological function relies heavily on structure-function relationships and the knowledge of how molecular structure and electronic properties of the metal centers influence the reactivity in metalloenzymes. EPR spectroscopy has proven to be one of the most powerful techniques towards obtaining information about interactions between metal centers as well as defining ligand structures. SQR is an electron transport enzyme wherein the substrates, organic and metallic cofactors are held relatively far apart. Here, the proximity relationships of the metallic cofactors were studied through their weak spin-spin interactions by means of EPR power saturation and electron spin-lattice (T_1) measurements, when the enzyme was poised at designated reduction levels. Analysis of the electron T_1 measurements for the S-3 center when the b-heme is paramagnetic led to a detailed analysis of the dipolar interactions and distance determination between two interacting metal centers. Studies of ligand environment of the metal centers by electron spin echo envelope modulation (ESEEM) spectroscopy resulted in the identication of peptide nitrogens as coupled nuclei in the environment of the S-1 and S-3 centers.

Finally, an EPR model was developed to describe the ferromagnetically coupled trinuclear copper clusters in pMMO when the enzyme is oxidized. The Cu(II) ions in these clusters appear to be strongly exchange coupled, and the EPR is consistent with equilateral triangular arrangements of type 2 copper ions. These results offer the first glimpse of the magneto-structural correlations for a trinuclear copper cluster of this type, which, until the work on pMMO, has had no precedent in the metalloprotein literature. Such trinuclear copper clusters are even rare in synthetic models.

Mechanistic studies of reactions at the single-molecule level using microfluidics with applications in molecular diagnostics

Motivated by needs in molecular diagnostics and advances in microfabrication, researchers started to seek help from microfluidic technology, as it provides approaches to achieve high throughput, high sensitivity, and high resolution. One strategy applied in microfluidics to fulfill such requirements is to convert continuous analog signal into digitalized signal. One most commonly used example for this conversion is digital PCR, where by counting the number of reacted compartments (triggered by the presence of the target entity) out of the total number of compartments, one could use Poisson statistics to calculate the amount of input target.

However, there are still problems to be solved and assumptions to be validated before the technology is widely employed. In this dissertation, the digital quantification strategy has been examined from two angles: efficiency and robustness. The former is a critical factor for ensuring the accuracy of absolute quantification methods, and the latter is the premise for such technology to be practically implemented in diagnosis beyond the laboratory. The two angles are further framed into a “fate” and “rate” determination scheme, where the influence of different parameters is attributed to fate determination step or rate determination step. In this discussion, microfluidic platforms have been used to understand reaction mechanism at single molecule level. Although the discussion raises more challenges for digital assay development, it brings the problem to the attention of the scientific community for the first time.

This dissertation also contributes towards developing POC test in limited resource settings. On one hand, it adds ease of access to the tests by incorporating massively producible, low cost plastic material and by integrating new features that allow instant result acquisition and result feedback. On the other hand, it explores new isothermal chemistry and new strategies to address important global health concerns such as cyctatin C quantification, HIV/HCV detection and treatment monitoring as well as HCV genotyping.

Functional genomic studies of the structure and regulation of eukaryotic transcriptomes

The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.

The first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.

The second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.

The third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.

The fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).

The last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.

Temporary ponds in the UK; a critical biodiversity resource for freshwater plants and animals

The importance of ponds for biodiversity in Britain has been demonstrated by a number of studies. However, most of the research and interest has been directed at permanent waterbodies, and temporary ponds have been largely neglected. In this article the author present some preliminary findings from a project which aims to fill some of the many gaps in our knowledge of temporary ponds in Britain. The project, which runs for three years until the end of 2001, aims specifically to investigate the ecology of temporary ponds in England and Wales by describing (i) their wetland plant and macroinvertebrate communities, (ii) their physico-chemical characteristics, and (iii) their value as a biodiversity resource. The article focuses on the assessment of temporary ponds as a biodiversity resource and briefly considers aspects of species richness, rarity and distinctiveness. Where possible, temporary ponds are compared with other waterbody types, mainly permanent ponds from the National Pond Survey (NPS), to give the results a broader context.

Experimental studies of weak superconductivity

Experimental investigations were made of the nature of weak superconductivity in a structure having well-defined, controllable characteristics and geometry. Controlled experiments were made possible by using a thin-film structure which was entirely metallic and consisted of a superconducting film with a localized section that was weak in the sense that its transition temperature was depressed relative to the rest of the film. The depression of transition temperature was brought about by underlaying the superconductor with a normal metal.

The DC and AC electrical characteristics of this structure were studied. It was found that this structure exhibited a non-zero, time-average supercurrent at finite voltage to at least .2 mV, and generated an oscillating electric potential at a frequency given by the Josephson relation. The DC V-I characteristic and the amplitude of the AC oscillation were found to be consistent with a two- fluid (normal current-supercurrent) model of weak super-conductivity based on e thermodynamically irreversible process of repetitive phase-slip, and featuring a periodic time dependence in the amplitude of the superconducting order parameter.

The observed linewidth of the AC oscillation could be accounted for by incorporating Johnson noise in the two-fluid model.

Experimentally it was found that the behavior of a short (length on the order of the coherence distance) weak superconductor could be characterized by its critical current and normal-state resistance, and an empirical expression was obtained for the time dependence of the super-current and voltage.

It was found that the results could not be explained on the basis of the theory of the Josephson junction.

Gerando eus, tecendo redes e trançando nós: ditos e não ditos das professoras e estudantes negras nos cotidianos do curso de pedagogia

O presente trabalho objetiva compreender como a temática afrobrasileira era abordada nos cotidianos do Curso de Pedagogia, campus I, da Universidade Estadual da Paraíba. A questão norteadora da pesquisa foi descobrir como professoras e estudantes negras se sentem e enfocam essa temática nas suas redes de conhecimentos, práticas e relações. Para tanto, foi preciso adentrar na história do povo negro no Brasil a partir da luta dos movimentos sociais negros buscando entender as noções de raça, racismo, identidade e os limites da educação do/a negro/a ao longo da história brasileira. Neste percurso se dialoga com diversos estudiosos/as como: Santos (2002), Viana (2007), Oliveira (2003, 2006, 2007, 2008), Fernandes (2007), Domingues (2009), Munanga (2002, 2006, 2009), Veiga (2007, 2008)), Pereira (2008, 2009), Gomes (2003, 2006, 2008), Morin (2000), Hall (2003), Moreira e Câmara (2008), dentre outros. Exemplificando a luta e a resistência de mulheres negras são apresentadas as histórias de vida de quatro professoras e quatro estudantes negras, praticantes (CERTEAU, 2007), sujeitos da pesquisa, que expõem o processo de tessitura de suas identidades. Suas narrativas evidenciam indícios de racismo e discriminação que marcaram suas trajetórias, desde o seio familiar, fortalecendo-se na escola, na academia e, para algumas, chegando até o ambiente de trabalho. Uma das professoras é a pesquisadora que, à medida que narra a história de vida das praticantes, tece sua própria história, como mulheres costurando uma colcha de retalhos em que cada estampa retrata os momentos vividos. A metodologia de pesquisa nos/dos/com os cotidianos (ALVES, 2008) foi o caminho percorrido. Além da observação dos cotidianos, foram realizadas conversas (LARROSA, 2003) sobre as histórias de vida (BOSI, 2003; PORTELLI, 1997) e tecidas as narrativas a partir dessas vivências. Estas revelaram que tais mulheres, ao longo de suas histórias, viveram/vivem processos de afirmaçãonegação, visto que suas identidades não são fixas, mas negociações e renegociações (MUNANGA, 2010) que geram alegrias e conflitos. O entendimento do que é Ser Negra é experimentado por cada uma, sem um modelo padrão para suas existências. Elas foram se gerando nas relações com o/a outro/a, em cada contexto familiar, escolar, acadêmico e profissional. São senhoras de suas vidas e mesmo as mais jovens buscam fazer suas histórias com autonomia. São praticantes porque lutaram/lutam para conquistar um lugar na vida, utilizando astúcias e táticas para fabricar (CERTEAU, 2007) meios de enfrentamento das adversidades. As táticas do silêncio, do estudo e do trabalho revelaram que essas mulheres não ficaram invisíveis nem se colocaram como vítimas no espaço social. Assim, urge delinear a superação da visão da pessoa negra a partir dos traços físicos e reconhecer as raízes do povo brasileiro para compreender a história da negritude. A partir da ancestralidade, serão identificadas as origens do povo negro, que poderão trazer o passado de resistência e luta por liberdade, dignidade, cidadania que produzem o orgulho de ser negra. Orgulho que recupera a autoestima e a capacidade de organização e mobilização para combater o racismo e as desigualdades raciais. Os cursos de formação docente precisam abordar essa temática para capacitar os/as futuros/as professores/as nessa tarefa. Essa abordagem envolve o ensinaraprender, em que professores/as e estudantes assumem compromisso com uma educação crítica e inclusiva. A reflexão entre os/as docentes formadores/as nessa perspectiva plural e intercultural poderia ajudar na superação do eurocentrismo ainda presente nos conteúdos e nas mentalidades. Sob essa ótica, tecer um processo de formação docente no Curso de Pedagogia, comprometido com uma educação inclusiva, considera um contínuo fazerdesfazer, na tessitura de um diálogo permanente entre a práticateoriaprática, num movimento de pesquisaintervenção. Isso implica a reflexão da trajetória pessoal e coletiva e a articulação da ação pedagógica com um projeto político de transformação da sociedade excludente em sociedade plural e solidária. Os não ditos necessitam de aprofundamento em trabalhos posteriores, pois os silenciamentos se referem ao passado próximo e as relações interraciais na família, na academia e também nos ambientes de trabalho. Será que não existe mais racismo? Ou este foi naturalizado dificultando sua identificação nos cotidianos? Os limites do processo de introjeção do racismo, que provocam a invisibilidade de algumas praticantes, são novos desafios para estudos futuros. Assim, percebo que colcha de retalhos tecida ao longo desta trajetória não é justaposição como usualmente é entendida. Para além disto, significa a socialização das experiências vividas nos cotidianos das praticantes, a fim de inspirar atitudes de combate ao racismo que se ampliem para o contexto social.

Sobre bruxos e raças: Economias de objeto-relação e os custos da representação ou, como ser um anti-anti-fetichista da diferença

Por que tantos estudos acadêmicos afirmam a equivalência conceitual entre raça e bruxaria? A dissertação investiga as raízes e conseqüências deste gesto. Procura-se enfocar a maneira em que os dois objetos viram objetos de pesquisa enquanto representações. Uma comparação entre os programas de pesquisa reputadamente opostoscognitivista e sociocultural serve para delinear os pressupostos e limites de tal abordagem. Lançando mão de uma perspectiva pragmatista e fenomenológica, a primeira parte do trabalho revela as dificuldades de um programa representacionalista em dar conta da instância concreta de identificação. Sugere-se que isto é resultado da sua ênfase excessiva na classificação. Fazendo uso das promessas de uma antropologia simétrica, a segunda parte re-examina a insistência da ciência social em `des-naturalizar os objetos através de uma exposição crítica da sua produção. Como uma alternativa ao anti-fetichismo da crítica social, a conclusão propõe uma maneira de tratar a alteridade capaz de oferecer novos objetos de saber e alterar nossa orientação visà-vis eles. O estudo de identidade poderia então ir além dos conceitos de reconhecimento, fronteiras, e exclusão.

Protocols for conducting dolphin capture-release health assessment studies

Marine mammals, such as dolphins, can serve as key indicator species in coastal areas by reflecting the effects of natural and anthropogenic stressors. As such they are often considered sentinels of environmental and ecosystem health (Bossart 2006; Wells et al. 2004; Fair and Becker 2000). The bottlenose dolphin is an apex predator and a key component of many estuarine environments in the southeastern United States (Woodward-Clyde Consultants 1994; SCDNR 2005). Health assessments of dolphins are especially critical in areas where populations are depleted, show signs of epidemic disease and/or high mortality and/or where habitat is being altered or impacted by human activities. Recent assessments of environmental conditions in the Indian River Lagoon, Florida (IRL) and the estuarine waters surrounding Charleston, South Carolina (CHS) highlight the need for studies of the health of local bottlenose dolphins. While the condition of southeastern estuaries was rated as fair in the National Coastal Condition Report (U.S. EPA 2001), it was noted that the IRL was characterized by poorer than expected benthic communities, significant sediment toxicity and increased nutrient concentrations. Similarly, portions of the CHS estuary have sediment concentrations of aliphatic aromatic hydrocarbons, select inorganic metals, and some persistent pesticides far in excess of reported bioeffect levels (Hyland et al. 1998). Long-term trends in water quality monitoring and recent scientific research suggest that waste load assimilation, non-point source runoff impacts, contaminated sediments, and toxic pollutants are key issues in the CHS estuary system. Several ‘hot spots’ with high levels of heavy metals and organic compounds have been identified (Van Dolah et al. 2004). High concentrations of anthropogenic trace metals, polychlorinated biphenyls (PCB’s) and pesticides have been found in the sediments of Charleston Harbor, as well as the Ashley and Cooper Rivers (Long et al. 1998). Two superfund sites are located within the CHS estuary and the key contaminants of concern associated with these sites are: polycyclic aromatic hydrocarbons (PAH), lead, chromium, copper, arsenic, zinc and dioxin. Concerns related to the overall health of IRL dolphins and dermatologic disease observed in many dolphins in the area (Bossart et al. 2003) initiated an investigation of potential factors which may have impacted dolphin health. From May-August 2001, 35 bottlenose dolphins died in the IRL during an unusual mortality event (MMC 2003). Many of these dolphins were diagnosed with a variety of skin lesions including proliferative ulcerative dermatitis due to protozoa and fungi, dolphin pox and a vesicular dermatopathy of unknown etiology (Bossart et al. 2003). Multiple species from fish to dolphins in the IRL system have exhibited skin lesions of various known and unknown etiologies (Kane et al. 2000; Bossart et al. 2003; Reif et al. 2006). On-going photo-identification (photo-ID) studies have documented skin diseases in IRL dolphins (Mazzoil et al. 2005). In addition, up to 70% of green sea turtles in the IRL exhibit fibropapillomas, with the highest rates of occurrence being seen in turtles from the southern IRL (Hirama 2001).

Do we know the strength of the chorioamnion? A critical review and analysis

The chorioamnion is the membrane that surrounds the fetus during gestation. Normally, it must remain intact for the duration of pregnancy, 37-42 weeks, and only rupture during or just before labour and delivery of the fetus. In a significant number (3%) of all births, this does not happen, and membranes rupture before term, resulting in preterm birth and significant perinatal morbidity. It is known that the material properties of chorioamnion may play a major role in mechanical rupture; a number of studies have been undertaken to characterise the physical nature of chorioamnion and examine factors that may predispose to rupture. However, the existing literature is inconsistent in its choice of both physical testing methods and data analysis techniques, motivating the current review. Experimental data from a large number of chorioamnion mechanical studies were collated, and data were converted to standard engineering quantities. The failure strength of the chorioamnion membrane was found consistently to value approximately 0.9 MPa. It is hoped that past and future studies of membrane mechanics can provide insight into the role of chorioamnion in labour and delivery. In addition, biomechanical approaches can help elucidate the potential causes of early rupture, and suggest future protocols or treatments that could both diagnose and prevent its occurrence. © 2009 Elsevier Ireland Ltd.

Studies on the transportation of live clams

Live clams (Villorita cyprinoides) collected from their natural beds were packed in different ways like dry pack, tray pack, in oxygenated water (wet pack) and depurated samples in wet pack. It was found that the packaging in l kg lots in 200 gauge polythene bags with oxygen at a temperature of 20°C could keep them live for 4 days. In tray pack without oxygen and water they can be kept alive for 3 days at 20°C. Temperature seems to be the critical factor in the transportation of live clams. At room temperature both dry and wet pack can be kept for 24 h only. Depuration technique does not appear to be useful in prolonging the storage life of clams in live condition as percentage mortality is more at 48 h both at 20°C and room temperature compared to the non-depurated samples.

Towards probabilistic performance metrics for climate change impact studies

This paper explores the current state-of-the-art in performance indicators and use of probabilistic approaches used in climate change impact studies. It presents a critical review of recent publications in this field, focussing on (1) metrics for energy use for heating and cooling, emissions, overheating and high-level performance aspects, and (2) uptake of uncertainty and risk analysis. This is followed by a case study, which is used to explore some of the contextual issues around the broader uptake of climate change impact studies in practice. The work concludes that probabilistic predictions of the impact of climate change are feasible, but only based on strict and explicitly stated assumptions. © 2011 Elsevier B.V. All rights reserved.

A critical reassessment of the role of mitochondria in tumorigenesis

Background Mitochondrial DNA (mtDNA) is being analyzed by an increasing number of laboratories in order to investigate its potential role as an active marker of tumorigenesis in various types of cancer. Here we question the conclusions drawn in most of these investigations, especially those published in high-rank cancer research journals, under the evidence that a significant number of these medical mtDNA studies are based on obviously flawed sequencing results. Methods and Findings In our analyses, we take a phylogenetic approach and employ thorough database searches, which together have proven successful for detecting erroneous sequences in the fields of human population genetics and forensics. Apart from conceptual problems concerning the interpretation of mtDNA variation in tumorigenesis, in most cases, blocks of seemingly somatic mutations clearly point to contamination or sample mix-up and, therefore, have nothing to do with tumorigenesis. Conclusion The role of mitochondria in tumorigenesis remains unclarified. Our findings of laboratory errors in many contributions would represent only the tip of the iceberg since most published studies do not provide the raw sequence data for inspection, thus hindering a posteriori evaluation of the results. There is no precedent for such a concatenation of errors and misconceptions affecting a whole subfield of medical research.

Wetlands and riparian zones as buffers and critical habitats for biotic communities in Lake Victoria

Despite their ecological and socio-economic importance, Lake Victoria's adjoining "swamps" and lake interface are among the least investigated parts of the lake. The "swamps" a term commonly equated to "wastelands" and the difficult working environment they present in comparison to open water, are major factors for the low level of attention accorded to shoreline wetlands. Moreover, definitions of wetlands highlighted for example in the Ramsar Convention as "areas of marsh, fern, peatland or water, whether natural or artificial, permanent or temporary, with water that is static or flowing, fresh or brackish, or salt, including areas of marine water, the depth of which does not exceed six metres" (Ramsar, 1971) were designed to protect birds (water fowl) of international importance. The Ramsar definition, which also includes oceans, has till recently been of limited use for Lake Victoria, because itdoes not fully recognise wetlands in relation to other public concerns such as water quality, biodiversity and the tisheries that are of higher socioeconomic priority than waterfowl. Prior to 1992, fishery research on Lake Victoria included studies of inshore shallow habitats of the lake without specific reference to distance or the type of vegetation at the shore. Results of these studies also conveniently relied heavily on trawl and gill net data from the 5-10 m depth zones as the defining boundary of shallow inshore habitats. In Lake Victoria, such a depth range can be at least one kilometre from the lake interface and by the 10m depth contour, habitats are in the sub-littoral range. Findings from these studies could thus not be used to make direct inferences on the then assumed importance of Lake Victoria wetlands in general.

Simulation studies on the magnetization of (RE)BCO bulk superconductors using various split-coil arrangements

Simulation studies were conducted on the magnetization of (RE)BCO (RE-Ba-Cu-O, where RE represents a rare earth element) bulk superconductors using various split-coil arrangements by solving the critical state equation using the commercial software FlexPDE. A pair of coaxial coils of identical size is identified as an optimum arrangement for practical magnetization at 77K by the zero-field cooling technique. In general, the magnetization process is likely to be most effective when the outer radius of the coils lies between 100% and 50% of the sample radius. A relatively large coil pair is necessary for samples with either a smaller aspect ratio or larger values of J c0. Two different regimes of flux penetration are found to be involved in the magnetization process. For a sufficiently small sample, the penetration field is determined by flux propagation from beneath the coil to the centre of the sample; for a sufficiently large sample, the definitive propagation route is from beneath the coil to the periphery of the sample. Effective split-coil magnetization occurs only in the former regime, and both penetration regimes are completely different from that involved in the solenoidal-coil magnetization process. © 2012 IOP Publishing Ltd.

Theoretical and experimental studies on Jc and AC losses of 2G HTS coils

This paper begins with introducing the winding techniques of two superconducting double-pancake coils wound using 2G coated conductors. These winding techniques are able to guarantee a good performance for the superconducting coils. Then the coil critical currents were measured and compared with a simulation model. The results were consistent. Finally the coil AC losses were measured using an experimental circuit including a compensation coil. The simulation results are close to the experiment results. © 2010 IEEE.