Biochemical and structural characterization of residue 96 mutants of Plasmodium falciparum triosephosphate isomerase: active-site loop conformation, hydration and identification of a dimer-interface ligand-binding site

Plasmodium falciparum TIM (PfTIM) is unique in possessing a Phe residue at position 96 in place of the conserved Ser that is found in TIMs from the majority of other organisms. In order to probe the role of residue 96, three PfTIM mutants, F96S, F96H and F96W, have been biochemically and structurally characterized. The three mutants exhibited reduced catalytic efficiency and a decrease in substrate-binding affinity, with the most pronounced effects being observed for F96S and F96H. The k(cat) values and K-m values are (2.54 +/- 0.19) x 10(5) min(-1) and 0.39 +/- 0.049 mM, respectively, for the wild type; (3.72 +/- 0.28) x 10(3) min(-1) and 2.18 +/- 0.028 mM, respectively, for the F96S mutant;(1.11 +/- 0.03) x 10(4) min(-1) and 2.62 +/- 0.042 mM, respectively, for the F96H mutant; and (1.48 +/- 0.05) x 10(5) min(-1) and 1.20 +/- 0.056 mM, respectively, for the F96W mutant. Unliganded and 3-phosphoglycerate (3PG) complexed structures are reported for the wild-type enzyme and the mutants. The ligand binds to the active sites of the wild-type enzyme (wtPfTIM) and the F96W mutant, with a loop-open state in the former and both open and closed states in the latter. In contrast, no density for the ligand could be detected at the active sites of the F96S and F96H mutants under identical conditions. The decrease in ligand affinity could be a consequence of differences in the water network connecting residue 96 to Ser73 in the vicinity of the active site. Soaking of crystals of wtPfTIM and the F96S and F96H mutants resulted in the binding of 3PG at a dimer-interface site. In addition, loop closure at the liganded active site was observed for wtPfTIM. The dimer-interface site in PfTIM shows strong electrostatic anchoring of the phosphate group involving the Arg98 and Lys112 residues of PfTIM.

Association between ORMDL3, IL1RL1 and a deletion on chromosome 17q21 with asthma risk in Australia

Genome-wide association studies followed by replication provide a powerful approach to map genetic risk factors for asthma. We sought to search for new variants associated with asthma and attempt to replicate the association with four loci reported previously (ORMDL3, PDE4D, DENND1B and IL1RL1). Genome-wide association analyses of individual single nucleotide polymorphisms (SNPs), rare copy number variants (CNVs) and overall CNV burden were carried out in 986 asthma cases and 1846 asthma-free controls from Australia. The most-associated locus in the SNP analysis was ORMDL3 (rs6503525, P = 4.8 x 10(-)(7)). Five other loci were associated with P < 10(-)(5), most notably the chemokine CXC motif ligand 14 (CXCL14) gene (rs31263, P = 7.8 x 10(-)(6)). We found no evidence for association with the specific risk variants reported recently for PDE4D, DENND1B and ILR1L1. However, a variant in IL1RL1 that is in low linkage disequilibrium with that reported previously was associated with asthma risk after accounting for all variants tested (rs10197862, gene wide P = 0.01). This association replicated convincingly in an independent cohort (P = 2.4 x 10(-)(4)). A 300-kb deletion on chromosome 17q21 was associated with asthma risk, but this did not reach experiment-wide significance. Asthma cases and controls had comparable CNV rates, length and number of genes affected by deletions or duplications. In conclusion, we confirm the association between asthma risk and variants in ORMDL3 and identify a novel risk variant in IL1RL1. Follow-up of the 17q21 deletion in larger cohorts is warranted.

Molecular Pathways of Disturbed Sleep and Depression: Studies on Adenosine and Gene Expression Patterns

Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.

Water and thermal regimes for field pea in Australia and their implications for breeding.

There is a large gap between the refined approaches to characterise genotypes and the common use of location and season as a coarse surrogate for environmental characterisation of breeding trials. As a framework for breeding, the aim of this paper is quantifying the spatial and temporal patterns of thermal and water stress for field pea in Australia. We compiled a dataset for yield of the cv. Kaspa measured in 185 environments, and investigated the associations between yield and seasonal patterns of actual temperature and modelled water stress. Correlations between yield and temperature indicated two distinct stages. In the first stage, during crop establishment and canopy expansion before flowering, yield was positively associated with minimum temperature. Mean minimum temperature below similar to 7 degrees C suggests that crops were under suboptimal temperature for both canopy expansion and radiation-use efficiency during a significant part of this early growth period. In the second stage, during critical reproductive phases, grain yield was negatively associated with maximum temperature over 25 degrees C. Correlations between yield and modelled water supply/demand ratio showed a consistent pattern with three phases: no correlation at early stages of the growth cycle, a progressive increase in the association that peaked as the crop approached the flowering window, and a progressive decline at later reproductive stages. Using long-term weather records (1957-2010) and modelled water stress for 104 locations, we identified three major patterns of water deficit nation wide. Environment type 1 (ET1) represents the most favourable condition, with no stress during most of the pre-flowering phase and gradual development of mild stress after flowering. Type 2 is characterised by increasing water deficit between 400 degree-days before flowering and 200 degree-days after flowering and rainfall that relieves stress late in the season. Type 3 represents the more stressful condition with increasing water deficit between 400 degree-days before flowering and maturity. Across Australia, the frequency of occurrence was 24% for ET1, 32% for ET2 and 43% for ET3, highlighting the dominance of the most stressful condition. Actual yield averaged 2.2 t/ha for ET1, 1.9 t/ha for ET2 and 1.4 t/ha for ET3, and the frequency of each pattern varied substantially among locations. Shifting from a nominal (i.e. location and season) to a quantitative (i.e. stress type) characterisation of environments could help improving breeding efficiency of field pea in Australia.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Using a structural approach to identify relationships between soil and erosion in a semi-humid forested area, South India

Biogeochemical and hydrological cycles are currently studied on a small experimental forested watershed (4.5 km(2)) in the semi-humid South India. This paper presents one of the first data referring to the distribution and dynamics of a widespread red soil (Ferralsols and Chromic Luvisols) and black soil (Vertisols and Vertic intergrades) cover, and its possible relationship with the recent development of the erosion process. The soil map was established from the observation of isolated soil profiles and toposequences, and surveys of soil electromagnetic conductivity (EM31, Geonics Ltd), lithology and vegetation. The distribution of the different parts of the soil cover in relation to each other was used to establish the dynamics and chronological order of formation. Results indicate that both topography and lithology (gneiss and amphibolite) have influenced the distribution of the soils. At the downslope, the following parts of the soil covers were distinguished: i) red soil system, ii) black soil system, iii) bleached horizon at the top of the black soil and iv) bleached sandy saprolite at the base of the black soil. The red soil is currently transforming into black soil and the transformation front is moving upslope. In the bottom part of the slope, the chronology appears to be the following: black soil > bleached horizon at the top of the black soil > streambed > bleached horizon below the black soil. It appears that the development of the drainage network is a recent process, which was guided by the presence of thin black soil with a vertic horizon less than 2 in deep. Three distinctive types of erosional landforms have been identified: 1. rotational slips (Type 1); 2. a seepage erosion (Type 2) at the top of the black soil profile; 3. A combination of earthflow and sliding in the non-cohesive saprolite of the gneiss occurs at midslope (Type 3). Types 1 and 2 erosion are mainly occurring downslope and are always located at the intersection between the streambed and the red soil-black soil contact. Neutron probe monitoring, along an area vulnerable to erosion types 1 and 2, indicates that rotational slips are caused by a temporary watertable at the base of the black soil and within the sandy bleached saprolite, which behaves as a plane of weakness. The watertable is induced by the ephemeral watercourse. Erosion type 2 is caused by seepage of a perched watertable, which occurs after swelling and closing of the cracks of the vertic clay horizon and within a light textured and bleached horizon at the top of black soil. Type 3 erosion is not related to the red soil-black soil system but is caused by the seasonal seepage of saturated throughflow in the sandy saprolite of the gneiss occurring at midslope. (c) 2006 Elsevier B.V. All rights reserved.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.

Antithyroid drugs and their analogues: synthesis, structure, and mechanism of action

Thyroid hormones are essential for the development and differentiation of all cells of the human body. They regulate protein, fat, and carbohydrate metabolism. In this Account, we discuss the synthesis, structure, and mechanism of action of thyroid hormones and their analogues. The prohormone thyroxine (14) is synthesized on thyroglobulin by thyroid peroxidase (TPO), a heme enzyme that uses iodide and hydrogen peroxide to perform iodination and phenolic coupling reactions. The monodeiodination of T4 to 3,3',5-triiodothyronine (13) by selenium-containing deiodinases (ID-1, ID-2) is a key step in the activation of thyroid hormones. The type 3 deiodinase (ID-3) catalyzes the deactivation of thyroid hormone in a process that removes iodine selectively from the tyrosyl ring of T4 to produce 3,3',5'-triiodothyronine (rT3). Several physiological and pathological stimuli influence thyroid hormone synthesis. The overproduction of thyroid hormones leads to hyperthyroidism, which is treated by antithyroid drugs that either inhibit the thyroid hormone biosynthesis and/or decrease the conversion of T4 to T3. Antithyroid drugs are thiourea-based compounds, which indude propylthiouracil (PTU), methimazole (MM I), and carbimazole (CBZ). The thyroid gland actively concentrates these heterocyclic compounds against a concentration gradient Recently, the selenium analogues of PTU, MMI, and CBZ attracted significant attention because the selenium moiety in these compounds has a higher nucleophilicity than that of the sulfur moiety. Researchers have developed new methods for the synthesis of the selenium compounds. Several experimental and theoretical investigations revealed that the selone (C=Se) in the selenium analogues is more polarized than the thione (C=S) in the sulfur compounds, and the selones exist predominantly in their zwitterionic forms. Although the thionamide-based antithyroid drugs have been used for almost 70 years, the mechanism of their action is not completely understood. Most investigations have revealed that MMI and PTU irreversibly inhibit TPO. PTU, MTU, and their selenium analogues also inhibit ID-1, most likely by reacting with the selenenyl iodide intermediate. The good ID-1 inhibitory activity of Pill and its analogues can be ascribed to the presence of the -N(H)-C(=O)- functionality that can form hydrogen bonds with nearby amino add residues in the selenenyl sulfide state. In addition to the TPO and ID-1 inhibition, the selenium analogues are very good antioxidants. In the presence of cellular reducing agents such as GSH, these compounds catalytically reduce hydrogen peroxide. They can also efficiently scavenge peroxynitrite, a potent biological oxidant and nitrating agent.

Technologies for Measuring Currents in Coastal Environments, Portland, Maine, October 26-28, 2005:workshop proceedings

The Alliance for Coastal Technologies (ACT) Workshop entitled "Technologies for Measuring Currents in Coastal Environments" was held in Portland, Maine, October 26-28, 2005, with sponsorship by the Gulf of Maine Ocean Observing System (GoMOOS), an ACT partner organization. The primary goals of the event were to summarize recent trends in nearshore research and management applications for current meter technologies, identify how current meters can assist coastal managers to fulfill their regulatory and management objectives, and to recommend actions to overcome barriers to use of the technologies. The workshop was attended by 25 participants representing state and federal environmental management agencies, manufacturers of current meter technologies, and researchers from academic institutions and private industry. Common themes that were discussed during the workshop included 1) advantages and limitations of existing current measuring equipment, 2) reliability and ease of use with each instrument type, 3) data decoding and interpretation procedures, and 4) mechanisms to facilitate better training and guidance to a broad user group. Seven key recommendations, which were ranked in order of importance during the last day of the workshop are listed below. 1. Forums should be developed to facilitate the exchange of information among users and industry: a) On-line forums that not only provide information on specific instruments and technologies, but also provide an avenue for the exchange of user experiences with various instruments (i.e. problems encountered, cautions, tips, advantages, etc). (see References for manufacturer websites with links to application and technical forums at end of report) b) Regional training/meetings for operational managers to exchange ideas on methods for measuring currents and evaluating data. c) Organize mini-meetings or tutorial sessions within larger conference venues. 2. A committee of major stakeholders should be convened to develop common standards (similar to the Institute of Electrical and Electronics Engineers (IEEE) committee) that enable users to switch sensors without losing software or display capabilities. (pdf contains 28 pages)

Development of Metalloenzyme Dioxygen Reduction Cathodes

The prime thrust of this dissertation is to advance the development of fuel cell dioxygen reduction cathodes that employ some variant of multicopper oxidase enzymes as the catalyst. The low earth-abundance of platinum metal and its correspondingly high market cost has prompted a general search amongst chemists and materials scientists for reasonable alternatives to this metal for facilitating catalytic dioxygen reduction chemistry. The multicopper oxidases (MCOs), which constitute a class of enzyme that naturally catalyze the reaction O₂ + 4H⁺ + 4e^- → 2H₂O, provide a promising set of biochemical contenders for fuel cell cathode catalysts. In MCOs, a substrate reduces a copper atom at the type 1 site, where charge is then transferred to a trinuclear copper cluster consisting of a mononuclear type 2 or “normal copper” site and a binuclear type 3 copper site. Following the reduction of all four copper atoms in the enzyme, dioxygen is then reduced to water in two two-electron steps, upon binding to the trinuclear copper cluster. We identified an MCO, a laccase from the hyperthermophilic bacterium Thermus thermophilus strain HB27, as a promising candidate for cathodic fuel cell catalysis. This protein demonstrates resilience at high temperatures, exhibiting no denaturing transition at temperatures high as 95°C, conditions relevant to typical polymer electrolyte fuel cell operation.

In Chapter I of this thesis, we discuss initial efforts to physically characterize the enzyme when operating as a heterogeneous cathode catalyst. Following this, in Chapter II we then outline the development of a model capable of describing the observed electrochemical behavior of this enzyme when operating on porous carbon electrodes. Developing a rigorous mathematical framework with which to describe this system had the potential to improve our understanding of MCO electrokinetics, while also providing a level of predictive power that might guide any future efforts to fabricate MCO cathodes with optimized electrochemical performance. In Chapter III we detail efforts to reduce electrode overpotentials through site-directed mutagenesis of the inner and outer-sphere ligands of the Cu sites in laccase, using electrochemical methods and electronic spectroscopy to try and understand the resultant behavior of our mutant constructs. Finally, in Chapter IV, we examine future work concerning the fabrication of enhanced MCO cathodes, exploring the possibility of new cathode materials and advanced enzyme deposition techniques.

Cloning and analysis of laccases from acinetobacter baumannii isolates

Laccases (benzenediol : oxygen oxi doreductases; EC 1.10.3.2) are wide spread i n nature. They are usually found in higher plants and fungi (Thurston 19 94; Mayer and Staples 2002), but recently some bacterial laccases have also been found . The first laccase studied was from Rhus vernicifera in 1883, a Japanese lacquer tree, fr om which the name laccase was derived (Yoshida , 1883). These enzymes belong to the group of bl ue multi - copper oxidases (MCOs) . They usually contain four copper atoms located in three distinct sites. Each site reacts differently to light. The Type 1 (T1) site copper atom absorbs intensely at 600 nm and emits the blue light , the Type 2 (T2) site copper atom is not visible in the absorption spectr um and last, the Type 3 (T3) site has two c opper atoms and absorbs at 330 nm ( Santhanam et al . , 2011; Quintanar et al . , 2007 ) . The protei n structure acts as a complex ligand for the catalytic coppers, providing them the right structure where changes between the reduction states are thermodynamically possible (Dub é , 2008 ) . These enzymes oxidize a surprisingly wide variety of organic and inorganic compounds like, diphenols, polyphenols, substituted phenols, diamines and a romatic amines, with concomitant reduction of molecular oxygen to water (Thurston , 1

The control of indirect effects of biomanipulation

Results from long-term investigations on biomanipulation show that indirect effects are at least as important as direct effects are for the stability of biomanipulation. Three types of indirect effects can be distinguished: (1) a change in quantity or quality of the resource base, (2) behavioural change of the prey, and (3) development of anti-predator traits. Although indirect effects of type (2), (e.g. a change in the pattern of vertical migration of zooplankton), and type (3), (e.g. development of helmets and neck teeth in Daphnia), are important mechanisms, the most essential indirect effects regarding biomanipulation belong to type (1). An example of the latter will be demonstrated: the complex of indirect effects of enhanced grazing by large herbivores on the phosphorus metabolism of the lake. It is concluded that control of the indirect effects is absolutely necessary to stabilize biomanipulation measures, but this is much more difficult than the control of direct effects and needs deeper insights into the structuring mechanisms of food webs. Proper management of fish stocks, in combination with the control of phosphorus load and/or the physical conditions, seems to be the most promising way of controlling the indirect effects of biomanipulation.

Estudo estrutural e funcional das proteínas ligadoras de ácidos graxos (FABP- Fatty Acid Binding Proteins) de Fasciola hepatica

As proteínas ligadoras de ácidos graxos (Fatty Acid Binding Proteins, FABPs) de parasitos têm um papel importante no processo de infecção por estes organismos. Por este motivo, estas proteínas são antígenos candidatos para vacina contra a infecção por Schistosoma mansoni e Fasciola hepatica. No presente trabalho foram caracterizadas FABPs de F. hepatica e comparadas com a proteína Sm14 de S. mansoni, a FABP de parasito melhor caracterizada, mediante análise de sequências e estruturas modeladas. Também foram clonadas, expressas e purificadas as FABPs tipo 1 e tipo 3 de F. hepatica. Os resultados do presente estudo indicam que a FABP tipo 3 de F. hepatica é relacionada estrutural, imunológica e funcionalmente com a Sm14, um candidato vacinal amplamente estudado. Devido à importância da Sm14 como alvo para o desenvolvimento de vacina para a esquistossomose, as características apresentadas pela FhFABP3 de F. hepatica apontam esta proteína como um candidato importante também para o desenvolvimento de uma vacina contra a fasciolose

Sujeitos nulos, tópicos e a satisfação do EPP na aquisição de segunda língua: a influência do português brasileiro no inglês

Esta dissertação aborda a questão da transferência entre línguas na aquisição de segunda língua/língua estrangeira (L2/FL), mais especificamente, a influência do Português Brasileiro (PB) como língua materna (L1) na aquisição de inglês como L2/FL no que diz respeito ao preenchimento da posição de sujeito pronominal. Esse fenômeno é investigado à luz da teoria linguística gerativista nos moldes do Programa Minimalista (CHOMSKY, 1995) e da psicolinguística, no âmbito das questões de aquisição de L2/FL, ciência responsável por fornecer modelos procedimentais de como a produção e a compreensão de sentenças podem ser entendidas. A discussão sobre o modo como se dá a aquisição de L2 tem se mostrado complexa na literatura. O PB e o inglês diferem em relação à satisfação do traço EPP, responsável pelo preenchimento da posição de sujeito sintático nas línguas naturais. O PB tem se aproximado do inglês quanto ao preenchimento de sujeitos referenciais, mas não no que concerne aos sujeitos expletivos, apresentando ainda construções de tópico-sujeito, características que podem interferir na aquisição do valor paramétrico negativo para sujeitos nulos no inglês. A fim de investigar as mudanças que vêm afetando o PB nesse âmbito e observar o quanto aprendizes de inglês como FL falantes de PB se mostram sensíveis à agramaticalidade de sentenças com sujeito nulo no inglês em diferentes contextos, foram realizados dois experimentos, ambos com uma tarefa de julgamento de gramaticalidade. O experimento piloto foi realizado com aprendizes dos níveis básico e avançado e apresentava dois tipos distintos de sentenças (Tipo 1: sujeito nulo e Tipo 2: tópico + sujeito nulo); e um experimento final com aprendizes dos níveis básico, intermediário e avançado, com três tipos de sentenças (Tipo 1: sujeito nulo, Tipo 2: tópico + sujeito nulo e Tipo 3: conjunção + sujeito nulo). Dada a complexidade da gramática do PB, nossa hipótese de trabalho é de que não se observe uma transferência total, mas o surgimento de uma interlíngua que ora se aproxima, ora se afasta da gramática-alvo, refletindo a sobrecarga de processamento que lidar com as duas gramáticas impõe. Os resultados sustentam a hipótese ao indicar que (i) o valor do parâmetro do sujeito nulo parece ser transferido da L1 para a L2, uma vez que foi encontrado um alto número de respostas incorretas; (ii) a interferência se dá mais fortemente no âmbito dos sujeitos expletivos; (iii) há interferência de restrições gerais da gramática da L1 (restrições a V1) na L2; e (iv) a interferência diminui em função do aumento da proficiência em L2. Além disso, nas sentenças do tipo 2, parece haver uma possível interferência do PB que acaba por mascarar a omissão do expletivo, o que indica uma dificuldade de integração de informações provenientes das limitações decorrentes da necessidade de processar duas línguas em momentos específicos de modo a evitar a interferência da língua indesejada, no caso a L1, que por ainda ser dominante exige mais recursos para ser inibida (SORACE, 1999, 2011).