Paleoceanographic and paleoclimatic evolution of the Aegean Sea since the last interglacial (~130 ka BP) with special emphasis on sapropel formation

Five long piston cores collected from different subbasins of the Aegean Sea constitute the primary source of data for this PhD thesis. This study is the first to document a continuous paleoceanographic and paleoclimatic record of the Aegean Sea since the last interglacial. The chronostratigraphic reconstructions of the cored sediments based on organic carbon contents, stratigraphic position of known ash layers and oxygen isotopic curve matching collectively demonstrate the presence of sapropel S1 and MISS sapropels S3, S4 and S5 in the Aegean Sea subbasins. Generally, the organic carbon (TOC wt%) contents in sapropels range between 0.8% and 2% with highest concentrations of 9-13% in sapropels S4 and S5. Average sedimentation rates range between 4.7 and 11.8 cmlka with highest rates being observed in Euboea and North Ikaria basins (9.8 and 11.8 cm lka, respectively). The timing of the onset of sapropels S4 and S5 mostly predate those in the eastern Mediterranean with ages ranging from 106.4-105.6 and 128.6-128.4 ka BP, respectively. On the other hand, the initiation of the onset of sapropel S3 (i.e., 83.2-80.4 ka BP) seems to agree with its Mediterranean counterparts, which highlights the heterogeneity of the Aegean Sea subbasins in terms of rapid vs. lagged response to changing climatic conditions. The sapropel initiations appear to be synchronous across the Aegean Sea; whereas, the terminations display a wider temporal variability implying that the cessation of sapropels is controlled both by the amplitude of paleoclimatic changes and the physiography/location ofthe subbasins. Quantitative variations in the planktonic faunal assemblages exhibit a sequence of bioevents during the last -130,000 years which allow identification of four major biozones. The distributional patterns of the most significant taxa demonstrate similar trends among all core localities suggesting that the major changes in the planktonic foraminifera assemblages have taken place rather synchronously in the Aegean Sea. Sapropels S3, S4 and S5 were deposited under similar hydrographic conditions during which a distinct deep chlorophyll maximum (DCM) layer was established. This situation points to a stratified water column and increased export productivity during times of sapropel formation. On the other hand, the faunal contrast between Sl and older sapropels indicates that the former was developed in the absence of a DCM layer, lacking a deep phytoplankton assemblage. Under such conditions, oxygen advection via intermediate water flow must have been significantly reduced which implies significant stagnation. Sapropels are interpreted to have been deposited under normal marine conditions with temporary establishment of semi-euxinic bottom water conditions. Both marine and terrestrial organic matter contributed equally to MISS sapropels. In addition, organic carbon isotopic values across sapropels are more depleted than those in the eastern Mediterranean which, in tum, suggests enhanced riverine input during their deposition. Primary productivity calculations show that, particularly for sapropels with very high TOC values, both preservation and increased productivity are imperative in order to deposit sapropels with very high organic carbon contents (i.e., up to 13%).

RNA Virus Evolution: a Cross-scale, Disease Dynamic Perspective

RNA viruses are an important cause of global morbidity and mortality. The rapid evolutionary rates of RNA virus pathogens, caused by high replication rates and error-prone polymerases, can make the pathogens difficult to control. RNA viruses can undergo immune escape within their hosts and develop resistance to the treatment and vaccines we design to fight them. Understanding the spread and evolution of RNA pathogens is essential for reducing human suffering. In this dissertation, I make use of the rapid evolutionary rate of viral pathogens to answer several questions about how RNA viruses spread and evolve. To address each of the questions, I link mathematical techniques for modeling viral population dynamics with phylogenetic and coalescent techniques for analyzing and modeling viral genetic sequences and evolution. The first project uses multi-scale mechanistic modeling to show that decreases in viral substitution rates over the course of an acute infection, combined with the timing of infectious hosts transmitting new infections to susceptible individuals, can account for discrepancies in viral substitution rates in different host populations. The second project combines coalescent models with within-host mathematical models to identify driving evolutionary forces in chronic hepatitis C virus infection. The third project compares the effects of intrinsic and extrinsic viral transmission rate variation on viral phylogenies.

Middle Miocene evolution and stable isotope ratios of Globorotalia (Fohsella) in the western equatorial Pacific

Evolution of the planktic foraminiferal lineage Globorotalia (Fohsella) occurred during the Miocene between 23.7 and 11.8 Ma and forms the basis for stratigraphic subdivision of the early middle Miocene (Zones N 10 through N 12). Important morphologic changes within the G. (Fohsella) lineage included a marked increase in test size, a transition from a rounded to an acute periphery, and the development of a keel in later forms. We found that the most rapid changes in morphology of G. (Fohsella) occurred between 13 and 12.7 Ma and coincided with an abrupt increase in the delta18O ratios of shell calcite. Comparison of isotopic results of G. (Fohsella) with other planktic foraminifers indicate that delta18O values of the lineage diverge from surface-dwelling species and approach deep-dwelling species after 13.0 Ma, indicating a change in depth habitat from the surface mixed layer to intermediate depth near the thermocline. Isotopic and faunal evidence suggests that this change in depth stratification was associated with an expansion of the thermocline in the western equatorial Pacific. After adapting to a deeper water habitat at 13.0 Ma, the G. (Fohsella) lineage became extinct abruptly at 11.8 Ma during a period when isotopic and faunal evidence suggest a shoaling of the thermocline. Following the extinction of G. (Fohsella), the ecologic niche of the lineage was filled by the Globorotalia (Menardella) group, which began as a deep-water form and later evolved to an intermediate-water habitat. We suggest that the evolution of G. (Fohsella) and G. (Menardella) were tightly linked to changes in the structure of the thermocline in the western equatorial Pacific.

Phocid seal leptin: tertiary structure and hydrophobic receptor binding site preservation during distinct leptin gene evolution

The cytokine hormone leptin is a key signalling molecule in many pathways that control physiological functions. Although leptin demonstrates structural conservation in mammals, there is evidence of positive selection in primates, lagomorphs and chiropterans. We previously reported that the leptin genes of the grey and harbour seals (phocids) have significantly diverged from other mammals. Therefore we further investigated the diversification of leptin in phocids, other marine mammals and terrestrial taxa by sequencing the leptin genes of representative species. Phylogenetic reconstruction revealed that leptin diversification was pronounced within the phocid seals with a high dN/dS ratio of 2.8, indicating positive selection. We found significant evidence of positive selection along the branch leading to the phocids, within the phocid clade, but not over the dataset as a whole. Structural predictions indicate that the individual residues under selection are away from the leptin receptor (LEPR) binding site. Predictions of the surface electrostatic potential indicate that phocid seal leptin is notably different to other mammalian leptins, including the otariids. Cloning the grey seal leptin binding domain of LEPR confirmed that this was structurally conserved. These data, viewed in toto, support a hypothesis that phocid leptin divergence is unlikely to have arisen by random mutation. Based upon these phylogenetic and structural assessments, and considering the comparative physiology and varying life histories among species, we postulate that the unique phocid diving behaviour has produced this selection pressure. The Phocidae includes some of the deepest diving species, yet have the least modified lung structure to cope with pressure and volume changes experienced at depth. Therefore, greater surfactant production is required to facilitate rapid lung re-inflation upon surfacing, while maintaining patent airways. We suggest that this additional surfactant requirement is met by the leptin pulmonary surfactant production pathway which normally appears only to function in the mammalian foetus.

The interplay between tectonics, sediment dynamics and gateways evolution in the Danube system from the Pannonian Basin to the western Black Sea

Understanding the natural evolution of a river–delta–sea system is important to develop a strong scientific basis for efficient integrated management plans. The distribution of sediment fluxes is linked with the natural connection between sediment source areas situated in uplifting mountain chains and deposition in plains, deltas and, ultimately, in the capturing oceans and seas. The Danube River–western Black Sea is one of the most active European systems in terms of sediment re-distribution that poses significant societal challenges. We aim to derive the tectonic and sedimentological background of human-induced changes in this system and discuss their interplay. This is obtained by analysing the tectonic and associated vertical movements, the evolution of relevant basins and the key events affecting sediment routing and deposition. The analysis of the main source and sink areas is focused in particular on the Miocene evolution of the Carpatho-Balkanides, Dinarides and their sedimentary basins including the western Black Sea. The vertical movements of mountains chains created the main moments of basin connectivity observed in the Danube system. Their timing and effects are observed in sediments deposited in the vicinity of gateways, such as the transition between the Pannonian/Transylvanian and Dacian basins and between the Dacian Basin and western Black Sea. The results demonstrate the importance of understanding threshold conditions driving rapid basins connectivity changes superposed over the longer time scale of tectonic-induced vertical movements associated with background erosion and sedimentation. The spatial and temporal scale of such processes is contrastingly different and challenging. The long-term patterns interact with recent or anthropogenic induced modifications in the natural system and may result in rapid changes at threshold conditions that can be quantified and predicted. Their understanding is critical because of frequent occurrence during orogenic evolution, as commonly observed in the Mediterranean area and discussed elsewhere.

Rapid hydrothermal cooling above the axial melt lens at fast-spreading mid-ocean ridge

Axial melt lenses sandwiched between the lower oceanic crust and the sheeted dike sequences at fast-spreading mid-ocean ridges are assumed to be the major magma source of oceanic crust accretion. According to the widely discussed "gabbro glacier'' model, the formation of the lower oceanic crust requires efficient cooling of the axial melt lens, leading to partial crystallization and crystal-melt mush subsiding down to lower crust. These processes are believed to be controlled by periodical magma replenishment and hydrothermal circulation above the melt lens. Here we quantify the cooling rate above melt lens using chemical zoning of plagioclase from hornfelsic recrystallized sheeted dikes drilled from the East Pacific at the Integrated Ocean Drilling Program Hole 1256D. Weestimate the cooling rate using a forward modelling approach based on CaAl-NaSi interdiffusion in plagioclase. The results show that cooling from the peak thermal overprint at 1000-10506 degrees C to 6006 degrees C are yielded within about 10-30 years as a result of hydrothermal circulation above melt lens during magma starvation. The estimated rapid hydrothermal cooling explains how the effective heat extraction from melt lens is achieved at fast-spreading mid-ocean ridges.

Impacts of high-nitrate freshwater inputs on macrotidal ecosystems. I. Seasonal evolution of nutrient limitation for the diatom-dominated phytoplankton of the Bay of Brest (France)

The chemical factors (inorganic nitrogen, phosphate, silicic acid) that potentially or actually control primary production were determined for the Bay of Brest, France, a macrotidal ecosystem submitted to high-nitrate-loaded freshwater inputs (winter nitrate freshwater concentrations >700 mu M, Si:N molar ratio as low as 0.2, i.e. among the lowest ever published). Intensive data collection and observations were carried out from February 1993 to March 1994 to determine the variations of physical [salinity, temperature, photosynthetically active radiation (PAR), freshwater discharges] and chemical (oxygen and nutrients) parameters and their impacts on the phytoplankton cycle (fluorescence, pigments, primary production). With insufficient PAR the winter stocks of nutrients were almost nonutilized and the nitrate excess was exported to the adjacent ocean, due to rapid tidal exchange. By early April, a diatom-dominated spring bloom developed (chlorophyll a maximum = 7.7 mu g l(-1); primary production maximum = 2.34 g C m(-2) d(-1)) under high initial nutrient concentrations. Silicic acid was rapidly exhausted over the whole water column; it is inferred to be the primary limiting factor responsible for the collapse of the spring bloom by mid-May. Successive phytoplankton developments characterized the period of secondary blooms during summer and fall (successive surface chlorophyll a maxima = 3.5, 1.6, 1.8 and 1.0 mu g l(-1); primary production = 1.24, 1.18 and 0.35 g C m(-2) d(-1)). Those secondary blooms developed under lower nutrient concentrations, mostly originating from nutrient recycling. Until August, Si and P most likely limited primary production, whereas the last stage of the productive period in September seemed to be N limited instead, this being a period of total nitrate depletion in almost the whole water column. Si limitation of spring blooms has become a common feature in coastal ecosystems that receive freshwater inputs with Si:N molar ratios <1. The peculiarity of Si Limitation in the Bay of Brest is its extension through the summer period.

Applied evolution: An integrated approach to studying life history traits in response to drug selection

The use of chemical control measures to reduce the impact of parasite and pest species has frequently resulted in the development of resistance. Thus, resistance management has become a key concern in human and veterinary medicine, and in agricultural production. Although it is known that factors such as gene flow between susceptible and resistant populations, drug type, application methods, and costs of resistance can affect the rate of resistance evolution, less is known about the impacts of density-dependent eco-evolutionary processes that could be altered by drug-induced mortality. The overall aim of this thesis was to take an experimental evolution approach to assess how life history traits respond to drug selection, using a free-living dioecious worm (Caenorhabditis remanei) as a model. In Chapter 2, I defined the relationship between C. remanei survival and Ivermectin dose over a range of concentrations, in order to control the intensity of selection used in the selection experiment described in Chapter 4. The dose-response data were also used to appraise curve-fitting methods, using Akaike Information Criterion (AIC) model selection to compare a series of nonlinear models. The type of model fitted to the dose response data had a significant effect on the estimates of LD50 and LD99, suggesting that failure to fit an appropriate model could give misleading estimates of resistance status. In addition, simulated data were used to establish that a potential cost of resistance could be predicted by comparing survival at the upper asymptote of dose-response curves for resistant and susceptible populations, even when differences were as low as 4%. This approach to dose-response modeling ensures that the maximum amount of useful information relating to resistance is gathered in one study. In Chapter 3, I asked how simulations could be used to inform important design choices used in selection experiments. Specifically, I focused on the effects of both within- and between-line variation on estimated power, when detecting small, medium and large effect sizes. Using mixed-effect models on simulated data, I demonstrated that commonly used designs with realistic levels of variation could be underpowered for substantial effect sizes. Thus, use of simulation-based power analysis provides an effective way to avoid under or overpowering a study designs incorporating variation due to random effects. In Chapter 4, I 3 investigated how Ivermectin dosage and changes in population density affect the rate of resistance evolution. I exposed replicate lines of C. remanei to two doses of Ivermectin (high and low) to assess relative survival of lines selected in drug-treated environments compared to untreated controls over 10 generations. Additionally, I maintained lines where mortality was imposed randomly to control for differences in density between drug treatments and to distinguish between the evolutionary consequences of drug treatment versus ecological processes affected by changes in density-dependent feedback. Intriguingly, both drug-selected and random-mortality lines showed an increase in survivorship when challenged with Ivermectin; the magnitude of this increase varied with the intensity of selection and life-history stage. The results suggest that interactions between density-dependent processes and life history may mediate evolved changes in susceptibility to control measures, which could result in misleading conclusions about the evolution of heritable resistance following drug treatment. In Chapter 5, I investigated whether the apparent changes in drug susceptibility found in Chapter 4 were related to evolved changes in life-history of C. remanei populations after selection in drug-treated and random-mortality environments. Rapid passage of lines in the drug-free environment had no effect on the measured life-history traits. In the drug-free environment, adult size and fecundity of drug-selected lines increased compared to the controls but drug selection did not affect lifespan. In the treated environment, drug-selected lines showed increased lifespan and fecundity relative to controls. Adult size of randomly culled lines responded in a similar way to drug-selected lines in the drug-free environment, but no change in fecundity or lifespan was observed in either environment. The results suggest that life histories of nematodes can respond to selection as a result of the application of control measures. Failure to take these responses into account when applying control measures could result in adverse outcomes, such as larger and more fecund parasites, as well as over-estimation of the development of genetically controlled resistance. In conclusion, my thesis shows that there may be a complex relationship between drug selection, density-dependent regulatory processes and life history of populations challenged with control measures. This relationship could have implications for how resistance is monitored and managed if life histories of parasitic species show such eco-evolutionary responses to drug application.

Near-infrared Instrumentation For Rapid-response Astronomy

Ɣ-ray bursts (GRBs) are the Universe's most luminous transient events. Since the discovery of GRBs was announced in 1973, efforts have been ongoing to obtain data over a broader range of the electromagnetic spectrum at the earliest possible times following the initial detection. The discovery of the theorized ``afterglow'' emission in radio through X-ray bands in the late 1990s confirmed the cosmological nature of these events. At present, GRB afterglows are among the best probes of the early Universe (z ≳ 9). In addition to informing theories about GRBs themselves, observations of afterglows probe the circum-burst medium (CBM), properties of the host galaxies and the progress of cosmic reionization. To explore the early-time variability of afterglows, I have developed a generalized analysis framework which models near-infrared (NIR), optical, ultra-violet (UV) and X-ray light curves without assuming an underlying model. These fits are then used to construct the spectral energy distribution (SED) of afterglows at arbitrary times within the observed window. Physical models are then used to explore the evolution of the SED parameter space with time. I demonstrate that this framework produces evidence of the photodestruction of dust in the CBM of GRB 120119A, similar to the findings from a previous study of this afterglow. The framework is additionally applied to the afterglows of GRB 140419A and GRB 080607. In these cases the evolution of the SEDs appears consistent with the standard fireball model. Having introduced the scientific motivations for early-time observations, I introduce the Rapid Infrared Imager-Spectrometer (RIMAS). Once commissioned on the 4.3 meter Discovery Channel Telescope (DCT), RIMAS will be used to study the afterglows of GRBs through photometric and spectroscopic observations beginning within minutes of the initial burst. The instrument will operate in the NIR, from 0.97 μm to 2.37 μm, permitting the detection of very high redshift (z ≳ 7) afterglows which are attenuated at shorter wavelengths by Lyman-ɑ absorption in the intergalactic medium (IGM). A majority of my graduate work has been spent designing and aligning RIMAS's cryogenic (~80 K) optical systems. Design efforts have included an original camera used to image the field surrounding spectroscopic slits, tolerancing and optimizing all of the instrument's optics, thermal modeling of optomechanical systems, and modeling the diffraction efficiencies for some of the dispersive elements. To align the cryogenic optics, I developed a procedure that was successfully used for a majority of the instrument's sub-assemblies. My work on this cryogenic instrument has necessitated experimental and computational projects to design and validate designs of several subsystems. Two of these projects describe simple and effective measurements of optomechanical components in vacuum and at cryogenic temperatures using an 8-bit CCD camera. Models of heat transfer via electrical harnesses used to provide current to motors located within the cryostat are also presented.

Wheat proteins evolution and environmental triggers of Wheat-related diseases

Cereals, and in particular wheat, have always been recognized as a fundamental food worldwide. In particular, the success of wheat is linked with unique properties of the gluten protein fraction used in bread making process to obtain products that are widely used in traditional and modern diets. The rapid increase in the world population let to a parallel increases in food production, particularly of wheat. Increasing yield potential and selection of cultivars much more resistant to plant disease and to environmental factors could have negatively affected the quality of the grain. Moreover, the “green revolution” was characterized by a widespread use of agricultural chemicals and by industrialization of food production that led to a huge rise in the consumption of refined products. Modern baking practices have shortened bread leavening, increased the use of chemical/yeast leavening agents and there is well-documented scientific evidence of the negative effects of ultra-processed food in human healthy. All this changes profoundly modified the human diet and, as a result, may have affected Gluten-related disease (GRDs) that has arisen in the whole word populations. Gluten-related diseases (GRDs) are multifactorial pathologies in which environmental factors and genetic background contribute to a low-grade chronic inflammation of the gastrointestinal tract. Here, I investigated the potential pro-inflammatory effect of different wheat varieties and whether bread making processing are involved in the onset or worsening of gut inflammation. In vitro, ex vivo and in vivo studies conducted throughout my Phd period have shown a pro-inflammatory effect of wheat especially marked in modern varieties and a higher inflammatory response linked to the use of common raising agent as Saccharomyces Cerevisiae and to the addiction of chemical bakery improver substances.

Prognostic Value Of Dna And Mrna E6/e7 Of Human Papillomavirus In The Evolution Of Cervical Intraepithelial Neoplasia Grade 2.

This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL). This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months. Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant. The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.

Effects Of Therapeutic Approach On The Neonatal Evolution Of Very Low Birth Weight Infants With Patent Ductus Arteriosus.

To analyze the effects of treatment approach on the outcomes of newborns (birth weight [BW] < 1,000 g) with patent ductus arteriosus (PDA), from the Brazilian Neonatal Research Network (BNRN) on: death, bronchopulmonary dysplasia (BPD), severe intraventricular hemorrhage (IVH III/IV), retinopathy of prematurity requiring surgical (ROPsur), necrotizing enterocolitis requiring surgery (NECsur), and death/BPD. This was a multicentric, cohort study, retrospective data collection, including newborns (BW < 1000 g) with gestational age (GA) < 33 weeks and echocardiographic diagnosis of PDA, from 16 neonatal units of the BNRN from January 1, 2010 to Dec 31, 2011. Newborns who died or were transferred until the third day of life, and those with presence of congenital malformation or infection were excluded. Groups: G1 - conservative approach (without treatment), G2 - pharmacologic (indomethacin or ibuprofen), G3 - surgical ligation (independent of previous treatment). Factors analyzed: antenatal corticosteroid, cesarean section, BW, GA, 5 min. Apgar score < 4, male gender, Score for Neonatal Acute Physiology Perinatal Extension (SNAPPE II), respiratory distress syndrome (RDS), late sepsis (LS), mechanical ventilation (MV), surfactant (< 2 h of life), and time of MV. death, O2 dependence at 36 weeks (BPD36wks), IVH III/IV, ROPsur, NECsur, and death/BPD36wks. Student's t-test, chi-squared test, or Fisher's exact test; Odds ratio (95% CI); logistic binary regression and backward stepwise multiple regression. Software: MedCalc (Medical Calculator) software, version 12.1.4.0. p-values < 0.05 were considered statistically significant. 1,097 newborns were selected and 494 newborns were included: G1 - 187 (37.8%), G2 - 205 (41.5%), and G3 - 102 (20.6%). The highest mortality was observed in G1 (51.3%) and the lowest in G3 (14.7%). The highest frequencies of BPD36wks (70.6%) and ROPsur were observed in G3 (23.5%). The lowest occurrence of death/BPD36wks occurred in G2 (58.0%). Pharmacological (OR 0.29; 95% CI: 0.14-0.62) and conservative (OR 0.34; 95% CI: 0.14-0.79) treatments were protective for the outcome death/BPD36wks. The conservative approach of PDA was associated to high mortality, the surgical approach to the occurrence of BPD36wks and ROPsur, and the pharmacological treatment was protective for the outcome death/BPD36wks.

Evolution Of The Deaths Registry System In Brazil: Associations With Changes In The Mortality Profile, Under-registration Of Death Counts, And Ill-defined Causes Of Death.

This paper examines the spatial pattern of ill-defined causes of death across Brazilian regions, and its relationship with the evolution of completeness of the deaths registry and changes in the mortality age profile. We make use of the Brazilian Health Informatics Department mortality database and population censuses from 1980 to 2010. We applied demographic methods to evaluate the quality of mortality data for 137 small areas and correct for under-registration of death counts when necessary. The second part of the analysis uses linear regression models to investigate the relationship between, on the one hand, changes in death counts coverage and age profile of mortality, and on the other, changes in the reporting of ill-defined causes of death. The completeness of death counts coverage increases from about 80% in 1980-1991 to over 95% in 2000-2010 at the same time the percentage of ill-defined causes of deaths reduced about 53% in the country. The analysis suggests that the government's efforts to improve data quality are proving successful, and they will allow for a better understanding of the dynamics of health and the mortality transition.

Digestive Peptidase Evolution In Holometabolous Insects Led To A Divergent Group Of Enzymes In Lepidoptera.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

A Rapid And Simple Capillary Electrophoresis Method For Indirect Determination Of The Biocide 2,2-dibromo-3-nitrilo-propionamide (dbnpa) In Cooling Waters.

A capillary zone electrophoresis (CE) method was developed for the determination of the biocide 2,2-dibromo-3-nitrilo-propionamide (DBNPA) in water used in cooling systems. The biocide is indirectly determined by CE measurement of the concentration of bromide ions produced by the reaction between the DBNPA and bisulfite. The relationship between the bromide peak areas and the DBNPA concentrations showed a good linearity and a coefficient of determination (R(2)) of 0.9997 in the evaluated concentration range of 0-75 μmol L(-1). The detection and quantification limits for DBNPA were 0.23 and 0.75 μmol L(-1), respectively. The proposed CE method was successfully applied for the analysis of samples of tap water and cooling water spiked with DBNPA. The intra-day and inter-day (intermediary) precisions were lower than 2.8 and 6.2%, respectively. The DBNPA concentrations measured by the CE method were compared to the values obtained by a spectrophotometric method and were found to agree well.