Theoretical models of planetary system formation II. Post-formation evolution

Aims. We extend the results of planetary formation synthesis by computing the long-term evolution of synthetic systems from the clearing of the gas disk into the dynamical evolution phase. Methods. We use the symplectic integrator SyMBA to numerically integrate the orbits of planets for 100 Myr, using populations from previous studies as initial conditions. Results. We show that within the populations studied, mass and semimajor axis distributions experience only minor changes from post-formation evolution. We also show that, depending upon their initial distribution, planetary eccentricities can statistically increase or decrease as a result of gravitational interactions. We find that planetary masses and orbital spacings provided by planet formation models do not result in eccentricity distributions comparable to observed exoplanet eccentricities, requiring other phenomena, such as stellar fly-bys, to account for observed eccentricities.

An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle

BACKGROUND: Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work. RESULTS: NanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo. CONCLUSIONS: PbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate requirements of NanoLuc are different from those of firefly luciferase, dual bioluminescence imaging for the simultaneous characterization of two lines, or two separate biological processes, is possible, as demonstrated in this work.

Neuroimmune Interactions: a Bidirectional Flow that Leads to Health and Disease

The Vedic philosophy deals with harmony and balance between the mind and the body as well as interactions with nature. This ancient approach to health and well-being is being more and more appreciated in part as we understand the intimate relationship between the immune system, our major defense mechanism and the nervous system. Like other organ systems, the immune system is dependent on the central nervous system (CNS) and the endocrine system in its role for effective defense against foreign and domestic invaders.

Relevant drug to drug interactions between itraconazole and non-nucleoside reverse transcriptase inhibitors in HIV-infected patients on maintenance therapy for disseminated histoplasmosis

Objective. Itraconazole is recommended life-long for preventing relapse of disseminated histoplasmosis in HIV-infected patients. I sought to determine if serum itraconazole levels are affected by the type of Highly Active Anti-Retroviral Therapy (NNRTI or PI) being taken concomitantly to treat HIV. ^ Design. Retrospective cohort. ^ Methods. De-identified data were used from an IRB-approved parent study which identified patients on HAART and maintenance itraconazole for confirmed disseminated histoplasmosis between January 2003 and December 2006. Available itraconazole blood levels were abstracted as well as medications taken by each patient at the time of the blood tests. Mean itraconazole levels were compared using the student's t-test. ^ Results. 11 patients met study criteria. Patient characteristics were: median age 36, 91% men, 18% white, 18% black, 55% Hispanic and 9% Asians, median CD4 cell count 120 cells/mm3. 14 blood levels were available for analysis—8 on PI, 4 on NNRTI and 2 on both. 8/8 itraconazole levels obtained while taking concomitant PI were therapeutic (>0.4 μg/mL) in contrast to 0/4 obtained while taking NNRTI. Two patients switched from NNRTI to PI and reached therapeutic levels. Mean levels on NNRTI (0.05 μg/mL, s.d. 0.0) and on PI (2.45 μg/mL, s.d. 0.21) for these two patients were compared via a paired t-test (t = 16.00, d.f. = 1, P = 0.04). Remaining patient levels were compared using an unpaired t-test. Mean itraconazole on concomitant PI (n = 6) was 1.37 μg/mL (s.d. 0.74), while the mean on concomitant NNRTI was 0.05 μg/mL (s.d. 0.0), t = 2.39, d.f. = 6, P = 0.05. ^ Conclusions. Co-administration of NNRTI and itraconazole results in significant decreases in itraconazole blood levels, likely by inducing the CYP3A4 enzyme system. Itraconazole drug levels should be monitored in patients on concomitant NNRTI. PI-based HAART may be preferred over NNRTI-based HAART when using itraconazole to treat HIV-infected patients with disseminated histoplasmosis. ^

Characterization of the interactions between fibronectin and the Borrelia burgdorferi lipoprotein, BBK32

The findings presented in this dissertation detail the complex interaction between BBK32 and fibronectin and describe novel consequences of the interaction. BBK32 is a fibronectin-binding protein on Borrelia burgdorferi, the causative agent of Lyme disease. We found that BBK32 contains multiple fibronectin-binding motifs, recognizing the fibronectin N-terminal domain (NTD) and the gelatin binding domain (GBD) in an anti-parallel order, where corresponding sites in BBK32 and fibronectin are aligned so that there is a one-to-one interaction between the proteins. While characterizing this interaction, we discovered that binding of BBK32 to the GBD inhibits the migration stimulating factor's (MSF) motogenic activity. In the presence of BBK32, endothelial cells do not migrate in response to increasing concentrations of MSF or the GBD. MSF is found under wound healing conditions, and inhibition of its activity may allow the tick-transmitted spirochetes to delay wound healing and to establish an infection. ^ Biophysical structural studies, designed to identify a mechanism of interaction, revealed that BBK32 binding to the NTD leads to the unfolding of plasma fibronectin, which exposes α5β1 integrin recognition motifs. Binding assays demonstrate that the BBK32-NTD interaction enhances the plasma fibronectin-α5β1 integrin interaction, which may allow B. burgdorferi to invade host cells, and thereby evade the host immune system. ^ We also determined that BBK32 binds fibronectin F3 modules, which leads to plasma fibronectin aggregation and induction of superfibronectin. The resulting superfibronectin is conformationally distinct from plasma and cellular fibronectin, and can inhibit endothelial cell proliferation. BBK32's active superfibronectin-forming motif has been located to a region between residues 160 and 175, which contains two sequence motifs that are also found in anastellin, the only other known superfibronectin-inducing protein. ^ A potential consequence of BBK32-induced superfibronectin formation was identified. BBK32-induced superfibronectin formation results in the exposure of α4β1 integrin recognition sequences in fibronectin. The α4β1 integrin is required for leukocyte transendothelial cell migration. BBK32-induced superfibronectin inhibits this activity. The inhibition of leukocyte recruitment to the infection site may slow the activity of the host immune system, and permit the spirochetes to establish an infection. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

The SasS -SasR two-component signal transduction system required for early Myxococcus xanthus multicellular *development

Initiation of Myxococcus xanthus multicellular development requires both nutrient limitation and high cell density. The extracellular signal, A signal, which consists of a set of amino acids at specific concentrations, serves as a cell density signal in M. xanthus early development. A reporter gene, designated 4521, that requires both starvation and A signal for developmental expression was used to identify mutations in the signal transduction pathways. A group of point mutations located in the chromosomal sasB locus that bypasses both requirements was previously isolated. One of these point mutations, sasB7, was mapped to the sasS gene, which is predicted to encode a transmembrane histidine protein kinase required for normal development. SasS is a positive regulator of 4521 and a candidate A signal sensor. This dissertation continues the characterization of the sasB locus, focusing on the sasR gene and the functional relationship of SasS and SasR. ^ The sasR gene is located 2.2-kb downstream of sasS. It is predicted to encode an NtrC-like response regulator, which belongs to the family of sigma54 transcriptional activators. SasR is a positive regulator of 4521 gene and is required for normal development. The sasR mutant displays phenotypes similar to that of sasS mutant. Both SasS and SasR are required for the A-signal-dependent 4521 expression. Genetic epistasis analysis indicates that SasR functions downstream of SasS. Biochemical studies show that SasS has autokinase activity, and phosphorylated SasS is able to transfer its phosphate to SasR. We propose that SasS and SasR form a two-component signal transduction system in the A signal transduction pathway. ^ To search for the genes regulated by SasS and SasR, expression patterns of a group of developmental genes were compared in wild-type and sasS null mutant backgrounds. SasS and SasR were found to positively regulate sasN and 4521. The sasN gene was previously identified as a negative regulator of 4521, located at about 170-bp downstream of sasR. It is required for normal fruiting body development. Based on the above data, a regulatory network consisting of sasS, sasR, sasN, and 4521 is hypothesized, and the interactions of the components in this network can now be further studied. ^

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^

Alkenone-derived sea surface temperature records of three sediment cores from the southern Benguela upwelling system

We present alkenone-derived Sea Surface Temperature (SST) records from three marine cores collected within the southern Benguela Upwelling System (BUS) spanning the last 3 ka. The SST evolution over the last 3 millennia is marked by aperiodic millennial-scale oscillations that broadly correspond to climatic anomalies identified over the North Atlantic region. The BUS SST data further suggest cooling and warming trends opposite to the temperature evolution in the Moroccan upwelling region and in Antarctica. In contrast, the last decades are marked by a cooling of unprecedented magnitude in both the Benguela and Moroccan upwelling systems, which is not observed in the Antarctic record. These contrasted responses in Atlantic upwelling systems triggered by natural and anthropogenic forcings shed light on how different climatic mechanisms are mediated by ocean-atmosphere interactions and transmitted to the geological records of past and present climate changes.

Age determination of sediment cores of the California Current System

Alkenone sea surface temperature (SST) records were generated from the Ocean Drilling Program's (ODP) Sites 1014 and 1016 to examine the response of the California Current System to global climate change during the last 136 ka. The temperature differences between these sites (Delta SST(NEP)=SST(ODP1014)-SST(ODP1016)) reflected the intensity of the California Current and varied between 0.4 and 6.1 °C. A high Delta SST(NEP) (weaker California Current) was found for late marine isotope stage (MIS) 2 and early MIS 5e, while a low Delta SST(NEP) (stronger California Current) was detected for mid-MIS 5e and MIS 1. Spectral analysis indicated that this variation pattern dominated 23- (precession) and 30-ka periods. Comparison of the Delta SST(NEP) and SST based on data from core MD01-2421 at the Japan margin revealed anti-phase variation; the high Delta SST(NEP) (weakening of the California Current) corresponded to the low SST at the Japan margin (the southward displacement of the NW Pacific subarctic boundary), and vice versa. This variation was synchronous with a model prediction of the tropical El Niño-Southern Oscillation behavior. These findings suggest that the intensity of the North Pacific High varied in response to precessional forcing, and also that the response has been linked with the changes of tropical ocean-atmosphere interactions.

Putting prey and predator into the CO2 equation-qualitative and quantitative effects of ocean acidification on predator-prey interactions

Little is known about the impact of ocean acidification on predator-prey dynamics. Herein, we examined the effect of carbon dioxide (CO(2)) on both prey and predator by letting one predatory reef fish interact for 24 h with eight small or large juvenile damselfishes from four congeneric species. Both prey and predator were exposed to control or elevated levels of CO(2). Mortality rate and predator selectivity were compared across CO(2) treatments, prey size and species. Small juveniles of all species sustained greater mortality at high CO(2) levels, while large recruits were not affected. For large prey, the pattern of prey selectivity by predators was reversed under elevated CO(2). Our results demonstrate both quantitative and qualitative consumptive effects of CO(2) on small and larger damselfish recruits respectively, resulting from CO(2)-induced behavioural changes likely mediated by impaired neurological function. This study highlights the complexity of predicting the effects of climate change on coral reef ecosystems.

Interactions among chronic and acute impacts on coral recruits: the importance of size-escape thresholds

Newly settled recruits typically suffer high mortality from disturbances, but rapid growth reduces their mortality once size-escape thresholds are attained. Ocean acidification (OA) reduces the growth of recruiting benthic invertebrates, yet no direct effects on survivorship have been demonstrated. We tested whether the reduced growth of coral recruits caused by OA would increase their mortality by prolonging their vulnerability to an acute disturbance: fish herbivory on surrounding algal turf. After two months' growth in ambient or elevated CO2 levels, the linear extension and calcification of coral (Acropora millepora) recruits decreased as CO2 partial pressure (pCO2) increased. When recruits were subjected to incidental fish grazing, their mortality was inversely size dependent. However, we also found an additive effect of pCO2 such that recruit mortality was higher under elevated pCO2 irrespective of size. Compared to ambient conditions, coral recruits needed to double their size at the highest pCO2 to escape incidental grazing mortality. This general trend was observed with three groups of predators (blenny, surgeonfish, and parrotfish), although the magnitude of the fish treatment varied among species. Our study demonstrates the importance of size-escape thresholds in early recruit survival and how OA can shift these thresholds, potentially intensifying population bottlenecks in benthic invertebrate recruitment.

Positron bunching and electrostatic transport system for the production and emission of dense positronium clouds into vacuum

We describe a system designed to re-bunch positron pulses delivered by an accumulator supplied by a positron source and a Surko-trap. Positron pulses from the accumulator are magnetically guided in a 0.085 T field and are injected into a region free of magnetic fields through a μ -metal field terminator. Here positrons are temporally compressed, electrostatically guided and accelerated towards a porous silicon target for the production and emission of positronium into vacuum. Positrons are focused in a spot of less than 4 mm FWTM in bunches of ∼8 ns FWHM. Emission of positronium into the vacuum is shown by single shot positron annihilation lifetime spectroscopy.

Ecosystem regimes and responses in a coupled ancient lake system from MIS 5b to present: the diatom record of lakes Ohrid and Prespa

We reconstruct the aquatic ecosystem interactions since the last interglacial period in the oldest, most diverse, hydrologically connected European lake system, by using palaeolimnological diatom and selected geochemistry data from Lake Ohrid “DEEP site” core and equivalent data from Lake Prespa core, Co1215. Driven by climate forcing, the lakes experienced two adaptive cycles during the last 92 ka: "interglacial and interstadial" and "glacial" cycle. The short-term ecosystems reorganizations, e.g. regime shifts within these cycles substantially differ between the lakes, as evident from the inferred amplitudes of variation. The deeper Lake Ohrid shifted between ultra oligo- and oligotrophic regimes in contrast to the much shallower Lake Prespa, which shifted from a deeper, (oligo-) mesotrophic to a shallower, eutrophic lake and vice versa. Due to the high level of ecosystem stability (e.g. trophic state, lake level), Lake Ohrid appears relatively resistant to external forcing, such as climate and environmental change. Recovering in a relatively short time from major climate change, Lake Prespa is a resilient ecosystem. At the DEEP site, the decoupling between the lakes' response to climate change is marked in the prolonged and gradual changes during the MIS 5/4 and 2/1 transitions. These response differences and the lakes' different physical and chemical properties may limit the influence of Lake Prespa on Lake Ohrid. Regime shifts of Lake Ohrid due to potential hydrological change in Lake Prespa are not evident in the data presented here. Moreover, a complete collapse of the ecosystems functionality and loss of their diatom communities did not happen in either lake for the period presented in the study.

Comparative responses of two dominant Antarctic phytoplankton taxa to interactions between ocean acidification, warming, irradiance, and iron availability

We investigated the responses of the ecologically dominant Antarctic phytoplankton species Phaeocystis antarctica (a prymnesiophyte) and Fragilariopsis cylindrus (a diatom) to a clustered matrix of three global change variables (CO2, mixed-layer depth, and temperature) under both iron (Fe)-replete and Fe-limited conditions based roughly on the Intergovernmental Panel on Climate Change (IPCC) A2 scenario: (1) Current conditions, 39 Pa (380 ppmv) CO2, 50 µmol photons/m**2/s light, and 2°C; (2) Year 2060, 61 Pa (600 ppmv) CO2, 100 µmol photons/m**2/s light, and 4°C; (3) Year 2100, 81 Pa (800 ppmv) CO2, 150 µmol photons/m**2/s light, and 6°C. The combined interactive effects of these global change variables and changing Fe availability on growth, primary production, and cell morphology are species specific. A competition experiment suggested that future conditions could lead to a shift away from P. antarctica and toward diatoms such as F. cylindrus. Along with decreases in diatom cell size and shifts from prymnesiophyte colonies to single cells under the future scenario, this could potentially lead to decreased carbon export to the deep ocean. Fe : C uptake ratios of both species increased under future conditions, suggesting phytoplankton of the Southern Ocean will increase their Fe requirements relative to carbon fixation. The interactive effects of Fe, light, CO2, and temperature on Antarctic phytoplankton need to be considered when predicting the future responses of biology and biogeochemistry in this region.