The Ontogeny of Mucosal and Systemic Antibody Responses to HIV-1 Infection

The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies.

After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.

Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies.

In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies.

Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.

Mapping the semantic structure of cognitive neuroscience.

Cognitive neuroscience, as a discipline, links the biological systems studied by neuroscience to the processing constructs studied by psychology. By mapping these relations throughout the literature of cognitive neuroscience, we visualize the semantic structure of the discipline and point to directions for future research that will advance its integrative goal. For this purpose, network text analyses were applied to an exhaustive corpus of abstracts collected from five major journals over a 30-month period, including every study that used fMRI to investigate psychological processes. From this, we generate network maps that illustrate the relationships among psychological and anatomical terms, along with centrality statistics that guide inferences about network structure. Three terms--prefrontal cortex, amygdala, and anterior cingulate cortex--dominate the network structure with their high frequency in the literature and the density of their connections with other neuroanatomical terms. From network statistics, we identify terms that are understudied compared with their importance in the network (e.g., insula and thalamus), are underspecified in the language of the discipline (e.g., terms associated with executive function), or are imperfectly integrated with other concepts (e.g., subdisciplines like decision neuroscience that are disconnected from the main network). Taking these results as the basis for prescriptive recommendations, we conclude that semantic analyses provide useful guidance for cognitive neuroscience as a discipline, both by illustrating systematic biases in the conduct and presentation of research and by identifying directions that may be most productive for future research.

Dramatic variability of the carbonate system at a temperate coastal ocean site (Beaufort, North Carolina, USA) is regulated by physical and biogeochemical processes on multiple timescales.

Increasing atmospheric carbon dioxide (CO2) from anthropogenic sources is acidifying marine environments resulting in potentially dramatic consequences for the physical, chemical and biological functioning of these ecosystems. If current trends continue, mean ocean pH is expected to decrease by ~0.2 units over the next ~50 years. Yet, there is also substantial temporal variability in pH and other carbon system parameters in the ocean resulting in regions that already experience change that exceeds long-term projected trends in pH. This points to short-term dynamics as an important layer of complexity on top of long-term trends. Thus, in order to predict future climate change impacts, there is a critical need to characterize the natural range and dynamics of the marine carbonate system and the mechanisms responsible for observed variability. Here, we present pH and dissolved inorganic carbon (DIC) at time intervals spanning 1 hour to >1 year from a dynamic, coastal, temperate marine system (Beaufort Inlet, Beaufort NC USA) to characterize the carbonate system at multiple time scales. Daily and seasonal variation of the carbonate system is largely driven by temperature, alkalinity and the balance between primary production and respiration, but high frequency change (hours to days) is further influenced by water mass movement (e.g. tides) and stochastic events (e.g. storms). Both annual (~0.3 units) and diurnal (~0.1 units) variability in coastal ocean acidity are similar in magnitude to 50 year projections of ocean acidity associated with increasing atmospheric CO2. The environmental variables driving these changes highlight the importance of characterizing the complete carbonate system rather than just pH. Short-term dynamics of ocean carbon parameters may already exert significant pressure on some coastal marine ecosystems with implications for ecology, biogeochemistry and evolution and this shorter term variability layers additive effects and complexity, including extreme values, on top of long-term trends in ocean acidification.

The island-scale internal wave climate of Moorea, French Polynesia

Analysis of five-year records of temperatures and currents collected at Moorea reveal strong internal wave activity at predominantly semi-diurnal frequencies impacting reef slopes at depths 30m around the entire island. Temperature changes of 1.5C to 3C are accompanied by surges of upward and onshore flow and vertical shear in onshore currents. Superimposed on annual temperature changes of approximately 3C, internal wave activity is high from Oct-May and markedly lower from Jun-Sep. The offshore pycnocline is broadly distributed with continuous stratification to at least 500m depth, and a subsurface fluorescence maximum above the strong nutricline at approximately 200m. Minimum buoyancy periods range from 4.8 to 6min, with the maximum density gradient occurring at 50 to 60m depth in summer and deepening to approximately 150 to 200m in winter. The bottom slope angle around all of Moorea is super-critical relative to the vertical stratification angle suggesting that energy propagating into shallow water is only a portion of total incident internal wave energy. Vertical gradient Richardson numbers indicate dominance by density stability relative to current shear with relatively limited diapycnal mixing. Coherence and lagged cross-correlation of semi-diurnal temperature variation indicate complex patterns of inter-site arrival of internal waves and no clear coherence or lagged correlation relationships among island sides. Semi-diurnal and high frequency internal wave packets likely arrive on Moorea from a combination of local and distant sources and may have important impacts for nutrient and particle fluxes in deep reef environments. © 2012 American Geophysical Union. All Rights Reserved.

Modeling the dynamics of Magnetic Semilevitation Melting

In semilevitation melting, a cylindrical metal ingot is melted by a coaxial a.c. induction coil. A watercooled solid base supports the ingot, while the top and side free surface is confined by the magnetic forces as the melting front progresses. The dynamic interplay between gravity, hydrodynamic stress, and the Lorentz force in the fluid determines the instantaneous free surface shape. The coupled nonstationary equations for turbulent flow, heat with phase change, and high-frequency electromagnetic field are solved numerically for the axisymmetric time-dependent domain by a continuous mesh transformation, using a pseudospectral method. Results are obtained for the two actually existing coil configurations and several validation cases.

Droplet generation with modulated AC electromagnetic field at nozzle exit

A method of droplet generation based on applying a modulated AC high frequency magnetic field in the localized region of capillary breakup is considered as ans alternative to traditional methods for high temperature liquid melt droplet serial production by pressure variation. The method is based on a pseudo-spectral approximation with a coordinate transformation adaptin to the developing free surface. The electromagnetic field is recomputed continuously with the domain shape change. Practical application cases for liquid silicon droplets of 0.5 - 2 mm diameter are considered in detail.

Identifying the damage mechanisms in paper using the acoustic emission monitoring technique

This paper investigates the use of the acoustic emission (AE) monitoring technique for use in identifying the damage mechanisms present in paper associated with its production process. The microscopic structure of paper consists of a random mesh of paper fibres connected by hydrogen bonds. This implies the existence of two damage mechanisms, the failure of a fibre-fibre bond and the failure of a fibre. This paper describes a hybrid mathematical model which couples the mechanics of the mass-spring model to the acoustic wave propagation model for use in generating the acoustic signal emitted by complex structures of paper fibres under strain. The derivation of the mass-spring model can be found in [1,2], with details of the acoustic wave equation found in [3,4]. The numerical implementation of the vibro-acoustic model is discussed in detail with particular emphasis on the damping present in the numerical model. The hybrid model uses an implicit solver which intrinsically introduces artificial damping to the solution. The artificial damping is shown to affect the frequency response of the mass-spring model, therefore certain restrictions on the simulation time step must be enforced so that the model produces physically accurate results. The hybrid mathematical model is used to simulate small fibre networks to provide information on the acoustic response of each damage mechanism. The simulated AEs are then analysed using a continuous wavelet transform (CWT), described in [5], which provides a two dimensional time-frequency representation of the signal. The AEs from the two damage mechanisms show different characteristics in the CWT so that it is possible to define a fibre-fibre bond failure by the criteria listed below. The dominant frequency components of the AE must be at approximately 250 kHz or 750 kHz. The strongest frequency component may be at either approximately 250 kHz or 750 kHz. The duration of the frequency component at approximately 250 kHz is longer than that of the frequency component at approximately 750 kHz. Similarly, the criteria for identifying a fibre failure are given below. The dominant frequency component of the AE must be greater than 800 kHz. The duration of the dominant frequency component must be less than 5.00E-06 seconds. The dominant frequency component must be present at the front of the AE. Essentially, the failure of a fibre-fibre bond produces a low frequency wave and the failure of a fibre produces a high frequency pulse. Using this theoretical criteria, it is now possible to train an intelligent classifier such as the Self-Organising Map (SOM) [6] using the experimental data. First certain features must be extracted from the CWTs of the AEs for use in training the SOM. For this work, each CWT is divided into 200 windows of 5E-06s in duration covering a 100 kHz frequency range. The power ratio for each windows is then calculated and used as a feature. Having extracted the features from the AEs, the SOM can now be trained, but care is required so that the both damage mechanisms are adequately represented in the training set. This is an issue with paper as the failure of the fibre-fibre bonds is the prevalent damage mechanism. Once a suitable training set is found, the SOM can be trained and its performance analysed. For the SOM described in this work, there is a good chance that it will correctly classify the experimental AEs.

Droplet formation with electromagnetic pulse force

Methods for serial generation of droplets from a liquid jet are shortly reviewed. A method of liquid metal droplet generation based on AC high frequency magnetic field is considered in detail. Numerical model for direct simulation of the time dependent droplet generation process is presented. Computed examples demonstrate the liquid silicon droplet formation for the cases of 500-1500 μm diameter.

Dynamic melting model for small samples in cold crucible

Purpose – A small size cold crucible offers possibilities for melting various electrically conducting materials with a minimal wall contact. Such small samples can be used for express contamination analysis, preparing limited amounts of reactive alloys or experimental material analyses. Aims to present a model to follow the melting process. Design/methodology/approach – The presents a numerical model in which different types of axisymmetric coil configurations are analysed. Findings – The presented numerical model permits dynamically to follow the melting process, the high-frequency magnetic field distribution change, the free surface and the melting front evolution, and the associated turbulent fluid dynamics. The partially solidified skin on the contact to the cold crucible walls and bottom is dynamically predicted. The segmented crucible shape is either cylindrical, hemispherical or arbitrary shaped. Originality/value – The model presented within the paper permits the analysis of melting times, melt shapes, electrical efficiency and particle tracks.

ALES: a multi-mission adaptive sub-waveform retracker for coastal and open ocean altimetry

Satellite altimetry has revolutionized our understanding of ocean dynamics thanks to frequent sampling and global coverage. Nevertheless, coastal data have been flagged as unreliable due to land and calm water interference in the altimeter and radiometer footprint and uncertainty in the modelling of high-frequency tidal and atmospheric forcing. Our study addresses the first issue, i.e. altimeter footprint contamination, via retracking, presenting ALES, the Adaptive Leading Edge Subwaveform retracker. ALES is potentially applicable to all the pulse-limited altimetry missions and its aim is to retrack both open ocean and coastal data with the same accuracy using just one algorithm. ALES selects part of each returned echo and models it with a classic ”open ocean” Brown functional form, by means of least square estimation whose convergence is found through the Nelder-Mead nonlinear optimization technique. By avoiding echoes from bright targets along the trailing edge, it is capable of retrieving more coastal waveforms than the standard processing. By adapting the width of the estimation window according to the significant wave height, it aims at maintaining the accuracy of the standard processing in both the open ocean and the coastal strip. This innovative retracker is validated against tide gauges in the Adriatic Sea and in the Greater Agulhas System for three different missions: Envisat, Jason-1 and Jason-2. Considerations of noise and biases provide a further verification of the strategy. The results show that ALES is able to provide more reliable 20-Hz data for all three missions in areas where even 1-Hz averages are flagged as unreliable in standard products. Application of the ALES retracker led to roughly a half of the analysed tracks showing a marked improvement in correlation with the tide gauge records, with the rms difference being reduced by a factor of 1.5 for Jason-1 and Jason-2 and over 4 for Envisat in the Adriatic Sea (at the closest point to the tide gauge).

Difference equation approach to two-thermocouple sensor characterisation in constant velocity flow environments

Thermocouples are one of the most popular devices for temperature measurement due to their robustness, ease of manufacture and installation, and low cost. However, when used in certain harsh environments, for example, in combustion systems and engine exhausts, large wire diameters are required, and consequently the measurement bandwidth is reduced. This article discusses a software compensation technique to address the loss of high frequency fluctuations based on measurements from two thermocouples. In particular, a difference equation sDEd approach is proposed and compared with existing methods both in simulation and on experimental test rig data with constant flow velocity. It is found that the DE algorithm, combined with the use of generalized total least squares for parameter identification, provides better performance in terms of time constant estimation without any a priori assumption on the time constant ratios of the thermocouples.