The effect of some triazole derivatives as inhibitors for the corrosion of mild steel in 1 M hydrochloric acid

Corrosion inhibition by some new triazole derivatives on mild steel in 1 M hydrochloric acid solutions has been investigated by weight loss test, electrochemical measurement, scanning electronic microscope analysis and quantum chemical calculations. The results indicate that these compounds act as mixed-type inhibitors retarding the anodic and cathodic corrosion reactions and do not change the mechanism of either hydrogen evolution reaction or mild steel dissolution. The studied compounds following the Langmuir adsorption isotherm, and the thermodynamic parameters were determined and discussed. The effect of molecular structure on the inhibition efficiency has been investigated by ab initio quantum chemical calculations. The electronic properties such as highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO) energy levels, energy gap (LUMO-HOMO), dipole moment and molecular orbital densities were calculated. (C) 2009 Published by Elsevier B.V.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mm borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic N134 cells induced by arsenic trioxide (AS(2)O(3)) at low concentration (1-2 mu m). Buthionine sulfoximine (BSO), in combination with AS(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of AS(2)O(3) and hydrogen peroxide (H2O2) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

How Useful is Lifelong Evolution for Robotics

Walker,J. and Wilson,M.S., 'How Useful is Lifelong Evolution for Robotics', Proceedings of the 7th International Conference on Simulation of Adaptive Behaviour, ed Hallam,B. and Floreano,D. and Hallam,J. and Hayes,G. and Meyer,J.A., pp 347-348, 2002, MIT Press

Singular perturbation of quantum stochastic differential equations with coupling through an oscillator mode

Gough, John; Van Handel, R., (2007) 'Singular perturbation of quantum stochastic differential equations with coupling through an oscillator mode', Journal of Statistical Physics 127(3) pp.575-607 RAE2008

A study of the oxygen electrochemistry of ruthenium and iridium

This thesis is concerned with an investigation of the anodic behaviour of ruthenium and iridium in aqueous solution and particularly of oxygen evolution on these metals. The latter process is of major interest in the large-scale production of hydrogen gas by the electrolysis of water. The presence of low levels of ruthenium trichloride ca. 10-4 mol dm-3 in acid solution give a considerable increase in the rate of oxygen evolution from platinum and gold, but not graphite, anodes. The mechanism of this catalytic effect was investigated using potential step and a.c. impedance technique. Earlier suggestions that the effect is due to catalysis by metal ions in solution were proved to be incorrect and it was shown that ruthenium species were incorporated into the surface oxide film. Changes in the oxidation state of these ruthenium species is probably responsible for the lowering of the oxygen overvoltage. Both the theoretical and practical aspects of the reaction were complicated by the fact that at constant potential the rates of both the catalysed and the uncatalysed oxygen evolution processes exhibit an appreciable, continuous decrease with either time or degree of oxidation of the substrate. The anodic behaviour of iridium in the oxide layer region has been investigated using conventional electrochemical techniques such as cyclic voltammetry. Applying a triangular voltage sweep at 10 Hz, 0.01 to 1.50V increases the amount of electric charge which the surface can store in the oxide region. This activation effect and the mechanism of charge storage is discussed in terms of both an expanded lattice theory for oxide growth on noble metals and a more recent theory of irreversible oxide formation with subsequent stoichiometry changes. The lack of hysteresis between the anodic and cathodic peaks at ca. 0.9 V suggests that the process involved here is proton migration in a relatively thick surface layer, i.e. that the reaction involved is some type of oxide-hydroxide transition. Lack of chloride ion inhibition in the anodic region also supports the irreversible oxide formation theory; however, to account for the hydrogen region of the potential sweep a compromise theory involving partial reduction of the outer regions of iridium oxide film is proposed. The loss of charge storage capacity when the activated iridium surface is anodized for a short time above ca. 1.60 V is attributed to loss by corrosion of the outer active layer from the metal surface. The behaviour of iridium at higher anodic potentials in acid solution was investigated. Current-time curves at constant potential and Tafel plots suggested that a change in the mechanism of the oxygen evolution reaction occurs at ca. 1.8 V. Above this potential, corrosion of the metal occurred, giving rise to an absorbance in the visible spectrum of the electrolyte (λ max = 455 nm). It is suggested that the species involved was Ir(O2)2+. A similar investigation in the case of alkaline electrolyte gave no evidence for a change in mechanism at 1.8 V and corrosion of the iridium was not observed. Oxygen evolution overpotentials were much lower for iridium than for platinum in both acidic and alkaline solutions.

Optimisation of immune effector function within the tumour microenvironment

Cancer represents a leading of cause of death in the developed world, inflicting tremendous suffering and plundering billions from health budgets. The traditional treatment approaches of surgery, radiotherapy and chemotherapy have achieved little in terms of cure for this deadly disease. Instead, life is prolonged for many, with dubious quality of life, only for disease to reappear with the inevitable fatal outcome. “Blue sky” thinking is required to tackle this disease and improve outcomes. The realisation and acceptance of the intrinsic role of the immune system in cancer pathogenesis, pathophysiology and treatment represented such a “blue sky” thought. Moreover, the embracement of immunotherapy, the concept of targeting immune cells rather than the tumour cells themselves, represents a paradigm shift in the approach to cancer therapy. The harnessing of immunotherapy demands radical and innovative therapeutic endeavours – endeavours such as gene and cell therapies and RNA interference, which two decades ago existed as mere concepts. This thesis straddles the frontiers of fundamental tumour immunobiology and novel therapeutic discovery, design and delivery. The work undertaken focused on two distinct immune cell populations known to undermine the immune response to cancer – suppressive T cells and macrophages. Novel RNAi mediators were designed, validated and incorporated into clinically relevant gene therapy vectors – involving a traditional lentiviral vector approach, and a novel bacterial vector strategy. Chapter 2 deals with the design of novel RNAi mediators against FOXP3 – a crucial regulator of the immunosuppressive regulatory T cell population. Two mediators were tested and validated. The superior mediator was taken forward as part of work in chapter 3. Chapter 3 deals with transposing the RNA sequence from chapter 2 into a DNA-based construct and subsequent incorporation into a lentiviral-based vector system. The lentiviral vector was shown to mediate gene delivery in vitro and functional RNAi was achieved against FOXP3. Proof of gene delivery was further confirmed in vivo in tumour-bearing animals. Chapter 4 focuses on a different immune cell population – tumour-associated macrophages. Non-invasive bacteria were explored as a specific means of delivering gene therapy to this phagocytic cell type. Proof of delivery was shown in vitro and in vivo. Moreover, in vivo delivery of a gene by this method achieved the desired immune response in terms of cytokine profile. Overall, the data presented here advance exploration within the field of cancer immunotherapy, introduce novel delivery and therapeutic strategies, and demonstrate pre-clinically the potential for such novel anti-cancer therapies.

Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

Characterisation of bacteriophage-host interactions in Lactococcus lactis

Lactococcus lactis is used extensively world-wide for the production of fermented dairy products. Bacteriophages (phages) infecting L. lactis can result in slow or incomplete fermentations, or may even cause total fermentation failure. Therefore, bacteriophages disrupting L. lactis fermentation are of economic concern. This thesis employed a multifaceted approach to investigate various molecular aspects of phage-host interaction in L. lactis. The genome sequence of an Irish dairy starter strain, the prophage-cured L. lactis subsp. cremoris UC509.9, was studied. The 2,250,427 bp circular chromosome represents the smallest among its sequenced lactococcal equivalents. The genome displays clear genetic adaptation to the dairy niche in the form of extensive reductive evolution. Gene prediction identified 2066 protein-encoding genes, including 104 which showed significant homology to transposase-specifying genes. Over 9 % of the identified genes appear to be inactivated through stop codons or frame shift mutations. Many pseudogenes were found in genes that are assigned to carbohydrate and amino acid transport and metabolism orthologous groups, reflecting L. lactis UC509.9’s adaptation to the lactose and casein-rich dairy environment. Sequence analysis of the eight plasmids of L. lactis revealed extensive adaptation to the dairy environment. Key industrial phenotypes were mapped and novel lactococcal plasmid-associated genes highlighted. In addition to chromosomally-encoded bacteriophage resistance systems, six functional such systems were identified, including two abortive infection systems, AbiB and AbiD1, explaining the observed phage resistance of L. lactis UC509.9 Molecular analysis suggests that the constitutive expression of AbiB is not lethal to cells, suggesting the protein is expressed in an un/inactivated form. Analysis of 936 species phage sk1-escape mutants of AbiB revealed that all such mutants harbour mutations in orf6, which encodes the major capsid protein. Results suggest that the major capsid protein is required for activation of the AbiB system, although this requires furrther investigations. Temporal transcriptomes of L. lactis UC509.9 undergoing lytic infection with either one of two distinct bacteriophages, Tuc2009 and c2, was determined and compared to the transcriptome of uninfected UC509.9 cells. Whole genome microarrays performed at various time-points post-infection demonstrated a rather modest impact on host transcription. Alterations in the UC509.9 transcriptome during lytic infection appear phage-specific, with a relatively small number of differentially transcribed genes shared between infection with either Tuc2009 or c2. Transcriptional profiles of both bacteriophages during lytic infection was shown to generally correlate with previous studies and allowed the confirmation of previously predicted promoter sequences. Bioinformatic analysis of genomic regions encoding the presumed cell wall polysaccharide (CW PS) biosynthesis gene cluster of several strains of L. lactis was performed. Results demonstrate the presence of three dominant genetic types of this gene cluster, termed type A, B and C. These regions were used for the development of a multiplex PCR to identify CW PS genotype of various lactococcal strains. Analysis of 936 species phage receptor binding protein phylogeny (RBP) and CW PS genotype revealed an apparent correlation between RBP phylogeny and CW PS type, thereby providing a partial explanation for the observed narrow host range of 936 phages. Further analysis of the genetic locus encompassing the presumed CW PS biosynthesis operon of eight strains identified as belonging to the CW PS C (geno)type, revealed the presence of a variable region among the examined strains. The obtained comparative analysis allowed for the identification of five subgroups of the C type, named C1 to C5. We purified an acidic polysaccharide from the cell wall of L. lactis 3107 (C2 subtype) and confirmed that it is structurally different from the CW PS of the C1 subtype L. lactis MG1363. Combinations of genes from the variable region of C2 subtype were amplified from L. lactis 3107 and introduced into a mutant of the C1 subtype L. lactis NZ9000 (a direct derivative of MG1363) deficient in CW PS biosynthesis. The resulting recombinant mutant synthesized a CW PS with a composition characteristic for that of the C2 subtype L. lactis 3107 and not the wildtype C1 L. lactis NZ9000. The recombinant mutant exhibited a changed phage resistance/sensitivity profile consistent with that of L. lactis 3107, which unambiguously demonstrated that L. lactis 3107 CW PS is the host cell surface receptor of two bacteriophages belonging to the P335 species as well as phages that are member of the 936 species. The research presented in this thesis has significantly advanced our understanding of L. lactis bacteriophage-host interactions in several ways. Firstly, the examination of plasmidencoded bacteriophage resistance systems has allowed inferences to be made regarding the mode of action of AbiB, thereby providing a platform for further elucidation of the molecular trigger of this system. Secondly, the phage infection transcriptome data presented, in addition to previous work, has made L. lactis a model organism in terms of transcriptomic studies of bacteriophage-host interactions. And finally, the research described in this thesis has for the first time explicitly revealed the nature of a carbohydrate bacteriophage receptor in L. lactis, while also providing a logical explanation for the observed narrow host ranges exhibited by 936 and P335 phages. Future research in discerning the structures of other L. lactis CW PS, combined with the determination of the molecular interplay between receptor binding proteins of these phages and CW PS will allow an in depth understanding of the mechanism by which the most prevalent lactococcal phages identify and adsorb to their specific host.

Solo and Chamber Music of Pulitzer Prize-Winning Composers

The Pulitzer Prize in Music, established in 1943, is one of America's most prestigious awards. It has been awarded to fifty-three composers for a "distinguished musical composition of significant dimension by an American that has had its first performance in the United States during the year." Composers who have won the Pulitzer Prize are considered to be at the pinnacle of their creativity and have provided the musical world with classical music compositions worthy of future notice. By tracing the history of Pulitzer Prize-winning composers and their compositions, researchers and musicians enhance their understanding of the historical evolution of American music, and its impact on American culture. Although the clarinet music of some of these composers is rarely performed today, their names will be forever linked to the Pulitzer, and because of that, their compositions will enjoy a certain sense of immortality. Of the fifty-four composers who have won the award, forty-seven have written for the clarinet in a solo or chamber music setting (five or less instruments). Just as each Pulitzer Prize-winning composition is a snapshot of the state of American music at that time, these works trace the history of American clarinet musical development, and therefore, they are valuable additions to the clarinet repertoire and worthy of performance. This dissertation project consists of two recitals featuring the solo and chamber clarinet music of sixteen Pulitzer Prize-winning composers, extended program notes containing information on each composer's life, their music, the Pulitzer Prize-winning composition and the recital selection, and a complete list of all Pulitzer Prize-winning composers and their solo and chamber clarinet music. Featured Composers Dominick Argento, To Be Sung Upon the Water Leslie Bassett, Soliloquies William Bolcom, Little Suite of Four Dances Aaron Copland, As it Fell Upon a Day John Corigliano, Soliloquy Norman Dello Joio, Concertante Morton Gould, Benny's Gig Charles Ives, Largo Douglas Moore, Quintet for Clarinet and Strings George Perle, Three Sonatas Quincy Porter, Quintet for Clarinet and Strings Mel Powell, Clarinade Shulamit Ran, Private Game Joseph Schwantner, Entropy Leo Sowerby, Sonata Ernst Toch, Adagio elegiaco

Pharmacological targeting of the mitochondrial phosphatase PTPMT1.

The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 microM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic beta-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes.

Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis.

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

An active learning approach for rapid characterization of endothelial cells in human tumors.

Currently, no available pathological or molecular measures of tumor angiogenesis predict response to antiangiogenic therapies used in clinical practice. Recognizing that tumor endothelial cells (EC) and EC activation and survival signaling are the direct targets of these therapies, we sought to develop an automated platform for quantifying activity of critical signaling pathways and other biological events in EC of patient tumors by histopathology. Computer image analysis of EC in highly heterogeneous human tumors by a statistical classifier trained using examples selected by human experts performed poorly due to subjectivity and selection bias. We hypothesized that the analysis can be optimized by a more active process to aid experts in identifying informative training examples. To test this hypothesis, we incorporated a novel active learning (AL) algorithm into FARSIGHT image analysis software that aids the expert by seeking out informative examples for the operator to label. The resulting FARSIGHT-AL system identified EC with specificity and sensitivity consistently greater than 0.9 and outperformed traditional supervised classification algorithms. The system modeled individual operator preferences and generated reproducible results. Using the results of EC classification, we also quantified proliferation (Ki67) and activity in important signal transduction pathways (MAP kinase, STAT3) in immunostained human clear cell renal cell carcinoma and other tumors. FARSIGHT-AL enables characterization of EC in conventionally preserved human tumors in a more automated process suitable for testing and validating in clinical trials. The results of our study support a unique opportunity for quantifying angiogenesis in a manner that can now be tested for its ability to identify novel predictive and response biomarkers.

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.