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The effects of adenosine agonists and antagonists were examined on population excitatory postsynaptic potential (population EPSP) slopes, population spike (PS) amplitudes and the relationship between the two i.e. EPSP-spike (E-S) coupling. Activation or blockade of adenosine A2A receptors responses, evoked by stimulation in the stratum radiatum, had no effect on these parameters. However, activation of the adenosine A1 receptor using N6-cyclopentyladenosine (CPA) resulted in a significant decrease in both population EPSP and population spike. When adenosine A1 receptors were activated using CPA (50 nM) the decrease in population spike amplitude was greater than could be accounted for by the decrease in population EPSP, resulting in a dissociation in the EPSP-spike relationship as measured by a right-shift in the E-S curve or a decrease in the ratio PS/EPSP. When adenosine A1 and A2A receptors were activated at the same time using CPA and 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) respectively, the depression of the E-S relationship was significantly attenuated. This was due to a mainly postsynaptic effect whereby activation of A2A receptors significantly attenuated the postsynaptic elevation of action potential threshold by adenosine A1 receptor activation. The use of a variety of signal transduction modulators suggested that adenylate cyclase, protein kinases or nitric oxide were not involved in the interaction between A2 and A1 receptors. Blocking potassium channels with barium or glibenclamide reduced the inhibitory effects of CPA on population spike amplitude but not population EPSP slope and attenuated the inhibitory effects of adenosine A1 receptor activation on E-S coupling in a similar manner to adenosine A2A receptor activation. The increases in the ratio PS/EPSP seen when A2A receptors were activated with CGS 21680 or when potassium channels were blocked with barium were not significantly different, suggesting that a change in postsynaptic potassium conductance may underlie the antagonistic effect of adenosine A2A receptor activation on A1-mediated responses. Application of ATP 10 μM to hippocampal slices initially decreased population spike amplitude and then produced long-term potentiation (LTP). 2.5 μM ATP also resulted in LTP of responses although did not inhibit the potentials during perfusion. The stable analogue of ATP, αβ-methyleneATP had no long-term effects on potentials. Blocking P2 receptors (with suramin) or N-methyl-D-aspartate (NMDA) receptors prevented the induction of LTP. When adenosine A1 receptors were blocked or the slices superfused with adenosine deaminase no inhibition of responses was seen during perfusion with ATP, nor was LTP induced. Adenosine itself did not induce LTP. These results suggest that induction of LTP following ATP perfusion requires the activation of both P2 and A1 receptors. During superfusion with ZM 241385, ATP 10 μM still induced LTP but no longer caused the inhibition of responses during perfusion. Following electrically induced LTP (100 Hz for 1 second), perfusion of ATP caused no further potentiation of responses, suggesting that electrically and ATP induced LTP share common mechanisms. Perfusion of a preparation of 5'adenylic acid deaminase (AMPase) from Aspergillus species for 10 min resulted in an initial increase in population spike amplitude which gave way to a decrease in potential size and EPSP slope that remained depressed for at least 30 min following washout of the enzyme, resulting in long-term depression (LTD). A decrease in EPSP-spike coupling was observed 30 min following washout of enzyme. Superfusion of AMPase on slices in which transmission had been potentiated using high frequency stimulation resulted in depotentiation of responses. The LTD caused by AMPase could not be inhibited by the allosteric inhibitor of the enzyme 2,3-diphosphoglyceric acid. Further investigation also ruled out the involvement of nitric oxide, protein kinase, and cyclooxygenase in the activity of the AMPase. The use of AMPase extracted from rabbit muscle resulted in no LTD of responses. Analysis of the crude enzyme extract from Aspergillus revealed that the preparation was not pure. Separation of the various components in the crude preparation led to the discovery that the AMPase was not the active factor causing depression of evoked responses. Further purification of the active protein and subsequent sequencing showed that the protein was similar to a β-glucosidase. However, perfusion of β-glucosidase extracted from almonds did not result in LTD of responses. Overall, this thesis extends knowledge of how synaptic transmission can be modulated by purines, and raises the possibility that a new family of proteins, related structurally to glucosidases, may also affect synaptic plasticity.

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Understanding the mechanisms used by HIV-1 to evade antibody neutralisation may contribute to the design of a high-coverage vaccine. This thesis explores the mechanism used by a Tier 3 virus leading to its high antibody neutralisation resistance phenotype. This thesis also describes how the glycans at the base of the V3 loop contribute to (i) breadth and potency in a cohort of unselected HIV-1-infected individuals and (ii) the selective pressures resulting from the V3/glycans shielding the virus from neutralisation and the glycans themselves being targets of broad antibody responses. HIV-1 isolates that are highly resistant to broadly neutralising antibodies could limit the efficacy of an antibody-based vaccine. For this reason, it is important to understand the mechanisms behind high HIV-1 resistance to neutralising antibodies. Chapter 2 and Chapter 3 of this thesis describe virus 253-11, a highly neutralisation resistant virus, which is particularly resistant to commonly-elicited, anti-membrane proximal external region (MPER) antibodies in sera. To further understand its resistance, mutations in the MPER were introduced that are known to delay fusion following CD4-binding and thus increase the time the virus spends in the open conformation. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4-binding by destabilising the closed trimer structure. From these data, we hypothesized that the neutralisation resistance of 253-11 was due to an unusually tight, compact pre-fusion Env trimer that resists transient changes to the open conformation. The open conformation frequently exposes narrowly-neutralising antibody epitopes. Because the unliganded 253-11 Env presumably transitions infrequently into the open conformation, it would be able to evade these responses. 253-11 was sensitive to most but not all of the most potent broadly neutralising antibodies (bnAbs) tested, most likely because those broadly neutralising antibodies can access their epitopes in the pre-fusion Env conformation. To gain further information about the structure of the 253-11 Env, we designed a recombinant 253-11 SOSIP trimer and found it to be stable and predominantly adopt a closed conformation. The crystal structure of the SOSIP trimer revealed structural elements likely responsible for 253-11 Env compactness including the inward disposition of the heptad repeat helices and gp120 protomers towards the trimer axis. Taken together, the data from Chapter 2 and Chapter 3 highlight an underappreciated Env compactness mechanism of HIV-1 resistance to neutralising antibodies and these data may be useful in HIV-1 immunogen design research. Previous candidate HIV vaccines have failed to induce wide-coverage neutralising antibodies capable of substantially protecting vaccinees. A key approach in HIV immunogen development has been to define and model epitopes recognised by anti-HIV bnAbs. Candidate immunogen models identified by bnAbs include the V3/glycans, the V2/apex and the MPER epitopes. Autoreactivity and polyreactivity of anti-V3/glycan and anti-MPER antibodies are thought to pose both direct and indirect barriers to achieving neutralisation breadth. Chapter 4 of this thesis explored which of these bnAb epitopes were associated with breadth and potency in a South African cohort of chronically HIV-infected individuals. The study found that antibodies targeting the V3/glycans were associated with breadth and potency. In contrast, antibodies to the V2/apex were not associated with neutralisation breadth/potency. This suggests that auto/polyreactivity are not critical factors in the development of breadth and potency and that the V3/glycans should remain a high-priority vaccine candidate. Since targeting the V3/glycans was associated with breadth and potency in this cohort, the study continued to look at this epitope to investigate the role of these glycans in neutralisation resistance of Tier 2 viruses. The HIV-1 Env is surrounded by glycans that often prevent antibody neutralisation, leading to the term the "glycan shield", however some bnAbs have evolved to recognise these carbohydrates. Chapter 4 of this thesis describes how the N-linked glycan at position N301 is critical for maintaining neutralisation resistance of one subtype C virus (Du156.12), but not for another subtype-matched virus (CAP45.2.00.G3). Thus, the loss of the N301 glycan may have a substantial antibody-related fitness cost for some viruses but not others. The N301 glycan, as well as glycans at positions 332 and 334, are the primary targets of the anti-V3/glycan class of neutralising antibodies, which may select for loss of the targeted glycan. The evidence presented in Chapter 4 suggests that in some viruses, loss of the N301 glycan may result in evasion of anti-V3/glycan antibody responses while maintaining overall neutralisation resistance. This phenomenon may impair efficacy of passively-infused anti-V3/glycan bnAbs or a therapeutic vaccine.

The innate immune system is the first line of defence against viral infections. Conventionally, innate immune activation begins with the detection of foreign nucleic acids by pattern recognition receptors (PRRs), which triggers a signalling cascade that culminates in the production of interferon (IFN) and other inflammatory cytokines and chemokines. Over the past few years, a number of studies have shown that IFN innate immune responses can also be triggered by stressors, such as membrane perturbations, cytoskeletal perturbations, oxidative stress, and endoplasmic reticulum (ER) stress 1–3. One way that some viruses provoke such stress responses is through membrane and cytoskeletal distortions during enveloped virus particle entry. In some cases, the glycoproteins responsible for virus particle entry can also trigger cell-cell fusion. The potential of cell-cell fusion to induce stress-based IFN responses analogous to those triggered by virus-cell fusion has not been addressed until very recently. To investigate if and how cell-cell fusion may induce antiviral mechanisms and IFN responses we used the reptilian reovirus p14 fusion associated small transmembrane (FAST) protein as a model of cell-cell fusion. We found that p14-mediated cell fusion led to the production of low level IFN and upregulation of interferon stimulated genes (ISGs) in a stimulator of interferon genes (STING) and interferon regulatory factor 3 (IRF3) dependent manner. We also observed that multinucleated cells experienced extensive DNA-damage that led to the accumulation of cytosolic DNA in the form of micronuclei. Micronuclei can be detected by cytosolic DNA PRRs like cyclic GMP-AMP synthase (cGAS) and signal IFN production through the cGAS-STING signalling axis. Additionally, early syncytia formation restricted replication of vesicular stomatitis virus (VSV), herpes simplex virus-1 (HSV), and vaccinia virus (VSV) in an IFN and IRF3 independent, and STING dependent manner, suggesting involvement of either a novel antiviral mechanism or suppression of virus replication and spread by biological changes in syncytial cells, such as cell cycle arrest. This study highlights a key role of DNA sensing pathways in the immune response to cell fusion associated stress and points out the importance of fusion kinetics in the selective advantage of syncytial viruses. Understanding the potential of syncytial cells to induce IFN responses and influence viral replication at a mechanistic level is beneficial to the design of improved oncolytic immunotherapy. / Thesis / Master of Science (MSc) / Viruses and their hosts continuously fight each other for survival. The host tries to protect itself from the virus by activating various features of its immune system, while the virus tries to block and evade detection by the immune system. One way that some viruses attempt to bypass the immune system and enhance spread involves expressing proteins that can merge together infected cells with neighboring uninfected cells. Cell-cell fusion disrupts the balanced environment within the cell, which is a form of stress that may activate immune responses. This work investigates if and how host cells may activate the immune system to respond and protect themselves from the cell merging activity of select viruses. We found that the stress associated with existing as a large, fused cell caused DNA damage and fragmentation. These DNA fragments could stimulate key immune sensors and initiate immune responses. We also observed an impaired ability of viruses to infect fused cells, but this restriction was not associated with typical immune responses, suggesting that some other biological change in fused cells created an environment that is not suitable for viral spread. Further investigation is required to fully understand this phenomenon; however, this study highlights some protective mechanisms of the host immune system in response to the stress of viral fusion protein induced cell-cell fusion.

This study investigated whether contingent-anxious conditioned stimulus termination was more important than temporally yoked termination in producing conservation or enhancement of learned fear. Thirty psychology students, twenty-six females and four males, were administered item thirty-nine from the Fear Survey Schedule and an avoidance test. After in vivo treatment exposure to a harmless snake, post-test measures identical to pretests revealed that contingent-anxious subjects retained significantly more fear (p <.05) on both indexes than temporally yoked subjects. No enhancement was found and only on the subjective measure did contingent-anxious subjects show fear conservation when contrasted with no-treatment controls (p >.05). Implications for "implosive" therapies were discussed.

Electrophysiologic responses are used to estimate hearing thresholds and fit hearing aids in young infants, but these estimates are not exact. An objective test of speech encoding could be used to validate infant fittings by showing that speech has been registered in the central auditory system. Such a test could also show the effects of auditory processing problems on the neural representation of speech. This thesis describes techniques for recording electrophysiologic responses to natural speech stimuli from the brainstem and auditory cortex. The first technique uses a Fourier analyzer to measure steady-state brainstem responses to periodicities and envelope changes in vowels, and the second uses a windowed cross-correlation procedure to measure cortical responses to the envelopes of sentences. Two studies were conducted with the Fourier analyzer. The first measured responses to natural vowels with steady and changing fundamentals, and changing formants. Significant responses to the fundamental were detected for all of the vowels, in all of the subjects, in 19 – 73 s (on average). The second study recorded responses to a vowel fundamental and harmonics. Vowels were presented in opposite polarities to distinguish envelope responses from responses to the spectrum. Significant envelope responses were detected in all subjects at the fundamental. Significant spectral responses were detected in most subjects at harmonics near formant peaks. The third study used cross-correlation to measure cortical responses to sentences. Significant envelope responses were detected to all sentences, at delays of roughly 180 ms. Responses were localized to the posterior auditory cortices. A model based on a series of overlapping transient responses to envelope changes could also account for the results, suggesting that the cortex either directly follows the speech envelope or consistently reacts to changes in this envelope. The strengths and weaknesses of both techniques are discussed in relation to their potential clinical applications.