The neurophysiology of the somatosensory system has been under investigation for decades. The coding properties of neural populations and single units in cortical and subcortical regions have been characterized extensively using electrophysiology. Tactile stimulation produces evoked responses in the thalamus and cortical regions dedicated to processing of sensory information. The ability to evoke naturalistic neural responses as well as tactile perceptual experiences by means of microstimulation in the absence of real stimulus presented to the periphery is imperative for the development of a sensory neuroprosthesis. Three specific aims were pursued in the present study: 1. The development and validation of a novel assessment technique to quantify sensory evoked responses. 2. The investigation of effects of ketamine anesthesia on the cortical evoked responses. 3. The assessment of the feasibility of microstimulation of Ventroposterior Lateral (VPL) nucleus of the thalamus as a way to produce naturalistic responses in the primary somatosensory cortex. Brief tactile stimulation of various hand locations produces characteristic responses in both thalamic and cortical multi-neuron populations. These responses were used to create somatotopic maps of the hand regions in awake animals with chronically implanted electrode arrays in the SI cortex and in anesthetized animals during acute preparations. In the first Aim, method based on the analysis of correlation was developed to compare the firing recorded on individual electrodes during different stimuli presentations to determine which neural populations showed most activation during an evoked response. The procedure required relatively short duration of stimulation sessions. The effects of anesthesia on neural coding were studied in the second Aim using ketamine anesthetized monkeys. Ketamine anesthesia is easily induced, relatively short lasting, and generally safe. In awake chronically implanted monkeys properties of neural responses to tactile stimulation were assessed. Receptive fields (RF) corresponding to five digits were identified using recordings from an electrode array placed in area 1 of SI. Cortical stimulation fields (SF) representing signal flow from each receptive field were identified. Neural responses under ketamine anesthesia were reassessed using the same mapping techniques. The responses recorded during the maintenance phase of anesthesia were similar to responses evoked under awake condition. Most changes in the neural responses occurred during induction and recovery phases. Overall, ketamine produced responses of shorter duration compared to the awake condition. In the final Aim, isofluorane/fentanyl anesthetized monkeys had microelectrode arrays acutely implanted in the VPL thalamus and area1, 3b, and 2 of SI. Recordings were used to estimate the sizes of SFs in the thalamus and cortex. Thalamic neural representations were associated with RFs that typically spanned several digits, while cortical recordings were associated with smaller RFs. Microstimulation of the VPL (1ms single pulses, 50A) resulted in the activation of somatotopically appropriate cortical neural ensembles. The responses in the VPL and cortex were characterized by the presence of High Frequency Oscillations (HFO) (up to 800Hz). The presence of the HFOs was observed following both tactile and electrical stimulation. In summary, techniques for analyzing evoked multiunit responses were developed and used to explore cortical and thalamic responses to tactile stimulation, the effects of ketamine anesthesia on such responses, and parameters for thalamic stimulation to simulate tactile “sensation” in cortex.

Co-application of the convulsant 4-aminopyridine (4-AP) and the GABAB receptor antagonist CGP 55845 to adult guinea pig hippocampal slices elicits giant GABA-mediated postsynaptic potentials (GPSPs) and interictal-like epileptiform discharges. In the first part of the thesis, I tested the effects of the group I metabotropic glutamate receptor (mGluR) subtype-selective antagonists on this synchronous activity. Electrophysiological field recordings were performed in the CA3 region of hippocampal slices from adult guinea pigs. The mGlu5 receptor antagonists MPEP and MTEP increased GPSP rate, but the mGlu1 receptor antagonist LY 367385 did not. Simultaneous block of both subtypes of group I mGluR did not decrease pre-existing epileptiform activity and also did not prevent the emergence of epileptiform activity. Thus, in the 4-AP/CGP 55845 model, enough glutamate was released to activate group I mGluRs and affect GPSP rate via mGlu5 receptors; however, this group I mGluR activation was not required for the generation of the epileptiform activity. Application of 4-AP (100 μM) in a solution containing reduced divalent cation concentrations (0.6mM Mg2+ and 1.2 mM Ca2+) to hippocampal-entorhinal-perirhinal slices of adult rat brain induced ictal-like epileptiform activity in entorhinal and perirhinal cortices as revealed by electrophysiological field potential recordings. The most exciting finding of the second part of my thesis was that epileptiform activity, including ictal-like activity, persisted after washing out the 4-AP. This persistence indicated that a change had occurred in the slice so that it was now “epileptic” in the absence of the convulsant 4-AP. The maintenance of the persistent ictal-like epileptiform activity required ionotropic glutamate-mediated synaptic transmission but not group I mGluR activation. The presence of hippocampus was not required for the induction of persistent ictal-like events in entorhinal cortex. The induction of persistent ictal-like epileptiform activity was dependent upon both NMDA and AMPA/kainate ionotropic glutamate receptor activation but not upon mGluR activation. Application of the protein synthesis inhibitor cycloheximide did not prevent the induction of persistent ictal-like activity. In conclusion, I have demonstrated both interictal-like and ictal-like activity that is not dependent upon group I mGluR activation and have characterized a new in vitro model of epileptogenesis that is clearly distinct from the group I metabotropic glutamate-dependent model.

The microsomal triglyceride transfer protein (MTP) transfers lipids (triacylglycerols, cholesteryl esters, and phospholipids) in vitro and is required for the secretion of apoB-lipoproteins in vivo. Inhibitors of MTP lipid transfer activity significantly increase the degradation of apoB and reduce its secretion. Therefore, transfer of lipids to apoB by MTP is believed to be essential for the assembly of apoB-lipoproteins. In the following studies we explored the structures and activities of evolutionarily distinct MTP homologs in order to better understand how MTP might be assisting apoB-lipoprotein assembly. We describe specific and sensitive in vitro assays to measure MTP cholesteryl ester and phospholipid transfer activities using fluorescent lipids. These activities are dependent on MTP concentration and demonstrate saturation kinetics. In contrast to the human MTP that transfers all lipids, we observed that the Drosophila MTP is deficient in the transfer of triacylglycerols and cholesteryl esters but does transfer phospholipids. Similarly the zebrafish MTP, like the human MTP, transfers triacylglycerols while the C. elegans MTP lacks this activity. Despite differences in their abilities to transfer lipids, all MTP orthologs assisted in the assembly and secretion of human apoB48-lipoproteins. Furthermore, the amounts of apoB secreted with the assistance of different MTP proteins were augmented by increasing triacylglycerol synthesis (the addition of oleic acid or expression of acyl-CoA:monoacylglycerol acyltransferase and acyl-CoA:diacylglycerol acyltransferase proteins) and decreased by treating the cells with triacsin C, an inhibitor of triacylglycerol synthesis. We subsequently showed that the secondary and tertiary structures in the orthologs were highly conserved while their primary amino acid sequences were less conserved. Interestingly the bC domains as well as helices 4-6 in the a-helical domains demonstrated greater conservation in vertebrate MTPs than the other structural domains. Together these data reveal that the phospholipid transfer activity is the most ancient activity associated with MTP and is sufficient to generate an apoB-lipoprotein particle. Triacylglycerol transfer activity was acquired during evolution and is specific to vertebrate MTP proteins. This activity is associated with greater conservation in the bC domain and helices 4-6 in the a-helical domain. Therefore, we propose that the amino acid content contained in the bC domain as well as helices 4-6 might be critical for the robust triacylglycerol transfer activity in vertebrates.

To prevent sexual transmission of HIV, a successful vaccine must elicit protective immune responses and durable immunologic memory that can respond rapidly upon virus exposure at mucosal sites. Thus, replication competent vesicular stomatitis virus (VSV) vectors are being investigated for HIV vaccine delivery with particular focus on the effect of delivery route on immune responses. VSV is a practical vector candidate because it naturally enters through mucosal sites, infects multiple cell types, and is highly immunogenic. Moreover, VSV typically does not cause disease in humans and pre-existing immunity is rare. Attenuated, replication-competent VSV vectors have been designed to co-express HIV Envelope (Env) trimers (from HIV-1 subtype B, strain JRFL) and the natural VSV attachment protein (G) derived from the Indiana (VSV-Env-GIN) or New Jersey serotype (VSV-Env-GNJ). To optimize Env trimer expression, several insert designs have been tested utilizing VSV or CD5 (human T-cell surface glycoprotein) signal sequences and different combinations of Env and VSV-G transmembrane (TM) and intracellular domains. Improved Env expression and vector genetic stability was observed when the CD5 signal and VSV-G TM sequences were used. Infected Vero cells were used to analyze cell surface expression and the antibody-binding profile of the Env trimer immunogen. Infected cells that expressed VSV G on their surface also expressed Env which was recognized by Env-specific broadly neutralizing antibodies b12, 2F5, 2G12, PGV04 and PGT126. Furthermore, the cells produced Env in a conformation that bound the antibody PGT145, which preferentially binds conformational determinants on trimeric Env. These results demonstrate that VSV can express membrane-bound trimeric Env in a conformation containing epitopes recognized by known broadly neutralizing antibodies. To investigate cellular and humoral responses elicited by vectors expressing HIV Env trimers, a heterologous prime-boost regimen was conducted in Balb/c mice with plasmid DNA prime and rVSV boost. DNA plasmids expressing EnvG were administered by intramuscular (IM) injection and electroporation (EP) with plasmid expressing IL-12 (pIL-12) at week 0 and 3 followed by a VSV-Env-GIN infection at week 6. The effect of administering the VSV boost by different routes also was investigated. A second study based on VSV-Env-GIN prime/VSV-Env-GNJ boost with both vectors administered by intranasal (IN) or IM route in different combinations administered at weeks 0 and 3 was used to study the immunogenicity of the two vectors without a DNA prime. Immune responses were analyzed by flow cytometry, ELISA, and competition binding assay at various times after the immunizations. Both studies elicited strong Env-specific antibody responses in the serum. pDNA+pIL-12 prime/ rVSV boost elicited Env-specific CD4+ T cells in the spleen and lung for the IN route. In the same pDNA+pIL-12 prime/ rVSV boost study, Env-specific CD8+ T cells were detected in the lungs for the DNA and IN groups. Notably, the IN route was able to elicit b6 and PGV04-like antibodies. These findings are of great interest as they seem to underline the importance of the quality of the elicited antibody response, which cannot be readily ascertained by measurement of the quantity of Env-specific antibodies. The titer of Env-specific antibodies has been used as a widespread measurement of vaccine efficacy in the HIV vaccine field. The results from the current studies point to the need for the characterization of these antibody responses and perhaps the quality of the antibody response should be used as the standard measurement of vaccine efficacy. The viral prime and viral boost study also elicited high Env-specific antibody responses in the serum but had a different cell-mediated response profile in that both CD4+ and CD8+ T cells were detected in the lungs but none in the spleen. The IM-IN route was able to elicit b6-like antibodies. The outcome of these studies highlights that VSV vectors expressing EnvG can elicit Env-specific humoral and cell-mediated immune responses in two different vaccine regimens. Even more importantly, these VSV-EnvG vectors were able to elicit antibodies similar to known broadly neutralizing antibodies. These findings give tremendous support to the use of EnvG as an immunogen in a live-replicating vector platform for future HIV vaccines.

This work comprises two studies involving bacteriophage T7 RNA polymerase (RNAP). In the first study, we explored the potential applications of T7 RNAP as a tightly regulated molecular motor in nanotechnology. In the second study, we investigated the mechanism by which T7 RNAP selects for correct nucleotide substrates during transcription. RNAP converts the chemical energy stored in ribonucleoside triphosphates (NTPs) into the mechanical work of transcription. Previous studies have shown that RNAP can exert forces up to 30 piconewtons (pN) as it moves along the DNA while copying the information in the DNA template into RNA. The forward motion of RNAP is dependent on the availability of the next incoming (correct) NTP that is encoded by the template strand of the DNA. Withholding a required NTP results in the formation of a stable halted elongation complex (EC) in which the enzyme remains bound to the DNA until transcription is resumed by addition of substrate. Immobilizing RNAP or the DNA template to which it is bound to a solid surface confers the ability to add and wash away different mixtures of substrates for individual transcription steps. In this way RNAP can be incrementally positioned along DNA with intervals as small as 1 base-pair (bp) or 0.34 nanometer (nm). To investigate the potential application of RNAP in nanotechnology a modified version of T7 RNAP that contains specific ligand binding domains fused to the amino-terminus of the enzyme was utilized to facilitate capture, controlled movement, and positioning of other biomolecules, and to construct and manipulate simple nanodevices. The ability to harness the linear and rotary forces exerted by single molecules of T7 RNAP was also examined. In the former case, fluorescence microscopy was used to observe unidirectional linear translocation of a nanodot cross-linked to T7 RNAP along immobilized T7 DNA. In the latter case, the ability of immobilized T7 RNAP to rotate a reporter bead, which was tethered to the downstream end of the DNA, was demonstrated using a magnetic tweezers apparatus. In the second part of this work, the fidelity of transcription by T7 RNAP was investigated. Sequence and structural comparisons indicate that T7 RNAP is related to members of a superfamily of nucleotide polymerases that include DNA directed DNA polymerases (DNAPs) of the pol I-type, mitochondrial RNA polymerases (mt RNAP), and reverse transcriptases (RT). These enzymes share a conserved structure around the catalytic site that resembles a partially closed right hand and includes fingers, palm and thumb subdomains. Structural studies indicate that T7 RNAP and bacterial DNAP I possess a similar catalytic pathway in which the active site undergoes a transition from a catalytically inactive “open” conformation to an active “closed” conformation following substrate binding. Kinetic and structural data indicate that the open to closed conformational change acts as a fidelity checkpoint mechanism, whereby a tight-fit geometry of the active site (in the closed conformation) precludes the acceptance of non-Watson-Crick base pairs. It has also been suggested that this mechanism might contribute to frameshift fidelity by regulating the transfer of the template DNA base (TSn) from a pre-insertion site (in the open configuration) to the insertion site (active site) in the closed configuration. Genetic analyses of T7 RNAP and DNAP I indicate that residues involved in fidelity are predominately located within the pre-insertion and insertion sites, respectively. Thus, it has been suggested that T7 RNAP may select the correct nucleotide in the pre-insertion site, prior to catalysis. Whether a similar mechanism might contribute to fidelity of DNAP I remains an open question. In a manner similar to that used in studies of DNAP to measure fidelity, primer/template (p/t) assemblies were used to investigate misincorporation by T7 RNAP. It was found that T7 RNAP can generate errors by a novel mechanism involving template strand re-alignment. Here, the template base immediately downstream from the active site (the TSn+1 base) is utilized during two consecutive nucleotide addition cycles. In the first cycle, the NTP complementary to TSn+1 (the +1 NTP) is incorporated due to transient formation of a –1 frameshift in which the proper template base (TSn) is presumably “flipped out” of the active site. In the second cycle, the template is re-aligned and the mismatched p/t is rapidly extended resulting in a substitution in the RNA. Changes in the organization of the transcription bubble and alanine substitution of residues Y639 and F644 near the active site were found to affect the rate of substitution errors. Lastly, T7 RNAP was found to generate identical substitution errors on templates in which transcription was initiated from a promoter suggesting that this phenomenon might be relevant to native transcription complexes within the cell.

Background: This dissertation addresses the critical public health issue of antiretroviral therapy (ART) adherence among people with HIV (PWH) who inject drugs (PWID) in Kazakhstan, a country experiencing one of the world's fastest-growing HIV epidemics. The research examines factors influencing ART adherence among PWID, with a particular focus on the impact of the COVID-19 pandemic. The central research questions explored are: "What multi-level factors are associated with ART adherence among PWID with HIV in serococordant and serodiscordant relationships?” and "What factors are associated with ART adherence among HIV-positive PWID during the COVID-19 pandemic?” Methods: This study investigated HIV medication adherence among people who inject drugs (PWID) using two complementary methodological approaches. First, qualitative analysis used in-depth interviews with 20 PWID living with HIV and 18 of their intimate partners, analyzing the dyadic data to understand relationship dynamics and shared experiences around medication adherence. Building on these qualitative insights, we then conducted a secondary-data analysis on a cross-sectional survey from October 2020 to August 2022 with 66 HIV-positive PWID and their treatment support partners (n=66). The survey data were analyzed using multilevel generalized linear mixed models with Bayesian estimation to examine factors associated with ≥90% and ≥80% adherence levels. The qualitative findings informed our selection of variables for the quantitative analysis and provided contextual depth to interpret the statistical results. Results: Qualitative results revealed multilevel barriers and facilitators of ART adherence at individual (e.g., substance use), interpersonal (e.g., social support), and structural levels (e.g., stigma, transportation). The manifestation of social support varied between seroconcordant and serodiscordant dyads. Quantitative findings showed low overall ART adherence, with 55.8% of participants achieving ≥80% adherence and only 14.7% reaching the ≥90% threshold. Key factors associated with higher adherence included: having a partner with HIV (AOR=14.94, 95% CI=1.51-420.77) for ≥90% adherence; lower conflict at home during the COVID-19 pandemic (AOR=22.77, 95% CI=1.62-841.84) for ≥90% adherence; and meeting with a professional to discuss alcohol or drug use (AOR=6.78, 95% CI=1.25-117.61) for ≥80% adherence. Factors associated with lower adherence included: poorer mental health (AOR=0.09, 95% CI=0.005, 0.76) for ≥80% adherence and experiencing negative emotional impacts of COVID-19 (AOR=0.18, 95% CI=0.002, 0.98) for ≥80% adherence. Conclusion: This dissertation concludes that relationship factors, social support, partner HIV status, mental health, and the personal impact of public health crises significantly influence ART adherence among PWID. These findings have important implications for public health policy and clinical practice in addressing the HIV epidemic in Kazakhstan and similar contexts, particularly during periods of mass disruption like the COVID-19 pandemic. Future research and interventions may benefit from integrating dyad approaches into ART adherence strategies, considering partner HIV status, prioritizing mental health support, and addressing the personal and household impacts of crises. Such comprehensive approaches could potentially improve health outcomes for PWH and reduce HIV transmission risk among PWID populations.

Glass ceramics’ mechanical properties, esthetics and biocompatibility show an important improvement compared to traditional dental ceramics. Materials such as lithium disilicate ceramics are increasingly popular in dentistry today, due to their improved mechanical performance and natural appearance, an improvement from previous systems based on zirconia. Previous research showed dental ceramics can be sintered with Microwave Hybrid Sintering (MHS), in a reduced time consuming process that may improve the mechanical properties of the material. The objective of this dissertation is to investigate if glass ceramics such as lithium disilicate can be sintered and improved with MHS. Materials and methods: Samples of lithium disilicate glass ceramics were sintered using MHS and conventional sintering, as described by Kashi et al. (2006), Pendola et al. (2013). The produced samples were tested (density, hardness, flexural strength) and analyzed (X-rays diffraction, SEM) to compare the mechanical properties and microstructure of the material with samples sintered using Conventional Sintering. Results: MHS process sintered the ceramic materials in shorter times when compared to conventional sintering. The mechanical properties of lithium disilicate glass ceramics appear to be improved using MHS. Hardness is increased in samples sintered using MHS (652 HV + 23, P<0.0001) when compared with conventional sintering (567 HV + 14) and flexural strength is also improved for MHS process (454 MPa + 51 vs 304 MPa + 75, P<0.0001). X-Ray Diffraction shows an increase of the crystalline phase in samples sintered with MHS, which may explain the improvement of the mechanical properties. SSALT shows a higher reliability for MHS ceramics when compared to Conventional Furnace Sintering processed ceramics. Conclusion: MHS can be a good alternative for sintering dental ceramics, including the most recent dental glass ceramics, by offering a firing process with reduced processing times and therefore lower energy consumption. MHS also produces an improvement on the mechanical properties of the material.

The role of endogenous amyloid beta-peptides as causal factors of neurodegenerative diseases is largely unknown. We have previously reported that interactions between Alzheimer's disease A beta P[1-40] peptide in solution and planar bilayer membranes made from anionic phospholipids lead to the formation of cation-selective channels. We now find and report here that the spontaneous insertion of free A beta P[1-40] across the bilayer can be detected as an increase in bilayer capacity. To this end we recorded the displacement currents across planar bilayers (50 mM KCl on both sides) in response to sudden displacements of the membrane potential, from -300 to 300 mV in 20-mV increments. To monitor the A beta P[1-40]-specific displacement currents, we added A beta P[1-40] (1-5 microM) to the solution on either side of the membrane and noted that the direction of the displacement current depended on the side with A beta P[1-40]. The size of the A beta P[1-40]-specific charge displaced during a pulse was always equal to the charge returning to the original configuration after the pulse, suggesting that the dipole molecules are confined to the membrane. As a rule, the steady-state distribution of the A beta P[1-40]-specific charges within the bilayer could be fit by a Boltzmann distribution. The potential at which the charges were found to be equally distributed (V(o)) were approximately -135 mV (peptide added to the solution in the compartment electrically connected to earth) and 135 mV (peptide added to the solution connected to the input of the amplifier). The A beta P[1-40]-specific transfer of charge reached a maximum value (Q(max)) when the electrical potential of the side containing the amyloid beta-protein was taken to either -300 or 300 mV. For a circular membrane of 25-microm radius ( approximately 2000 microm(2)), the total A beta P[1-40]-specific charge Q(max) was estimated as 55 fC, corresponding to some 170 e.c./microm(2). Regardless of the side selected for the addition of A beta P[1-40], at V(o) the charge displaced underwent an e-fold change for a approximately 27-mV change in potential. The effective valence (a) of the A beta P[1-40] dipole (i.e., the actual valence Z multiplied by the fraction of the electric field chi acting on the dipole) varied from 1 to 2 electronic charges. We also tested, with negative results, the amyloid peptide with the reverse sequence (A beta P[40-1]). These data demonstrate that A beta P[1-40] molecules can span the low dielectric domain of the bilayer, exposing charged residues (D(1), E(3), R(5), H(6), D(7), E(11), H(13), and H(14)) to the electric field. Thus the A beta P[1-40] molecules in solution must spontaneously acquire suitable conformations (beta-pleated sheet) allowing specific interactions with charged phospholipids. Interestingly, the domain from residues 676 to 704 in the APP(751) is homologous with the consensus sequence for lipid binding found in other membrane proteins regulated by anionic phospholipids.

CPEB-1 is a sequence-specific RNA binding protein that stimulates the polyadenylation-induced translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). Although CPEB-1 was identified originally in Xenopus oocytes, it has also been found at postsynaptic sites of hippocampal neurons where, in response to N-methyl-D-aspartate receptor activation, it is thought to induce the polyadenylation and translation of alphaCaMKII and perhaps other CPE-containing mRNAs. Because some forms of synaptic modification appear to be influenced by local (synaptic) protein synthesis, we examined long-term potentiation (LTP) in CPEB-1 knockout mice. Although the basal synaptic transmission of Schaffer collateral-CA1 neurons was not affected in the knockout mice, we found that there was a modest deficit in LTP evoked by a single train of 100 Hz stimulation, but a greater deficit in LTP evoked by one train of theta-burst stimulation. In contrast, LTP evoked by either four trains of 100 Hz stimulation or five trains of theta-burst stimulation were not or were only modestly affected, respectively. The deficit in LTP evoked by single stimulation in knockout mice appeared several minutes after tetanic stimulation. Long-term depression (LTD) evoked by 1 Hz stimulation was moderately facilitated; however, a stronger and more enduring form of LTD induced by paired-pulse 1 Hz stimulation was unaffected. These data suggest that CPEB-1 contributes in the translational control of mRNAs that is critical only for some selected forms of LTP and LTD.

Expression of VP16-CREB, a constitutively active form of CREB, in hippocampal neurons of the CA1 region lowers the threshold for eliciting the late, persistent phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This VP16-CREB-mediated L-LTP differs from the conventional late phase of LTP in not being dependent on new transcription. This finding suggests that in the transgenic mice the mRNA transcript(s) encoding the protein(s) necessary for this form of L-LTP might already be present in CA1 neurons in the basal condition. We used high-density oligonucleotide arrays to identify the mRNAs differentially expressed in the hippocampus of transgenic and wild-type mice. We then explored the contribution of the most prominent candidate genes revealed by our screening, namely prodynorphin, BDNF, and MHC class I molecules, to the facilitated LTP of VP16-CREB mice. We found that the overexpression of brain-derived neurotrophic factor accounts for an important component of this phenotype.