Toxoplasma gondii infects approximately one third of the population worldwide. There is strong evidence that a relationship between T. gondii titer and depressive symptoms exists. There is also evidence suggesting a protective effect of RhD positive blood factor on toxoplasma-induced behavioral and personality changes. This protective effect may influence the relationship between T. gondii and prenatal depressive symptoms. The purpose of this secondary data analysis was to examine the possible protective effect of RhD positive blood factor on prenatal depressive symptoms in 56 pregnant women with T. gondii infection. The cross-sectional design was utilized to answer the question “Does positive RhD blood factor provide a protective effect on prenatal depressive symptoms of patients infected with T. gondii when controlling for ethnicity, race, income, marital status, age and stress?” The conceptual model hypothesized that there was a relationship between socio-demographic variables (age, income, marital status, race, and ethnicity), stress, positive T. gondii titers, RhD positive blood factor, and prenatal depressive symptoms. Pearson correlations and multiple regression were utilized to explore the aims of this study demonstrated in the four statistical models. Significant relationship between stress and positive T.gondii seropositivity on prenatal depressive symptoms was identified. There was no significant relationship identified between RhD positive blood factor on the pregnant women infected with T. gondii which could be attributed to the small sample size.

La protéine RhBG humaine est un membre de la famille des protéines Rh (Rhésus), premiers canaux à gaz caractérisés chez les mammifères et par conséquent, de la superfamille Amt/Mep/Rh des transporteurs d'ammonium. Elle facilite le transport de la forme neutre de l'ammonium (NH3) et est ancrée à la membrane plasmique basolatérale de cellules épithéliales rénales, via l'ankyrine G, protéine adaptatrice du squelette membranaire dépendant de la spectrine. Nous avons montré que la mutation du motif d'ancrage à l'ankyrine G, localisé dans l'extrémité C-terminale intracytoplasmique de RhBG, retarde l'adressage, diminue la stabilité à la surface et abolit la fonction de transport de NH3 de la protéine RhBG dans les cellules épithéliales HEK293. Nous avons également montré que la tyrosine Y429, qui appartient au signal d'adressage basolatéral YED de RhBG, est phosphorylée in vitro par les kinases Src et Syk purifiées. D'autre part, le traitement de cellules HEK293 par le pervanadate, un inhibiteur de phosphatases sur phosphotyrosine (donc un activateur indirect des tyrosine kinases) diminue fortement l'activité de transport de la protéine native mais pas celle d'un mutant Y429A (bloquant sa phosphorylation). Ce résultat montre d'une part que Y429 est le seul résidu tyrosine de RhBG impliqué dans la régulation du transport de NH3, via une phosphorylation, d'autre part que la protéine RhBG est phosphorylée ex vivo sur cette tyrosine. Des mutations Y429D et Y429E, mimant une phosphorylation constitutive, abolissent le transport de NH3 et favorisent la solubilisation du RhBG membranaire. À l'inverse, des mutants Y429A et Y429F non phosphorylés et non phosphorylables se comportent comme la protéine RhBG sauvage. En revanche, des études en microscopie confocale montrent que seules les mutations Y429A et Y429F entraînent une dépolarisation de l'expression de RhBG dans des cellules MDCK polarisées, les mutants Y429D et Y429E étant normalement localisés dans le domaine basolatéral des cellules. L'ensemble de ces résultats démontre que l'adressage, l'ancrage au squelette sous-membranaire et la fonction de transport d'ammonium de RhBG sont régulés à la fois par la phosphorylation et la liaison au squelette membranaire de son domaine C-terminal cytoplasmique et nous a conduit à proposer un modèle dans lequel la phosphorylation de la tyrosine Y429 faisant partie du motif YED, serait nécessaire à l'adressage de RhBG vers la membrane basolatérale de cellules épithéliales polarisées, alors que la déphosphorylation de cette tyrosine permettrait l'ancrage de la protéine au squelette membranaire, via l'ankyrine G, et l'activation du canal à NH3 par un changement conformationnel potentiel de l'extrémité C-terminale.

<p>Brain-machine interfaces (BMIs) use neuronal activity to control external actuators. As such, they show great promise for restoring motor and communication abilities in persons with paralysis or debilitating neurological disorders.</p><p>While BMIs aim to enact normal sensorimotor functions, so far they have lacked afferent feedback in the form of somatic sensation. This deficiency limits the utility of current BMI designs and may hinder the translation of future clinical BMIs, which will need a means of delivering sensory signals from prosthetic devices back to the user. </p><p>This dissertation describes the development of brain-machine-brain interfaces (BMBIs) capable of bidirectional communication with the brain. The interfaces consisted of efferent and afferent modules. The efferent modules decoded motor intentions from the activity of populations of cortical neurons recorded with chronic multielectrode recording arrays. The activity of these ensembles was used to drive the movements of a computer cursor and a realistic upper-limb avatar. The afferent modules encoded tactile feedback about the interactions of the avatar with virtual objects through patterns of intracortical microstimulation (ICMS).</p><p>I first show that a direct intracortical signal can be used to instruct rhesus monkeys about the direction of a reach to make with a BMI. Rhesus monkeys placed an actuator over an instruction target and obtained, from the target's artificial texture, information about the correct reach path. Initially these somatosensory instructions took the form of vibrotactile stimulation of the hands. Next, ICMS of primary somatosensory cortex (S1) in one monkey and posterior parietal cortex (PPC) in another was substituted for this peripheral somatosensory signal. Finally, the monkeys made direct brain-controlled reaches using the activity of ensembles of primary motor cortex (M1) cells, conditional on the ICMS cues. The monkey receiving ICMS of S1 was able to achieve the same level of proficiency with ICMS as with the stimulus delivered to the skin of the hand. The monkey receiving ICMS of PPC was unable to perform the task above chance. This experiment indicates that ICMS of S1 can form the basis of an afferent prosthetic input to the brain for guiding brain-controlled prostheses.</p><p>I next show that ICMS of S1 can provide feedback about the interactions of a virtual-reality upper-limb avatar and virtual objects, enabling active touch. Rhesus monkeys initially controlled the avatar with the movements of their arms and used it to search through sets of up to three objects. Feedback in the form of temporal patterns of ICMS occurred whenever the avatar touched a virtual object. Monkeys learned to use this feedback to find the objects with particular artificial textures, as encoded by the ICMS patterns, and select those associated with reward while avoiding selecting the non-rewarded objects. Next, the control of the avatar was switched to direct brain-control and the monkeys continued to move the avatar with motor commands derived from the extracellular neuronal activity of M1 cells. The afferent and efferent modules of this BMBI were temporally interleaved, and as such did not interfere with each other, yet allowed effectively concurrent operation. Cortical motor neurons were measured while the monkey passively observed the movements of the avatar and were found to be modulated, a result that suggests that concurrent visual and artificial somatosensory feedback lead to the incorporation of the avatar into the monkey's internal brain representation.</p><p>Finally, I probed the sensitivity of S1 to precise temporal patterns of ICMS. Monkeys were trained to discriminate between periodic and aperiodic ICMS pulse trains. The periodic pulse-trains consisted of 200 Hz bursts at a 10 Hz secondary frequency. The aperiodic pulse trains had a distorted periodicity and consisted of 200 Hz bursts at a variable instantaneous secondary frequency. The statistics of the aperiodic pulse trains were drawn from a gamma distribution with equal mean inter-burst intervals to the periodic pulse trains. The monkeys were able to distinguish periodic pulse trains from aperiodic pulse trains with coefficients of variation of 0.25 or greater. This places an upper-bounds on the communication bandwidth that can be achieved with a single channel of temporal ICMS in S1.</p><p>In summary, rhesus monkeys were augmented with a bidirectional neural interface that allowed them to make reaches to objects and discriminate them by their textures--all without making actual movements and without relying on somatic sensation from their real bodies. Both action and perception were mediated by the brain-machine-brain interface. I probed the sensitivity of the afferent leg of the interface to precise temporal patterns of ICMS. Moreover, I describe evidence that the BMBI controlled avatar was incorporated into the monkey's internal brain representation. These results suggest that future clinical neuroprostheses could implement realistic feedback about object-actuator interactions through patterns of ICMS, and that these artificial somatic sensations could lead to the incorporation of the prostheses into the user's body schema.</p> / Dissertation

In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity. Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea. This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.

B virus diagnosis presents a challenge largely complicated by the asymptomatic infection of rhesus macaques, and extremely pathogenic fatal infections in humans. Humoral detection of antibodies is generally performed using whole virus antigen for which preparation requires strict biosafety measures and specialized BSL-4 facilities. As an alternative to utilizing B virus antigen, we describe the production of a truncated form of B virus envelope glycoprotein D, gD 287, in a baculovirus expression system, and evaluate its diagnostic potential as an antigen in recombinant ELISA. After purification and characterization, gD 287 was tested using 22 negative and 72 positive macaque sera samples previously classified using the traditional method. We find that sensitivity and specificity of the recombinant ELISA are dependent on antibody titer of tested serum and gD 287 shows good to excellent predictive potential for identification of positive sera with titers higher than 500.

In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity. Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea. This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.

In the present study, electrophysiological and behavioral effects of compromised Gama-Aminobutyric Acid (GABAergic) transmission were investigated in adult Rhesus macaque monkeys (N=2). GABAergic transmission was perturbed in the putamen by administration of a GABAa receptor antagonist, gabazine (10 and 500 μM), via a microdialysis-local field potential (MD-LFP) probe. Resultant changes in striatal local field potentials (LFPs) were measured as an assay of synchrony. Gabazine perfusion evoked discrete large amplitude spikes in LFPs in all subjects, and the frequency and shape of individual spikes were concentration-dependent. Pre-treatment with the GABAa receptor agonist, muscimol (100 μM) blocked the gabazine-induced events, confirming a role for GABAa receptors in the effects. Behavioral manifestations of gabazine treatment were observed only at the maximum concentration. Unusual facial movements suggested aberrant electrical activity was propagated from striatum to motor cortex, perhaps via reentrant circuits. These results support a role for GABAergic transmission in segregation of striatal circuits.

CD8+ T cells responses are critical in the immune defense against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection. A major challenge for vaccine development is that HIV/SIV can rapidly mutate to escape containment by the CD8+ T cell response. Therefore, optimal virus control by a vaccine will likely require clonally diverse CD8+ T cells capable of recognizing mutant viruses. Mucosal tissues play a fundamental role in early HIV/SIV pathogenesis by serving as the site for viral entry, CD4+ T cell depletion, and a reservoir for viral replication. Vaccine strategies that induce effective mucosal immunity will likely be critical for protection against HIV/SIV. We examined the SIV Gag p11C-specific CD8+ T cell responses in peripheral blood, gastrointestinal (GI) mucosal and lung mucosal tissues of rhesus monkeys expressing the MHC class I molecule Mamu-A*01. We first investigated the clonal composition of this cell population during the acute and chronic phases of SIVmac infection. We showed that there is a narrowing of the clonal repertoire from acute to chronic infection and the same clonal populations of virus-specific CD8+ T cells are present in the systemic and mucosal compartments of chronically SIV-infected animals. These data indicated that virus-specific CD8+ T cells establish broadly distributed immune responses. Next, we examined the clonal diversity of systemic and mucosal p11C-specific CD8+ T cells induced by prime-boost vaccination. We found that systemic prime-boost vaccination induced clonally diverse p11C-specific populations in mucosal tissues. There were high levels of clonal sharing between systemic and mucosal compartments soon after vaccination. However, later following vaccination there was decreased clonal sharing between the GI mucosa and the systemic circulation. We showed that this was due to limited trafficking of p11C-specific CD8+ T cells to the GI mucosa following vaccination. Overall, these studies indicate that following SIV infection and systemic vaccination the same p11C-specific clones are present in mucosal and systemic compartments. Moreover, the apparent immune compartmentalization is a consequence of differences in cell trafficking between systemic and mucosal CD8+ T cells. These observations have important implications for the design of HIV vaccines that generate effective mucosal immunity.

Natural killer (NK) cells can kill virus-infected cells without prior antigenic exposure, and are therefore important for controlling viral replication prior to the onset of adaptive immune responses. Primate NK cells express activating and inhibitory killer-cell immunoglobulin-like receptors (KIRs) that bind to specific major histocompatibility complex (MHC) class I molecules. The importance of KIR interactions with MHC class I in human immunodeficiency virus (HIV) pathogenesis is demonstrated by the association of select KIR and MHC class I genotypes with delayed progression to acquired immunodeficiency syndrome (AIDS).

In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity. Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea. This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.