Human immunodeficiency virus (HIV) infection of macrophages in brain and other tissues plays an important role in development of HIV-associated neurological disorders and other aspects of disease pathogenesis. Macrophages express low levels of CD4, and macrophage-tropic HIV strains express envelope glycoproteins (Envs) adapted to overcome this restriction to virus entry by mechanisms that are not well characterized. One mechanism that influences this phenotype is increased exposure of the CD4 or CCR5 binding site, which may increase dissociation of soluble gp120 (sgp120) from Env trimers based on structural models. Little is known about spontaneous sgp120 shedding from primary HIV Envs or its biological significance. In this dissertation, we identify genetic determinants in brain-derived Envs that overcome the restriction imposed by low CD4, examine spontaneous sgp120 shedding by these Envs, and explore the biological significance of these findings. Sequence analysis of the gp120 beta-3 strand of the CCR5-binding site bridging sheet identified D197, which eliminates an N-linked glycosylation site, as a viral determinant associated with brain infection and HIV-associated dementia (HAD), and position 200 as a positively-selected codon in HAD patients. Mutagenesis studies showed that D197 and T/V200 enhance fusion and infection of macrophages and other cells expressing low CD4 by enhancing gp120 binding to CCR5. Sgp120 shedding from primary brain and lymphoid Envs was highly variable within and between patients, representing a spectrum rather than a categorical phenotype. Brain Envs with high sgp120 shedding mediated enhanced fusion and infection with cells expressing low CD4. Furthermore, viruses expressing brain Envs with high sgp120 shedding had an increased capacity to induce lymphocyte activation during PBMC infection, despite similar levels of viral replication. Genetic analysis demonstrated greater entropy and positive selection in Envs with high versus low levels of sgp120 shedding, suggesting that diversifying evolution influences gp120-gp41 association. Finally, we examined V3 loop sequences from dual-tropic brain and lymphoid Envs and found that the frequency of R5X4 HIV-1 is underestimated by most predictive bioinformatic algorithms. Together, these studies provide a better understanding of how neurotropic HIV Envs adapt to target cells expressing low CD4, and possible roles of these viral adaptations in disease pathogenesis.

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: neuroblastoma; SHSY; Ependymin; Limulus Includes bibliographical references (p. 56-60).

ABSTRACT This thesis was divided into two parts. The purpose of part I was to clone and sequence the full-length ependymin gene from the invertebrate Limulus polyphemus, or portions of the gene, and to use RT-PCR to determine whether expression of this gene increases during leg regeneration. PCR was chosen as the method for obtaining the gene due to the success our lab had previously characterizing several ependymin genes using this approach. Three sets of primers were designed based on the conserved domains between teleost fish and three invertebrate ependymin sequences. “Sea primers" were designed based on the nucleotide sequence of the sea cucumber H. glaberrima for each conserved domain, and these primers produced all four of the expected size amplicons with Limulus DNA, but surprisingly only one such band with the sea cucumber Sclerodactyla briareus. The consensus primers (con-primers) were designed based on the most conserved nucleotide among all known ependymin species at each particular position in the conserved domains. Primers designated“5-11 primers" were designed based on the absolutely conserved domains among the three known invertebrate ependymins. Neither con-primers nor 5-11 primers produced any bands of the expected size; this was true for both species of DNA. One very strong band was produced using“5-11" primer pair 6/10 with both species. One of the bands from this reaction from Limulus was cloned and sequenced, and showed a very strong homology (88% over 292 bp) with mouse FGF-14, a neurotrophic factor involved in mouse neurogenesis. The expression of this gene during leg regeneration will be tested in future experiments. Limulus GAPDH was also cloned and sequenced, and a genomic intron was identified for the first time in this study. This Limulus housekeeping gene will be used in future studies for gene expression comparisons. The purpose of part two of this thesis was to study the up-regulation of growth-related genes induced by treatment of a human neuroblastoma SH-SY5Y cell line with a human ependymin peptide mimetic (hEPN-1), in an attempt to help provide a basis for using human EPN mimetics as therapeutics in stroke and neurodegenerative diseases. The sequence of this mimetic is derived from an area of human MERP-1 analogous to goldfish mimetic CMX-8933. The human mimetic was previously found to up-regulate growth related genes L-19, EF-2 and ATP Synthase in the mouse neuroblastoma cell line Nb2a (Saif, 2004). The expression levels of genes encoding ribosomal proteins and ribosomal RNA were studied using RT-PCR as hallmarks of proliferating cells. hEPN-1 was found to increase the expression of the nuclear-encoded ribosomal proteins S-19 and S-12, an average of 2.76 fold and 1.74 fold, with statistically significant p-values of 0.031 and 0.015 (<0.05), respectively. The expression levels of nuclear-encoded 5.8S ribosomal RNA (p = 0.018) and the mitochondrial-encoded 16S RNA (p = 0.046) were found to be increased an average of 14.04 fold and 3.91 fold, respectively. Thus, human ependymin mimetic hEPN-1 appears to stimulate growth-related genes, a property which can be useful to regenerate neuronal tissue after injury.

No description available.

Dietary intervention remains a popular, albeit challenging, approach for combating obesity. In recent years, dietary interventions that increase consumption of medium-chain triglycerides (MCT) instead of long-chain triglycerides (LCT) have gained attention. Pre-clinical research has demonstrated that rats fed a high-fat diet (HFD) induce adiposity, but a dietary shift from LCT to MCT suppresses this effect. To date, the extent to which this effect operates via suppressed hyperphagia is not fully understood. In the present study, we sought to determine how consuming a HFD composed of different fat types affects energy intake, adiposity, and hippocampal brain-derived neurotropic factor (BDNF) levels. Rats were assigned to one of four diet groups – rat chow (CHOW), LCT-enriched HFD (LCT-HFD), MCT-enriched HFD (MCT-HFD), or coconut oil-enriched HFD (COCO-HFD), which composes a mixture of LCT and MCT. In Experiment 1, all animals were given <i>ad libitum</i> access to their assigned diet, whereas in Experiments 2 and 3, HFD-subjects were pair-fed to CHOW to prohibit hyperphagia. In Experiments 1 and 2, subjects were aged 20-24 weeks, whereas in Experiment 3, subjects were aged 10-11 weeks. Across experiments, we found that the effect of MCT consumption on suppressing HFD-induced adiposity is causally related to suppressed HFD-induced hyperphagia. Additionally, we failed to detect an effect of HFD consumption on hippocampal BDNF. Therefore, our findings did not support or oppose the hypothesis that MCT consumption attenuates HFD-induced BDNF deficiency. Future studies should focus on determining the causal relationship between MCT consumption, energy expenditure, and HFD-induced adiposity.

Le virus de la maladie de Borna (BDV) est un virus persistant dans le système nerveux central responsable de troubles du comportement chez l’animal et possiblement chez l’homme. En utilisant des cellules progénitrices neurales humaines, des travaux antérieurs à mon arrivée au laboratoire ont montré que BDV altère la neurogenèse humaine. Les objectifs de ma thèse étaient d’identifier les déterminants viraux responsables de cette altération et de caractériser les mécanismes moléculaires impliqués. Nous avons montré que la phosphoprotéine (P) et la nucléoprotéine (N), mais pas la protéine X, induisent une inhibition spécifique de la neurogenèse humaine, la genèse des astrocytes n’étant pas altérée. Ensuite, focalisant notre étude sur P, nous avons montré qu’elle affectait particulièrement la genèse des neurones GABAergiques. La caractérisation moléculaire a ensuite révélé une diminution de l’expression de gènes impliqués dans la spécification (ApoE et Noggin) et dans la maturation (SCG10/Stathmin2 et TH) neuronale. En conclusion, nos résultats démontrent, pour la première fois, qu’une protéine virale perturbe la neurogenèse GABAergique humaine, un processus connu pour être dérégulé dans certaines maladies psychiatriques. Ils améliorent ainsi notre compréhension de la pathogenèse de ce virus persistant et de son rôle possible dans les maladies psychiatriques chez l’homme. / Borna disease virus (BDV) is a persistent neurotropic virus causing neurobehavioral disorders in animals and possibly humans. Using human neural progenitor cells, it had been shown, before my arrival in the laboratory, that BDV induces an alteration in human neurogenesis. Here, we aimed at identifying the viral determinants involved in BDV-induced impairment of neurogenesis and at characterizing the underlying molecular mechanisms. We demonstrated that the phosphoprotein (P) and the nucleoprotein (N), but not the X protein, reduce neurogenesis. Focusing on the role of P, we evidenced an impairment of GABAergic neurogenesis. Then, seeking for the molecular mechanisms responsible for P-induced inhibition of neurogenesis, we showed that it induces a decrease in the expression of cellular factors involved in either neuronal specification (ApoE, Noggin) or maturation (SCG10/Stathmin, TH). Thus, in this study, we demonstrated for the first time that a viral protein is capable of inhibiting GABAergic neurogenesis, a process that is dysregulated in some psychiatric diseases. Our results improve our understanding of the pathogenesis of this persistent neurotropic virus and of its possible role in psychiatric disorders.

Eine der häufigsten viralen Infektionskrankheiten im Kindesalter ist gegenwärtig die Parotitis epidemica. Mit dem Auftreten einer abakteriellen Meningitis bei Kindern als Folge einer Mumpsvirusinfektion ist in über 80% der Fälle zu rechnen. 1. In serologisch bestätigten Mumpsfällen wurden Liquorproben von Kindern mit einer Meningitis immunfluoreszenzoptisch untersucht. In den lympho-monozytären Liquorzellen konnte das Mumpsantigen in 64% der Fälle nachgewiesen werden. 2. Der immunfluoreszenzoptisch Nachweis des Mumpsantigens in Liquorzellen stellt eine Erweiterung der diagnostischen Möglichkeiten bei serösen Meningitiden des Kindes unklarer Äthiologie dar. 3. Während der Prüfung von Mumpsviren reagieren intracerebral infizierte Affen mit einer statistisch gesicherten Erhöhung der Liquorzellzahl. Bei Affen, die mit einer neuropathogenen Variante infiziert waren, sind die Liquorzellzahlen auch 28 Tage p.i. signifikant höher im Vergleich zu mit Impfstoff infizierten Tieren. Der Nachweis des Mumpsantigens in den Liquorzellen beweist die Spezifität der experimentellen Mumpsmeningitis bei Affen. Das virale Antigen wird nur innerhalb der ersten Versuchswoche in den Liquorzellen detektiert, nach 3 – 4wöchiger Versuchsdauer ist das Mumpsvirus im Liquor der Affen nicht mehr nachweisbar. Diese Befunde sind mit den Befunden bei natürlichen Mumpsmeningitiden der Kinder vergleichbar. 4. Minischweine und Katzen reagieren auf intracerebral appliziertes Mumpsvirus mit charakteristischen liquorzytologischen Veränderungen, die mit den Befunden beim Affen korrelieren. Sie reagieren auf intracerebral inokuliertes Mumpsvirus bereits in der ersten Woche mit einer deutlichen Liquorpleozytose. Die Höhe der Zellzahl im Liquor ist bei Mumpswildviren (α = 0,05) bedeutend größer als bei den Impfviren. Die Zellzahlerhöhung ist nach 8 Wochen nicht auf die Normalwerte zurückgegangen. 5. Der immunfluoreszenzoptische Nachweis des Mumpsantigens in den Liquorzellen der Minischweine und Katzen ist zeitlich begrenzt. Es ergaben sich Unterschiede zwischen Mumpsviren vom Wild- und Impftyp. Der Nachweis des viralen Antigens in den Liquorzellen war 4 Wochen p.i. nicht mehr möglich. 6. Das Differentialzellbild des Liquors ist bei allen Tiermodellen durch lympho-monozytäre Zellen gekennzeichnet, wobei monozytäre Zellformen zu jedem Zeitpunkt der Infektion überwiegen. 7. Vor der Anwendung von Lebendimpfstoffen beim Menschen ist eine tierexperimentelle Sicherheitsprüfung notwendig. Die Einbeziehung der Liquordiagnostik (Verlauf der Pleozytose und der fluoreszenzoptische Antigennachweis in den Liquorzellen) ist eine wesentliche Ergänzung zur Charakterisierung der neurotropen Eigenschaften des viralen Mumpsantigens. / One of the most common viral infectious diseases in childhood is currently parotitis epidemica. The occurrence of abacterial meningitis in children as a result of mumps virus infection can be expected in more than 80% of cases. 1. In serologically confirmed mumps cases, cerebrospinal fluid samples from children with meningitis were examined by immunofluorescence. Mumps antigen was detected in the lympho-monocytic cerebrospinal fluid (CSF) cells in 64% of cases. 2. Immunofluorescence detection of mumps antigen in CSF cells represents an extension of diagnostic possibilities in serous meningitis of the child of unclear etiology. 3. During mumps virus testing, intracerebrally infected monkeys respond with a statistically confirmed increase in CSF cell count. In monkeys infected with a neuropathogenic variant, CSF cell counts are significantly higher even 28 days p.i. compared with vaccine-infected animals. Detection of mumps antigen in CSF cells demonstrates the specificity of experimental mumps meningitis in monkeys. The viral antigen is detected in the CSF cells only within the first week of the experiment; after 3 - 4 weeks of the experiment, the mumps virus is no longer detectable in the CSF of the monkeys. These findings are comparable to the findings in natural mumps meningitis of children. 4. Minipigs and cats respond to intracerebrally applied mumps virus with characteristic liquor cytologic changes that correlate with the findings in monkeys. They respond to intracerebrally inoculated mumps virus with marked CSF pleocytosis as early as the first week. The level of cell number in CSF is significantly greater in wild mumps virus (α = 0.05) than in vaccine virus. The cell count increase did not return to normal values after 8 weeks. 5. Immunofluorescence detection of mumps antigen in CSF cells of minipigs and cats is temporal. Differences were found between wild-type and vaccine-type mumps viruses. Detection of viral antigen in CSF cells was no longer possible 4 weeks p.i.. 6. The differential cell pattern of CSF is characterized by lympho-monocytic cells in all animal models, with monocytic cell forms predominating at each time point of infection. 7. Animal safety testing is required prior the use of live vaccines in humans. The inclusion of CSF diagnostics (course of pleocytosis and fluorescence antigen detection in CSF cells) is an essential addition to characterize the neurotropic properties of the viral mumps antigen.

<p dir="ltr">Past research has extensively explored the benefits of acute aerobic exercise (AE) on memory and executive functions. Additionally, the cross-sectional relationship between muscle strength – a direct outcome of RE – and cognition is unknown, despite the simultaneous onset of muscle and cognitive decline in one’s thirties. However, the effects of acute resistance exercise (RE) on cognition remain understudied, despite the growing popularity of RE and evidence that RE may have distinct effects on cognition.. Therefore, the present study aimed to broaden our understanding of the connection between muscle strength and hippocampal-dependent memory and to investigate the influence of RE on memory and executive function.</p><p dir="ltr">A sample of 125 healthy young adults (18-50 years old) completed this study. On the first day of testing, subjects completed a cognitive battery testing aspects of hippocampal dependent memory, spatial abilities, and working memory, a maximal muscle strength testing session including handgrip strength and one-rep-max testing, and maximal aerobic capacity testing. Subjects completed a bioelectrical impedance assessment (BIA) body scan to measure body composition on Day 2. Day 3 consisted of a randomized controlled trial (RCT), where subjects completed either 42 minute moderate intensity RE (n = 62) or a seated rest (n = 61). Cognitive testing including a memory recognition task, an inhibitory control task, and a working memory task were performed both before and after the intervention. Subjects also completed lactate, blood pressure, and blood draw (only a subset of subjects (n = 59)) before and after intervention.</p><p dir="ltr">The results first revealed that after controlling for known covariates, those with greater handgrip strength performed better on mental rotation tasks (t = 2.14, p = 0.04, Δr2= 0.04), while those with higher upper-body relative strength did better on recognition (t = 2.78, p = 0.01, Δr2 = 0.06) and pattern separation (t = 2.03, p = 0.04, Δr2= 0.04) tasks. Further, while there was no acute effect of RE on memory performance, response times during measures of inhibitory control (t = 4.15, p < 0.01, d = 0.40) and working memory decreased after exercise (t = 7.01, p < 0.01, d = 0.46), along with decreases in P3 latency during the inhibitory control task (t =-5.99, p < 0.01, d = 0.58). Additionally, blood lactate (t =-17.18, p < 0.01, d = 2.06), serum brain derived neurotropic factor (BDNF) (t = -4.17, p < 0.01, d = 0.66), and systolic blood pressure (t = -10.58, p < 0.01, d = 0.99) all increased following RE, while diastolic blood pressure (t = 4.90, p < 0.01,d = 0.50) decreased. Notably, the change in systolic blood pressure (t = -2.83, p = 0.01, Δr2 = 0.06) was associated with improvements in behavioral measures of inhibitory control, changes in lactate (t = -2.26, p = 0.03, Δr2 = 0.04) and systolic blood pressure (t = -3.30, p < 0.01, Δr2 = 0.08) were also related to improved behavioral changes in working memory, and changes in lactate (t = -3.31, p < 0.01, Δr2= 0.08) and BDNF (t = -2.12, p = 0.04, Δr2= 0.08) related to faster P3 latency during inhibitory control. Importantly, these associations between physiological and cognitive changes were consistent across both exercise and rest groups, suggesting that physiological changes were linked to improved cognitive performance regardless of group assignment.</p><p dir="ltr">In conclusion, this study highlights the positive relationships between cross-sectional muscle strength and aspects of memory and spatial abilities, with distinct contributions from handgrip and upper body strength. Furthermore, acute RE was shown to enhance executive functions, particularly in terms of processing speed during inhibitory control (response time and P3 latency) and working memory (response time). This study suggests that RE can be a valid way to garner exercise-induced benefits on executive functions potentially through its influence on lactate, BDNF, and blood pressure, however, since these effects were evident regardless of intervention, more work is needed to determine if RE-induced changes have the same mechanisms. Overall, these findings underscore the potential benefits of muscle strength and RE on enhancing executive function in young and middle-aged adults.</p>