Thesis (M.S.)--Boston University

The prevalence of stress and anxiety disorders in modern society is increasing, but the development of new treatments decreasing due to high research costs and low success rates in clinical trials. The latest type of compounds introduced to treat anxiety and depression was the specific serotonin reuptake inhibitors (SSRI), which was introduced in 1987. Since then, no new class of compounds have been introduced, suggesting that the need to find alternative targets in treating mental disorders is needed. In this thesis I have used the zebrafish as a model organism to study the modulation of behaviours through intracellular signalling pathways, known to be involved in learning, memory and anxiety. First, using the pro-convulsant compound, pentylenetetrazole (PTZ), an automated tracking system was established to quantify and analyse swimming behaviour in larvae zebrafish. Pentylenetetrazole induces seizures in zebrafish at high concentrations, however this thesis identifies that the combination of a low level of PTZ and subjecting the fish to alternating cycles of light and dark induced a reversed response to light and dark. A group of compounds with known anti-seizure effects were subsequently screened, which found that a combinational treatment with diazepam and two types of neurosteroids reversed the PTZ-induced light dark response. Secondly, using the same automated analysis setup, the effect of cAMP modulators was studied on behaviour in zebrafish larvae. Our lab has previously established that Rolipram, a PDE4 inhibitor, causes anxiety thigmotaxis in zebrafish larvae. In this thesis we treated zebrafish larvae with Rolipram and other compounds modulating cAMP, which greatly increased the swimming activity, which was reversed by subsequently treating with PD0325901. To test if the pharmacological modulation of cAMP-levels through the inhibition of other PDEs would lead to increased locomotor activity, a small library of PDE inhibitors was screened, and 4 compounds were identified that caused an increase in locomotion – three of these compounds were PDE4-inhibitors. Finally, by using two behavioural assays, I found that in adult fish Rolipram cause anxiety-like phenotypes, which is also reversible by MAPK-inhibition.

No description available.

The free-living amoeba and causative agent of Primary Amoebic Meningoencephalitis, Naegleria fowleri, has three life stages: the trophozoite, the flagellate, and the cyst. This study examined the ability of the cyst to attach to, excyst upon, and destroy cell cultures grown to confluent monolayers, and to cause Primary Amoebic Meningoencephalitis in a murine animal model. The co-culture of cysts with P388D.1, CHME3, Vero, human nasal epithelial, and rat primary mixed glial cells resulted in destruction of the monolayer of all cell types once the cysts attached and excysted. One day post exposure to cysts, the mixed glial cells exhibited a two-fold increase in lactate dehydrogenase (LDH) release compared to cells without cysts, and on day eight post exposure, showed a nearly four-fold increase in LDH. In this study, the cysts of N. fowleri were shown not to be infective in vivo in a murine model using B6C3F1 male mice. The mediation of the encystment process by the intracellular concentration of the secondary messenger cAMP, as described in other closely related genera and species of amoeba, was also investigated. Encystment of N. fowleri was shown to be mediated at least in part by the secondary messenger cAMP by treating cultures of the trophozoite with 100 uM dipyridamole, an inhibitor of cAMP-specific phosphodiesterases. Dipyridamole (100 μM) increased the rate of encystment by nearly two-fold compared to 0.1% DMSO by the end of a five day period of observation. This suggests that cAMP is an essential mediator of the encystment process within Naegleria fowleri.

How plants from a common species pool form community has been considered from a variety of approaches. A promising approach involves the search for assembly rules based on plant functional traits. This approach has potential to provide insight into community and ecosystem processes In this research. a general and simple conceptual model based on life forms and independent of species is used as a framework for assessing the internal structure of plant communities. Plant functional traits are used to identify patterns within and between plant communities in the contrasting environments of Camp Williams, Utah, and Camp Grayling, Michigan. The conceptual model has three different functional types formed by one to three functional groups. A functional group, made up of species with similar life form, is analogous to a vegetation stratum. A functional type, consisting of one or more functional groups. is analogous to a community or vegetation type. Correspondence analysis (CA) and analysis of variance (ANOVA) indicate that richness, species diversity, and trait diversity are essentially independent of functional type and are, for example, fairly consistent regardless of climatic regime or structural complexity. Cover. on the other hand. increases with the number of functional groups in a functional type. Consistent patterns and trends for sets of functional traits support the view that assembly rules may account for internal structure in plant communities. The consistent association of sets of traits with functional groups even in taxonomically dissimilar communities suggests that the functional traits are related to fundamental ecological processes that shape these communities. Ambiguity in some of the results might be explained by extending the analysis to additional installations that replicate the climatic conditions found at Camp Williams and Camp Grayling.

Current vegetation classes were generated from remotely sensed data to provide coarse-level information for an ecosystem management plan developed at Camp Williams, Utah. Vegetation trend from 1973 - 1993 was also examined via satellite imagery. The data set consisted of Landsat Multispectral Scanner (MSS) and Thematic Mapper (TM) images from July or August of 1973, 1975, 1980, 1988, and 1993. Two approaches were used to detect vegetation change. The first approach determined overall and cover type trend from standard digital image differencing of soil-adjusted vegetation index (SAVI) images. The second approach used an unsupervised classification of a composite SAVI image of all dates. The first approach defined areas of increase, decrease, and no significant change in SAVI and differences in trend for tree versus shrub cover types. The second approach resulted in an ecological classification that defined new environmental patterns based on vegetation trend.

Zwischen Blutkompartiment und umliegenden Interstitium besteht eine Barriere, die durch eine einzelne Schicht aus Endothelzellen gebildet wird. Essentiell für diese Barriere, deren Funktion in der Begrenzung des Austausches von Flüssigkeit und gelösten Stoffen liegt, sind interzelluläre Junktionen, welche die Endothelzellen miteinander verbinden. Durch eine gestörte Funktion und Regulation der Endothelbarriere entstehen beim Menschen verschiedene Pathologien wie zum Beispiel Ödeme, hämorrhagischer Schlaganfall und vaskuläre Malformationen. Es ist bekannt, dass cAMP die Endothelbarriere zum Teil durch Aktivierung der kleinen GTPase Rac1 stabilisiert. Trotz der großen medizinischen Relevanz dieses Signalweges, sind die damit einhergehenden Effekte auf die interzellulären Kontakte auf ultrastruktureller Ebene weitgehend unbekannt. In mikrovaskulären Endothelzellkulturen kam es ähnlich wie in intakten Mikrogefäßen zur Stärkung der Barrierefunktion. So resultierte sowohl nach Behandlung mit Forskolin und Rolipram (F/R), welche zur Steigerung der intrazellulären cAMP-Spiegel führen, als auch nach Zugabe von 8-(4-chlorophenylthio)-2´-O-methyladenosin-3´,5´-cyclic monophosphorothioate (O-Me-cAMP), einem selektiven Aktivator des cAMP nachgeschalteten Epac/Rap1-Signalweges, ein Anstieg des TER; außerdem konnte durch beide Substanzen (F/R und O-Me-cAMP) die Aktivierung von Rac1 induziert werden. Desweiteren wurde eine verstärkte Intensität und Linearisierung des Immunfluoreszenzsignals der Zelljunktionsproteine VE-Cadherin und Claudin5 entlang der Zellgrenzen beobachtet. In der ultrastrukturellen Analyse der interzellulären Kontaktzonen-Architektur zeigte sich unter F/R- oder O-Me-cAMP-Exposition ein signifikanter Anstieg an komplexen Interdigitationen. Diese komplexen Strukturen waren dadurch charakterisiert, dass sich die Membranen benachbarter Zellen, die durch zahlreiche endotheliale Junktionen stabilisiert wurden, über vergleichsweise lange Distanzen eng aneinanderlegten, so dass ein deutlich verlängerter Interzellularspalt resultierte. Die Inhibition der Rac1-Aktivierung durch NSC-23766 verminderte die Barrierefunktion und blockierte effektiv die O-Me-cAMP-vermittelte Barrierestabilisierung und Reorganisation der Kontaktzone einschließlich der Junktionsproteine. Demgegenüber konnte die F/R-vermittelte Barrierestabilisierung durch NSC-23766 nicht beeinträchtigt werden. Parallel dazu durchgeführte Experimente mit makrovaskulären Endothelien zeigten, dass es in diesem Zelltyp unter Bedingungen erhöhter cAMP-Konzentrationen weder zur Rac1-Aktivierung noch zur Barrierestärkung oder Kontaktzonen-Reorganisation kam. Diese Ergebnisse deuten darauf hin, dass in mikrovaskulären Endothelien Rac1-vermittelte Änderungen der Kontaktzonen-Morphologie zur cAMP-induzierten Barrierestabilisierung beitragen. / Evidence exists that cAMP stabilizes the endothelial barrier in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2´-O-methyladenosine-3´,5´-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (Epac/Rap1) signaling enhanced transendothelial electrical resistance (TER) and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin5. Ultrastructural analysis of the intercellular contact zone morphology documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contacts displaying complex interdigitations of cell borders in which membranes of neighboring cells were closely apposed over comparatively long distances and which were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP whereas F/R-mediated barrier enhancement was not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement and contact zone reorganization. These results indicate that in microvascular endothelium Rac1-mediated alterations in contact zone architecture contributes to cAMP-induced barrier stabilization.

<p>The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca²⁺, which has been shown to be due to influx of extracellular Ca²⁺ as a primary response.</p><p>Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX₁ than on OX₂ receptors.</p><p>When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca²⁺-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX₁ receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency G_S-coupling and a high-potency PKC coupling.</p><p>In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca²⁺-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.</p>

The extent of damage in animal models of spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of the second messenger, cyclic adenosine monophosphate (cAMP), via administration of the phosphodiesterase 4 inhibitor, Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following thoracic contusive SCI injury in rat. Rolipram or vehicle control (10% ethanol) was given daily for 2 weeks post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons (CMAs) was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (2 h PI) increased neuronal survival; 0.5 mg- 1.0 mg/kg protected oligodendrocytes, 1.0 mg/kg produced optimal preservation of CMAs. Administration of 1.0 mg/kg Rolipram via different routes (intravenous [i.v.], subcutaneous [s.c.] or oral, 2 h PI) demonstrated that all routes allowed for significant protection following SCI; the i.v. route provided the best clinical translation. Examination of delayed treatment, initiated 1-48 h after SCI, revealed protective efficacy of Rolipram even when administered up to 48 h PI. With the optimal therapeutic protocol (1.0 mg/kg, i.v.), Rolipram reduced the levels of the chemokine, monocyte chemoattractant protein acutely post-injury and elevated the levels of the anti-inflammatory cytokine, interleukin-10, based on Enzyme-Linked ImmunoSorbent Assay (ELISA) results. Rolipram, when delivered within 48 h PI, was also able to significantly reduce the number of ED1-positive mononuclear phagocytes compared to vehicle-treated controls. This work supports the use of Rolipram as an acute neuroprotectant following SCI, defines an administration protocol, and investigates a potential mechanism for Rolipram-mediated protection.

One of the largest populations with whom social workers work are individuals with disabilities. Due to the increase of children with disabilities, it is important to evaluate the programs in which the children participate. Camp Koinonia is a program at the University of Tennessee that works with children with multiple disabilities for one week a year. The purpose of this study was to conduct a program evaluation on Camp Koinonia in 2009 (n = 109). This study assessed age,(continuous variable, n = 109) gender (male, n = 61; female n = 48), category of disability (cognitive, n = 50; physical, n = 59) and type of disability (cerebral palsy, n = 17; down syndrome, n = 21; autism spectrum disorder, n = 18; and mental retardation, n = 22). This study compared the previously mentioned variables to physical, cognitive, and emotional outcomes as measured by an altered version of the Functional Assessment of Characteristics for Therapeutic Recreation, Revised (FACTR-R). The results of reliability analysis found that each subscale of physical (a = .71), cognitive (a = .80), and emotional (a = .83) outcomes of the FACTR-R were reliable. A multiple groups confirmatory factor analysis was conducted on the FACTR-R which found that this measurement tool was a good tool to use. The results of this study found that the children with physical disabilities had statistically significant improvements with their physical outcomes. Specifically, the children with cerebral palsy had statistically significant improvements in physical outcomes. There were also statistically significant improvements in children with autism spectrum disorders and emotional outcomes. A discussion of limitations and recommendations for future research is presented.