Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

El Decabromodifenil èter (BDE-209) és un retardant de la flama àmpliament utilitzat i font de preocupació a causa de la toxicitat mostrada per altres Difenil Èters Polibromats (PBDEs). La presència de PBDEs en la llet materna fa preocupant la seva exposició durant el desenvolupament. Pensem que l’exposició primerenca a BDE-209 pot produir efectes a llarg termini i interactuar amb factors genètics, com el genotip de l’ApolipoproteinaE. Ratolins portadors de les diferents isoformeshumanes de l’ApoE foren tractats amb una dosi oral aguda de 0, 10 o 30 mg / kg de BDE-209 en el dia postnatal 10 i van ser avaluats per neurocomportament durant el desenvolupament, a l'edat adulta i la vellesa. L’exposició a BDE-209 indueix un retard en el desenvolupament físic i neuromotor i en la compactació de la mielina en els ratolins ApoE2, disminueix els nivells de tiroxina lliure en les femelles adultes i disminueix l'activitat en ratolins ApoE4. Els efectes més consistents durant tota la vida s'observen en ratolins ApoE3 i consisteixen en problemes d'aprenentatge als 4 mesos, i problemes d'aprenentatge i memòria i un augment de l'ansietat als 12 mesos. / Decabromodiphenyl Ether (BDE-209) is a flame retardant widely used and source of concern because ofthe toxicity showed by other Polibrominateddiphenyl ethers (PBDEs). The presenceof PBDEs in human’s breast milkmakes worrying its exposure during development. We hypothesised that an early exposure to BDE-209 can induce long-term impairments and interact with genetic factors, such as ApolipoproteinE genotype. Mice carrying the different Human ApoE isoforms treated with an acute oral dose of 0, 10 or 30 mg/kg of BDE-209 on postnatal day 10 were assessed for neurobehaviourduring development, in young adulthood and old age. BDE-209 exposure inducesadelay in physic and neuromotordevelopment and in myelin compaction in ApoE2 mice, decreases the levels of free thyroxin in adult femalesand decreases activity in ApoE4 mice. The most consistent effects across the lifespan are observed in ApoE3 mice and consist of impaired learning at 4 months, and impaired learning and memory and increased anxiety at 12 months.

User interface design is becoming more reliant on user emotional states to improve usability, adapt to the users state, and allow greater expressiveness. Historically, usability has relied on performance metrics for evaluation, but user experience, with an emphasis on aesthetics and emotions, has become recognized as important for improving user interfaces. Research is ongoing into systems that automatically adapt to users states such as expertise or physical impairments and emotions are the next frontier for adaptive user interfaces. Improving the emotional expressiveness of computers adds a missing element that exists in human face-to-face interactions. The first step of incorporating users emotions into usability evaluation, adaptive interfaces, and expressive interfaces is to sense and gather the users emotional responses. Affective computing research has used predictive modeling to determine user emotional states, but studies are usually performed in controlled laboratory settings and lack realism. Field studies can be conducted to improve realism, but there are a number of logistical challenges with field studies: user activity data is difficult to gather, emotional state ground truth is difficult to collect, and relating the two is difficult. In this thesis, we describe a software solution that addresses the logistical issues of conducting affective computing field studies and we also describe an evaluation of the software using a field study. Based on the results of our study, we found that a software solution can reduce the logistical issues of conducting an affective computing field study and we provide some suggestions for future affective computing field studies.

Many Americans are afflicted with inflammation of the colon. They are also at a higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has the potential to provide information on tissue structural properties that may be lost in the fixation and slicing procedures required for histopathology. Optical sectioning provides images in three dimensions capturing the organizational structure of cells and colon crypts throughout the entire colon. I have constructed a custom built fluorescence and reflectance confocal microscope for imaging molecular and morphological changes associated with development of inflammation in a mouse model. A confocal microscope is a point scanning system that removes out of focus light by placing a pinhole aperture in the conjugate image plane located in front of the detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system is approximately 3 μm with an axial resolution of 6 μm and 4 μm for reflectance and fluorescence mode, respectively. As colon tissue becomes inflamed, there is a distinct change in the structure and architecture of the tissue. The colon crypts are no longer uniform in size or distribution throughout the tissue. Having a large field of view of 1mm2 allows for many colon crypts to be visualized within a single frame. Histology was performed on the same tissue imaged for the inflammatory study confirming the constructed confocal microscope’s ability to characterize inflamed tissue and the potential use for guided biopsy. Mosaicing, or image tiling, is an imaging technique that stitches single frames together to produce a much larger field of view. An extended frame with 1 mm x 2 cm field of view is achieved within seconds. This extended frame would allow mosaicing of the entire mouse colon much faster than conventional methods without loss of resolution. The acquired confocal images of colon tissue demonstrate the microscope’s ability to resolve cell nuclei lining the colon crypts within a relatively large field of view.

The roles of genes responsible for the survival and persistence of Brucella in the host and the relationship between these genes and the disease were investigated via signature-tagged transposon mutagenesis. As much as 8% of the Brucella genome is important for survival of this organism in the host. This is an unusually high number and may help to explain the chronic or persistent nature of Brucella infections. Mutants attenuated in the mouse model were divided into two groups. The early mutants failed to establish infection or colonize the host. The late mutants colonized the host but failed to maintain infection. The vaccine potential of two mutants (virB10 and gcvH) that were unable to sustain infection was compared to that of a vaccine strain, S19. Survival of strain S19 in vivo was up to 12 weeks while virB10 and gcvH mutants were cleared from spleen at 8, and 24 weeks post-inoculation, respectively. Mice were vaccinated with individual mutants and then challenged with virulent S2308 at 8, 16, and 24 weeks postvaccination. As a result, protective immunity correlated with persistence of the mutant strain [gcvH>virB10]. These results suggest that survival is one of several factors that may influence protective immunity making it difficult to compare strains. For example, examination of host immune response revealed a similar pattern of host immune function (TH1 over TH2) in all mice except those vaccinated with virB10 mutant. Since gcvH mutant provided the best immunity, experiments were designed to explore its contribution of persistence to protection. In an effort to reduce non-specific activation induced by prolonged survival of gcvH mutant, protection was monitored after different periods of vaccination exposure followed with doxycycline treatment. In these studies, persistence of gcvH mutant enhanced protection against challenge. Overall, defined mutations in genes affecting survival may render mutants as vaccine candidates capable of stimulating protective immunity equal to or better than fortuitously isolated attenuated strains. Future studies should focus on characterization of these and other genes responsible for the persistence of Brucella to improve the safety and efficacy of live vaccines.

Not available / text

Not available / text

Not available / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Infundibular (arcuate) neurokinin B (NKB) neurons play a critical role in neuroendocrine control of reproduction. Specifically, a local network of arcuate neurons that co-express kisspeptin, neurokinin B, and dynorphin (so-called, KNDy neurons), has emerged as a potential pacemaker driving the pulsatile secretion of gonadotropin-releasing hormone (GnRH) that is required for normal reproduction. These neurons are the target of estrogen and may be an important link in estrogen negative feedback on GnRH functioning. KNDy neurons respond to estrogen withdrawal with dramatic changes in gene expression and somatic hypertrophy, an effect that is reversible by estradiol replacement. Studies addressing the effects of estrogen withdrawal and replacement on morphological and electrophysiological features of KNDy neurons have been hindered by the inability to target this subpopulation of neurons in the live tissue. This dissertation examines estrogen-induced changes in arcuate NKB circuitry and excitability and discusses its implications in reproductive axis. First, the novel Tac2-EGFP transgenic mouse model was characterized. The reproductive function, EGFP-ir distribution in the brain, and co-localization of EGFP with proNKB in the arcuate nucleus were examined and compared to littermate controls. Indices of reproductive function (puberty onset, estrous cyclicity, and LH pulsatility) were comparable between Tac2 and wildtype mice, suggesting that the transgenic animals have preserved estrogen negative feedback. The long-term estrogen withdrawal via ovariectomy and estradiol replacement model was used to examine electrophysiological and morphological changes in arcuate NKB neurons. We found that low-dose chronic estradiol replacement results in decreased excitability of arcuate NKB neurons, a finding that is consistent with the proposed role of this neuronal population in estrogen negative feedback on reproductive axis. Changes in excitability were seen despite the overall similarity in intrinsic properties of estradiol-treated and untreated ovariectomized mice. We also demonstrated for the first time that single arcuate NKB neurons form a local network by way of recurrent collaterals. Axonal targets of single NKB neurons included the internal zone of the median eminence, ependymal layer of the 3rd ventricle, and sites lateral and dorsal to the borders of the arcuate nucleus. Long-term treatment with estradiol resulted in decreased somatic volume and decreased dendritic spine density. Together, these data demonstrate that low-dose chronic estradiol replacement in ovariectomized mice resulted in morphological plasticity of arcuate NKB neurons that was accompanied by changes in excitability of this neuronal population, supporting the role of these neurons in estrogen negative feedback on GnRH secretion.