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CD4+ T cells and macrophages are the principal targets of HIV-1. They can be productively infected with the virus and transfer virions to contacting bystander cells. It has been suggested that soon after initial infection, free virions and virus-bearing or infected T cells and macrophages can enter the brain, triggering a cascade of inflammatory signals and recruitment of other immune cells. Chronic inflammation and increased viral antigens in the brain lead to HIV-1 associated neuropathy. Once free virions or infected cells enter the central nervous system, the first type of brain cells that they are likely to encounter are astrocytes, which extend endfeet around the blood vessels. These cells have been observed to contain virions and viral products, but their permissivity to productive infection has not been clearly demonstrated. By contrast, productive infection of resident microglia and perivascular macrophages is well established. Here, I investigate the permissivity of astrocytes to HIV-1 infection and found no evidence of infection by the free route. However, I found that astrocytes intimately contact HIV-1 infected macrophages and CD4+ T cells and, in some cases, extend filopodial membrane toward the infected cell. In astrocyte-T cell contact sites, termed synapses, virions appear to move along the astrocytic filopodia from the T cell to the astrocyte. In this case, the target cell mediated viral transfer across the intercellular gap. HIV-1-infected macrophages released virus that associated with astrocytes, remaining either on the surface of the astrocytes or within intracellular compartments. HIV-1 bound to astrocytes could be transmitted efficiently to permissive cells in trans. However, astrocyte-associated virus was sensitive to inhibitors including proteases and neutralizing antibodies, suggesting a surface-accessible compartment. This work provides insight into mechanisms of HIV-1 spread in the brain from infected CD4+ T cells and macrophages to astrocytes and their potential as virus reservoirs. I also optimized high resolution, correlative focused ion beam scanning electron microscopy technology to answer fundamental biological questions. I demonstrate the application of the technology to study skeletal muscle cell differentiation mechanisms. I combine the power of genetic mapping with structural analysis to qualitatively and quantitatively describe cellular states and functions. Using semi-automatic image processing analysis, I was able to compute high volumes of data and generate statistics that relate quantitative measurements of cellular structures to functions. The toolset developed here will be instrumental in studying cells and tissues in both research and clinical applications.

African trypanosomiasis is a protozoan infection affecting tens of thousands of people and millions of livestock animals across sub-Saharan Africa. In humans the disease is fatal without chemotherapeutic intervention and in animals it causes a severe anaemia that greatly impairs productivity. Available drug compounds are difficult to administer and unacceptably toxic. A natural product, ascofuranone, inhibits a key trypanosome specific respiratory enzyme, trypanosome alternative oxidase, and was shown over a decade ago to be trypanocidal using both in vitro and in vivo experiments. The compound suffers from rapid metabolism and contains several functionalities undesirable in a drug compound. Despite the promising activity the lack of applicable synthetic methods available hampered the development of chemotherapeutics from ascofuranone. In this work, novel synthetic routes were completed to explore the lead compound. New synthetic methods were successfully developed using palladium catalysed Suzuki couplings and Lewis acid catalysed rearrangements. Ortho-lithiation approaches also afforded potent novel inhibitors. Of particular note is a benzisoxazole, which is expected to alleviate many of the metabolic issues associated with ascofuranone. Alternate heterocycle analogues were explored and an interesting indazole analogue obtained. Finally, chemical methods were developed towards the benzisoxazole and indazole motifs with carboxylic acids, amenable to diversification by amide coupling. A preliminary range of novel amide containing 5, 6-heterocycles were synthesized to begin SAR exploration of these structures.

The study of antibody responses in prominent aquaculture species such as the rainbow trout, Oncorhynchus mykiss, can facilitate vaccine development and contribute to producing useful paradigms of adaptive immunity in lower vertebrates. Thus, it is essential to identify genes responsible for antibody responses. In the mouse model, hybridoma technology allows for the association of monoclonal antibodies possessing various affinities for antigen with specific VH sequences, gene family utilization, and other molecular events (i.e. somatic hypermutation) that occur during the specific immune response. The absence of a comparable hybridoma technology in piscine systems has limited similar studies of fish immunogenetics to date. Molecular and serological experiments were performed in an attempt to obtain information regarding somatic mutation and VH gene utilization for trout antibodies without reliance on hybridoma technology. PCR primers recognizing consensus sequences of FR1 and FR3 were used to amplify antibody VH sequences from panned, antigen-specific B cells. to follow the development of the expressed VH repertoire, lymphocytes were obtained at weeks 0, 5, 10, and 20 post primary immunization with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) or infectious hematopoietic necrosis virus (IHNV). Lymphocytes were also collected 10 weeks post secondary immunization (week 35). These studies were conducted in parallel with serological analyses of plasma antibodies obtained from the same sample in order to correlate molecular data with serological data from individual trout. Antigen-specific lymphocytes were processed to isolate RNA templates to produce cDNA which was cloned and sequenced. This sequence analysis allowed us to report, for the first time, the temporal accumulation of potential somatic variants that correlate to the development of new, high affinity antibody subpopulations during the immune response, some with the emergence of new antibody heavy chain isoelectropherotypes as identified by 2D-IEF/SDS-PAGE. Southern analysis and gene titration using various antigen-specific cDNA probes allowed us to correlate trout antibodies possessing various affinities for antigen with specific VH sequence and gene family utilization. Thus, trout Ig VH gene family utilization appears to follow the mouse model of differential use for specific immune response. These results reveal a capability for fine-tuning the piscine immune response previously not recognized.

Leishmania infantum chagasi (Lic) is a pathogenic protozoan parasite and one of the etiological agents of human visceral leishmaniasis (VL). VL is a potentially deadly disease characterized by variable fevers, cachexia, hepatosplenomegaly, and global immune suppression. Many questions regarding the pathogenesis of VL and the mechanisms of host defense during Lic infection remain to be elucidated. The primary focus of this thesis is the relationship between Lic and the mammalian immune system. We studied parasite-host interactions during Lic infection at the molecular, cellular, and organismal level. We generated transgenic parasites that expressed firefly luciferase and/or fluorescent proteins to expand our capacity to detect, observe, and quantify the parasites in a variety of experimental settings with modern analytical methodologies. Using luciferase-expressing Leishmania, we developed an experimental infection model in which parasites were detected and the relative parasite burden in specific anatomical locations could be quantified in a live animal host using bioluminescence imaging. This method allowed the parasite burden to be assessed in the same host throughout the course of infection. Utilizing this model we have made some intriguing observations relating to the kinetics and distribution of the parasite burden over time. The parasite burden was observed primarily in the liver and bone marrow over the first few weeks and then shifts to the spleen and bone marrow. To gain a better understanding of the initial parasite-host immune interactions in vivo, we studied the early inflammatory response after intradermal (i.d.) inoculation. We observed a rapid and abundant influx of neutrophils into the inoculated ears. The neutrophil influx was transient, dose dependent and specific for the local inoculation site. While there was not a significant neutrophil influx into the draining lymph nodes (dLN), there was an increase in the total cellularity and a striking increase in the relative proportion of B cells to T cells over the first week after intradermal parasite challenge. By inoculating transgenic mCherry-Lic we found that neutrophils were the primary parasite-laden host cell in the dermal tissue during the first day, but macrophages harbored most of the parasites by 2 days. Neutrophil depletion using low-dose antibody treatment resulted in a reduced rate of parasite uptake initially at the site of inoculation, but no significant change in the dLN dynamics. We further examined the parasite-host relationship by studying molecular signaling and cellular interactions between Leishmania and human neutrophils. We investigated the nature of the chemotactic activity of Leishmania-conditioned growth medium for human neutrophils by testing physical properties of the activity and ruled out some of the major Leishmania surface molecules as potential candidates. We aim to identify the agent(s) responsible for the activity in on-going studies. To this end, we are collaborating with a group at the NIH and testing biochemical purification/separation samples. We conclude that intradermal Lic challenge induces a rapid innate immune response at the local site of infection, that neutrophils sense Leishmania-derived factors leading to directed migration, and that neutrophils function as a primary site for Leishmania entry into the mammalian host.

The principal motivations for studying epidemics and their dynamics are understanding the biological characteristics of the epidemic agents and reducing the economical and social costs originating from epidemic outbreaks. The most commonly used epidemic models have important assumptions such as the law of mass action, and the latent and infectious periods being exponentially distributed with xed parameters. Under this kind of suppositions the models are analyzed with well known algorithms as the Euler and Euler-Maruyama, and methodology and results from the theory of Markovian processes. However, these assumptions are selected largely for their analytic convenience and in many cases are far from describing the agent's transmissibility attributes in the population and its biological characteristics in a host. The epidemic models studied here relax two important epidemic assumptions. The first to be relaxed is the one that susceptible individuals are equally likely to acquire the disease. A structure for the kind of individual contacts that can result in the infection transmission is incorporated in the population. This contact structure can be non-homogeneous and it is modeled as a random graph whose edges describe the contacts between individuals. The second assumption that is generalized, is the distribution of the latent and infectious period in the host individuals. This research work allows the latent and infectious period to have a distribution other than the exponential and hence the epidemic process is more general than a Markovian process. As in most stochastic models, the infectious contact is modeled as a random variable with Poisson distribution. However, to introduce the individual variations, the transmission rate is assumed to be a non negative random variable. This work extends the epidemic models suggested by Newman (2002) in two directions. The first, studies the hierarchical networks that have a more complex network structure, involving the interaction of populations. The second direction examines the evolution in times for outbreaks in networks. In this work, results for discrete and continuous time are obtained. The results for the continuous time model considers the infectious process to be a bivariate Markovian process. However, the results for the final outbreaks size and the developed simulation program include the general case were the latent and infectious period can have a distribution other than exponential. This research work also analyze the effect of four control measures in the contact structure, and using the simulation program and Monte Carlo-likelihood methodology, it estimates the parameters for measles and influenza. The results here obtained can be directly applied to study the dynamics of other kind of “agents” such as information and ideas. For example, the dynamics can involve the spread of computer viruses, rumors, eating habits and personal positions regarding a fact or idea.

The principal motivations for studying epidemics and their dynamics are understanding the biological characteristics of the epidemic agents and reducing the economical and social costs originating from epidemic outbreaks. The most commonly used epidemic models have important assumptions such as the law of mass action, and the latent and infectious periods being exponentially distributed with xed parameters. Under this kind of suppositions the models are analyzed with well known algorithms as the Euler and Euler-Maruyama, and methodology and results from the theory of Markovian processes. However, these assumptions are selected largely for their analytic convenience and in many cases are far from describing the agent's transmissibility attributes in the population and its biological characteristics in a host. The epidemic models studied here relax two important epidemic assumptions. The first to be relaxed is the one that susceptible individuals are equally likely to acquire the disease. A structure for the kind of individual contacts that can result in the infection transmission is incorporated in the population. This contact structure can be non-homogeneous and it is modeled as a random graph whose edges describe the contacts between individuals. The second assumption that is generalized, is the distribution of the latent and infectious period in the host individuals. This research work allows the latent and infectious period to have a distribution other than the exponential and hence the epidemic process is more general than a Markovian process. As in most stochastic models, the infectious contact is modeled as a random variable with Poisson distribution. However, to introduce the individual variations, the transmission rate is assumed to be a non negative random variable. This work extends the epidemic models suggested by Newman (2002) in two directions. The first, studies the hierarchical networks that have a more complex network structure, involving the interaction of populations. The second direction examines the evolution in times for outbreaks in networks. In this work, results for discrete and continuous time are obtained. The results for the continuous time model considers the infectious process to be a bivariate Markovian process. However, the results for the final outbreaks size and the developed simulation program include the general case were the latent and infectious period can have a distribution other than exponential. This research work also analyze the effect of four control measures in the contact structure, and using the simulation program and Monte Carlo-likelihood methodology, it estimates the parameters for measles and influenza. The results here obtained can be directly applied to study the dynamics of other kind of “agents” such as information and ideas. For example, the dynamics can involve the spread of computer viruses, rumors, eating habits and personal positions regarding a fact or idea.

The research desires to evaluate the preventing policies on emerging infectious diseases by system dynamics, and takes the SARS situation in Taiwan for example. According to epidemiology and everything about SARS, we build the model of SARS transmission and prevention. Therefore we can simulate the situation and policies, and find the effective policies. After the simulation and the evaluation, we find that most SARS patients at later stage are affected in hospital. For the reason, the most effective policies are the ¡uPolicy about enhancing protection abilities in hospital¡v and ¡uPolicy about reducing the interaction with people in hospital¡v. Furthermore, the effectiveness of ¡uQuarantine policies¡v is not stronger than the above policies. The most important thing is that we discover Taiwan is very lucky, because the infectivity is very low (about 3.7%). If the infectivity of SARS were as high as 10% and we still took the same policies as we took in 2003, the situation would be terrible. Anyway, when we confront this kind of emerging infectious diseases, the better way is taking policies in hospital intently.

No description available.