This study examined the effect of preweanling methyphenidate exposure on later drug reward. We examined the induction, extinction, and reinstatement of morphine induced conditioned place preference (CPP) in rats that received methylphenidate pretreatment during the preweanling period.

This thesis examines the development of vivisection as a method of physiological research between 1875 and 1901 in Great Britain, by examining some of the arguments, discussions, and ideas put forth by physiologists for the utilisation of vivisection in their research. Because this study operates within the context of medical history, questions of legitimacy, scientific development, and professional image are lifted. The development of vivisection during this period took place with a larger shift in scientific practice playing out in the background, where experimentalism began overtaking the previously more analytical approach to medicine and the sciences. The First Royal Commission on Vivisection in 1875 marks the beginning of this study, and the discussions within allow for a more nuanced picture of the professional debates on the practice, where both proponents and sceptics at times found common ground. Technological and societal aspects were central to much of the argumentation for the further development of vivisection, with technology easing the practical aspects of the method, and the concept of the "gentleman" allowing British "vivisectors" to argue against charges of cruelty, pointing rather to continental schools of physiology as the culprits, whilst lifting the "humanity" behind animal experimentation in Great Britain. In conjunction with pointing out the importance of the method for the development of medical science, the Cruelty to Animals Act and the lobbying on behalf of the professional journals British Medical Journal and The Lancet helped legitimise the practice in Great Britain. The Act allowed vivisection under set circumstances, and the two journals served as megaphones for scientific development on behalf of vivisection, at times even openly criticising sceptical opinions. At the same time, some saw experimental research through vivisection as merely one aspect of medical practice. One which needed to gain foothold in order to help advance medical science for the larger benefit of all humanity.

Introdução: Os traumas arteriais e venosos são responsáveis por expressiva morbimortalidade, e, em determinados territórios, a técnica de restauração aberta acrescenta riscos elevados ao paciente, que podem ser minimizados com o uso de técnicas endovasculares. Objetivos: O objetivo deste estudo foi criar um modelo experimental de trauma vascular periférico penetrante em que se avalia a viabilidade do reparo endovascular em lesões da parede arterial com diferentes extensões cincunferenciais. Método: Vinte porcos brancos machos foram divididos em quatro grupos, de acordo com a extensão circunferencial do trauma arterial: sem lesão arterial (Grupo 1); lesão arterial com extensão circunferencial <50% (Grupo 2); lesão arterial com extensão circunferencial >50%, variando entre 50-80% (Grupo 3); e secção completa (Grupo 4). A artéria carótida comum esquerda foi dissecada com controle arterial proximal e distal, procedimento que se seguiu de secção controlada da parede arterial, fechamento dos planos e compressão manual por dez minutos, seguida de tratamento endovascular com introdução de stent revestido ViabahnTM (5mm x 50 mm) por via de acesso femoral. Resultados: A viabilidade e a reprodutibilidade do modelo experimental proposto foram confirmadas pelo sucesso no tratamento de todos os animais sem trauma e nos animais com lesões <50%. Sucesso da técnica endovascular também foi observado em quatro dos cinco animais com lesões >50% e, em um animal com secção completa. Variáveis como a duração do procedimento, parâmetros ultrassonográficos e arteriográficos, e flutuação dos sinais vitais foram devidamente monitoradas. Conclusões: O reparo endovascular do trauma arterial periférico em animais de experimentação mostrou-se factível com limitação dependendo da extensão circunferencial da lesão. Este modelo experimental, envolvendo técnicas endovasculares, indicou etapas importantes a serem consideradas em outros estudos nestes animais e com a utilização destes materiais. / Background: Additional surgical trauma often increases the risk of major morbidity and mortality associated with vascular injury, and endovascular repair could have many advantages in such situations. Objectives: The aim of this study was to create an experimental animal model of penetrating peripheral artery injury and to evaluate the feasibility of endovascular repair in different degrees of circumferential injury. Methods: Twenty white male domestic pigs were divided into four groups according to the circumferential extent of arterial injury: no injury; circumferential injury extent < 50% or > 50%, ranging between 50- 80%, and complete sectioning. Left common carotid artery was dissected with proximal and distal artery control followed by controlled section of the arterial wall. Local manual compression was applied for 10 minutes followed by endovascular treatment with a 5 x 50 mm ViabahnTM covered stent using the femoral approach. Results: The feasibility and reproducibility of the proposed experimental model was confirmed by the successful treatment of all animals with no injury and with injuries with a circumferential extent < 50%. Success was also achieved in four of the 5 animals in the group with injuries of circumferential extent > 50%, and in one pig in the complete section group. Additional variables were monitored, such as duration of procedure, ultrasound and arteriography parameters and fluctuation of vital signs. Conclusions: Endovascular repair of arterial injury is possible depending on circumferential extension of arterial lesion. This experimental model, involving endovascular techniques, shows important steps to consider in further studies in these animals and use of these materials.

Este trabalho tem como objetivo descrever as principais doenças diagnosticadas em caprinos no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul no período de 2000 a 2016. No primeiro artigo foi realizado um estudo retrospectivo das causas de morte em caprinos diagnosticadas de 2000 a 2016. Do total de 322 caprinos necropsiados neste período, 290 (90%) obtiveram um diagnostico conclusivo. Dos casos conclusos, 167 (57,6%) corresponderam a enfermidades de origem infecciosa e toxi-infecciosas e 123 (42,4%) enquadrados em causas não infecciosas. Entre as doenças infecciosas foram contabilizados 54 casos de origem bacteriana, 60 casos com envolvimento parasitário, 14 casos de origem viral, além de 39 casos toxi-infecciosos. As doenças de caráter não infeccioso foram ainda agrupadas em doenças metabólicas (44 casos), intoxicações por plantas ou substâncias tóxicas (36), deficiências minerais e nutricionais (20), neoplasias e distúrbios no desenvolvimento (5). A hemoncose, eimeriose, pleuropneumonias e a enterotoxemia foram as doenças mais frequentemente diagnosticadas neste período. O segundo artigo descreve um surto de tuberculose em caprinos jovens. Onze de um total de 15 caprinos, de cinco a 15 dias de idade, foram positivos ao teste de tuberculina. Na necropsia, o parênquima pulmonar de todos os caprinos positivos apresentavam nódulos de 0,3 a 10 cm de diâmetro, coloração brancacenta a amarelada, ocasionalmente, também observados no fígado e baço Os linfonodos retrofaríngeos, mediastínicos e traqueobrônquicos apresentavam-se acentuadamente aumentados de tamanho e aspecto caseoso. Na avaliação histológica, a lesão era caracterizada por intensa necrose caseosa, com áreas de mineralização distrófica, associados a acentuado infiltrado inflamatório granulomatoso. A coloração de Ziehl-Neelsen e a marcação por imuno-histoquímica anti-complexo Micobacterium tuberculosis evidenciou discreta a moderada quantidade de bacilos álcool-ácido resistentes. O cultivo microbiológico e a análise molecular confirmaram o agente etiológico M. bovis. O terceiro artigo descreve dermatite e hepatopatia tóxica crônica natural e experimental em caprinos associadas ao consumo de farelo de arroz desengordurado. Caprinos jovens, de um a quatro meses de idade, apresentavam alopecia e formações crostosas na pele, apatia, emagrecimento, prurido discreto e, vinham a óbito em um período de 30-40 dias. À necropsia, o fígado apresentava irregularidades na superfície capsular, coloração alaranjada a avermelhada, além de rins com múltiplas áreas circulares brancacentas na superfície capsular. À análise microscópica, acentuada atrofia de hepatócitos em região periportal hepática e moderada degeneração hepatocelular microvacuolar. No estudo experimental comprovou-se a etiologia dos casos, através da manifestação de lesões de pele, hepática e renais similares ao dos casos naturais, entretanto em menor intensidade. / This work aims to describe the main diseases diagnosed in goats in the Sector of Veterinary Pathology of the Federal University of Rio Grande do Sul from 2000 to 2016. The first article describes the main causes of death in goats diagnosed between 2000 and 2016. A conclusive diagnosis was obtained in 290 (90%) cases from a total of 322 goats necropsied. Of these cases, 167 (57.6%) corresponded to infectious and toxi-infectious diseases, and 123 (42.4%) included non-infectious causes. Among the infectious diseases 54 cases were of bacterial origin, 60 cases were caused by parasite agents, 14 cases of viral origin, and 39 toxi-infectious cases. Non-infectious diseases were also grouped into metabolic diseases (44 cases), poisoning by plants or toxic substances (36), mineral and nutritional deficiencies (20), neoplasms and developmental disorders (5). Haemonchosis, eimeriosis, pleuropneumonia and enterotoxemia remain as one of the major control obstacles in goat farms. The second article describes an outbreak of tuberculosis in goat kids. Eleven of a total of 15 kids, from 5 to 15 days old, were positive to tuberculin. At necropsy, the pulmonary parenchyma of all positive goats had white to yellowish nodules of 0.3 to 10 cm in diameter, that were occasionally also observed in the liver and spleen The retropharyngeal, mediastinal and tracheobronchial lymph nodes were markedly enlarged and with a caseous aspect. Histologically, the lesion was characterized by an intense caseous necrosis, with areas of dystrophic mineralization, associated to a marked granulomatous inflammatory infiltrate. Ziehl-Neelsen histochemistry exam and immunohistochemical anti-Micobacterium tuberculosis complex evidenced mild to moderate amount of bacilli. Microbiological culture and molecular analysis confirmed M. bovis as the etiological agent. The third article describes a natural and an experimental toxic liver disease associated with the consumption of defatted rice bran in goats. These presented with alopecia and crusted formations on the skin, apathy, weight loss, mild pruritus, and death within a period of 30-40 days. At necropsy, the liver presented multifocal to coalescing orange to reddish irregular areas on the capsular surface, and the kidneys presented multiple white circular areas on the capsular surface. Microscopic analysis revealed a marked hepatocyte atrophy at the hepatic periportal region, and a moderate microvacuolar hepatocellular degeneration. In the experimental study, the etiology of the cases was demonstrated through the manifestation of lower intensity skin, liver and kidney lesions similar to those of the natural cases.

While substrate-borne vibrations are utilized by different reptile species, true conspecific communication via biotremors has not yet been demonstrated in reptiles. This study follows a preliminary report that the veiled chameleon (Chamaeleo calyptratus) could produce biotremors in communicative contexts. I tested chameleon behavioral sensitivity to vibrations by placing them on a dowel attached to a shaker emitting vibrations of 25, 50, 150, 300, and 600 Hz and then measured their changes in velocity before and after the stimulus. I then paired chameleons in various social contexts [anthropogenic disturbance (human disruption of animal); dominance (malemale; female-female C. calyptratus); courtship (male-female C. calyptratus); heterospecific (C. calyptratus + C. gracilis); and predator-prey (adult + juvenile C. calyptratus)] and used a video camera and accelerometers to record their behavior. This study demonstrates that chameleons produce biotremors and that receivers exhibit a freeze response when exposed to a simulated biotremor stimulus. Furthermore, veiled chameleons produce biotremors in anthropogenic disturbance, conspecific dominance and courtship contexts, and these biotremors are elicited by visual contact with another adult conspecific and heterospecifics. Overall, two classes of biotremor were identified, "hoots” and “rumbles,” which differ significantly in dominant frequency and waveform. No correlation was identified between animal size and dominant frequency of the biotremors they produced as biotremors originate from rapid muscle contractions. Juvenile chameleons of two months of age are able to produce biotremors, suggesting this behavior may have multiple functions. Overall, the data suggest that the veiled chameleon has the potential to utilize substrate-borne vibrational communication during conspecific and possibly heterospecific interactions.

a. Introduction The immune system must detect a wide variety of agents, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. However, the immune system needs to be well regulated since a disorder in an immune response can result in autoimmune diseases, tissue destruction, inflammatory diseases and cancer. Within the context of innate immunity, the mononuclear phagocyte system, cells comprising bone marrow progenitors, blood monocytes and tissue macrophages is acquiring great importance in the study of different pathologies and particularly the monocytes/macrophage functions. In this regard, recent studies demonstrate that monocytes present a heterogeneous population of innate cells. Monocyte was found leading to distinct cell populations with various subtypes with distinct functions. Two types of monocytes were identified in mice. Resident monocytes, with a CD11b+CCR2lowLy-6ClowCX3CR1high phenotype, migrate to uninjured tissues after emigration from bone marrow and differentiate into resident macrophages and dendritic cells. In contrast, a distinct inflamed monocyte subset with a CD11b+CCR2highLy-6ChighCX3CR1low phenotype infiltrates infected tissue and contributes to the development of inflammation. Currently, all studies performed with monocytes are done in transgenic models (i.e. CCR2-/-; GPF-CX3CR1 models) or with expensive techniques to study and acquire the maximum number of cell possible from mice blood. Monocytes constitute around 2% (100cells/μl) of the total peripheral blood leukocyte pool in mice, where only 1-5% are Ly-6Chigh monocytes. What makes difficult to study it. b. Objective 1. Development of an in vitro model that allow the generation of large amounts of Ly-6Chigh monocytes from bone marrow from mice. 2. Characterization of the phenotype of Ly-6Chigh monocyte generated in vitro. 3. Analyze the activation function of Ly-6Chigh monocyte generated in vitro. 4. Study the migration of Ly-6Chigh monocytes in to two inflammation models: - Skin (DNFB model) - Muscle (Notexin muscle model) 5. Analyze the therapeutic effect of Ly-6ChighCD11b+ monocytes injection in the resolution of inflammation in two experimental models of inflammation. c. Methodology and Results To achieve the first aim of our work, bone marrow cells were cultured with different grows factors and FCS at 37ºC in a humidified 5% CO2 atmosphere for 7 days when the population of floating cells was obtained and stained with Ly-6C and CD11b markers. This population was sorted for the acquirement of the Ly-6ChighCD11b+ cells. In order to characterize the phenotype of these cells we stained them with several markers. Our results demonstrated that this Ly-6Chigh enriched population is CD11b+CD62L+CCR2+ F4/80+CX3CR1low, presenting the same phenotype of the cells presents in the blood. To study the functional heterogeneity of enriched Ly-6Chigh cells, these cells were incubated with IFN-γ as typical classical stimuli and IL-4 as an alternative pathway stimulus. Ly-6Chigh cells incubated with IFN induced the expression of TNF and NOS2 with a characterized kinetics similar to macrophages. However, these cells increased arginase-1 levels when were stimulated with IL-4. Thus, the in vitro results have shown the plasticity and heterogeneity of monocytes as previously described by macrophages and thus, suggests us that these cells can also adapt to changing microenvironments as previously described. Further, to observe the migration capacity and functionality of these cells in vivo, we optimized two experimental model of inflammation. In the first model an ear skin irritation with 1%DNFB was induced. In the second model, muscle inflammation was developed by the injection of Notexin in the tibialis anterioris. In both models inflammation was induced and Ly-6Chigh enriched cells stained with an infrared fluorocrome were injected intravenous in mice and migration was observed by in vivo image at different days. Migratory capacity of Ly-6C cells to the inflamed tissues was appreciated in both models, corroborating with data previously described. To analyze the therapeutic effect of Ly-6ChighCD11b+ monocytes injection in the resolution of inflammation, RNA and histology cuts were obtained from both models. The results showed that Ly-6Chigh-injected mice express higher levels of anti-inflammatory genes such as mannose receptor, which corroborate with the histological images where animals treated with Ly-6Chigh cells recover before of the inflammatory process that untreated animals. d. Conclusion 1. We established a novel in vitro protocol to generate Ly-6ChighCD11b+ monocyte obtained from bone marrow of Balb/C mice. 2. The cells generated in vitro have the same phenotype of the Ly-6C from blood flow. 3. Cells Ly-6ChighCD11b+ monocyte present high plasticyty. 4. Ly-6ChighCD11b+ monocytes generated in vitro migrate in vivo. 5. Injection in acute and chronic in vivo inflammatory models of Ly-6ChighCD11b+ monocytes generated in vitro, display an improvement in the site of inflammation through the presentation of a more anti-inflammatory profile. / Monocitos circulantes proporcionan una defensa contra las infecciones y también a enfermedades autoinmunes. Recientemente dos tipos de monocitos fueran identificaron en la sangre periférica de ratones. El monocito ¿residente¿ con fenotipo CD11b+CCR2lowLy-6ClowCX3CR1high, que migran a tejidos no lesionados y se diferencian en macrófagos residentes y células dendríticas (DC). En contraste, un subconjunto distinto conocido como monocitos ¿inflamatorios¿, con un fenotipo CD11b+CCR2highLy-6ChighCX3CR1low son células que migran al tejido infectado y en lo cual contribuye al desarrollo de la inflamación. Monocitos Ly-6Chigh, el objetivo de nuestro trabajo, representan un 2-5% de los monocitos del torrente sanguínea de los ratones. Dado que estas células son de difícil obtención y que la cantidad obtenida de la sangre de ratones es muy baja, nuestro grupo desarrolló un nuevo sistema para generar monocitos Ly-6Chigh in vitro a partir de médula ósea de ratón, con el objetivo de estudiar sus funciones in vivo en dos modelos animales de inflamación. Nuestro laboratorio ha optimizado dos modelos animales capaces de inducir inflamación local en ratones Balb/c, inmunocompetentes. En el primer modelo, 1-fluoro-2 ,4-dinitrobenceno (DNFB) se aplicó tópicamente en la oreja derecha para crear en la piel condiciones que inducen la migración de estas células para el sitio de la irritación (modelo DNFB). En este modelo de piel, la inflamación en la oreja fue calculada atreves del peso neto, donde el peso de la oreja izquierda es restado del peso de la oreja derecha después de 24h y 48h de la inyección intravenosa (iv) de los monocitos Ly-6ChighCD11b+ en los ratones. En el segundo modelo, la inflamación es inducida atreves de la aplicación de una inyección de Notexin en el tibial anterior (TA) de la pierna derecha del animal la cual induce una inflamación muscular (modelo Notexin). Finalmente en ambos modelos, monocitos Ly-6ChighCD11b+ generados in vitro pre-tratados in vitro con citocinas pro- o anti-inflamatoria (IFN-¿ o IL-4) o no tratados, fueran inyectados iv en la colas de los ratones. Por otra parte, expresión génica fue medida mediante PCR cuantitativa en tiempo real, la migración celular fue evaluada in vivo atreves de imágenes realizadas por el equipo de IVIS, estudios de citometría de flujo y ensayos de histología también fueran realizados para evaluar la función de las células Ly-6Chigh en el sitio de inflamación. El principal objetivo de nuestro estudio es: 1. Desarrollo de un modelo in vitro que permita generar grandes cantidades de monocitos Ly-6Chigh a partir de médula ósea de ratones. 2. Caracterización del fenotipo de los monocitos Ly-6Chigh generados in vitro. 3. Analizar funciones de los monocitos Ly-6Chigh generados in vitro tras su activación in vitro. 4. Estudio de la capacidad migratoria de los monocitos Ly-6Chigh generados in vitro en dos modelos de inflamación. - Piel (modelo de DNFB en oreja). - Músculo (modelo Notexin muscular). 5. Analizar el efecto terapéutico de la inyección de monocitos Ly-6Chigh generados in vitro en la resolución de la inflamación en dos modelos experimentales de inflamación. En ambos modelos animales la inflamación local aumentó en función del número de monocitos Ly-6Chigh inyectados. Migración celular fue analizada por imágenes in vivo en ambos modelos, donde células Ly-6Chigh generated in vitro fluorescentes estaban presentes apenas en el tejido inflamado. Análisis de hematoxilina y eosina en cortes histológicos demostraron una mejoría del tejido de los animales tratados con monocitos Ly6Chigh. En resumen, los resultados obtenidos en esta Tesis Doctoral revelar un nuevo método para generar in vitro Ly-6Chigh monocitos de médula ósea de ratones, con una mejora en la eficiencia de la producción celular, que facilitan el estudio de estas células in vitro e in vivo. Además, también han demostrado la capacidad de las células Ly-6Chigh para cambiar el fenotipo de la estimulación in vitro verdadera y la capacidad de migrar, así como la heterogeneidad funcional en dos modelos de inflamación in vivo, lo que indica que estas células accionar de la misma manera como se las células proveniente de la sangre periférica. Además, hemos demostrado que Ly-6Chigh monocitos pueden ser pre-tratados con citoquinas en orther para retrasar o aumentar la reparación de tejidos (IFN-¿ o IL-4), respectivamente Todos estos resultados juntos sugieren que los monocitos Ly-6Chigh generado por nosotros in vitro son células funcionales que se pueden utilizar como una herramienta terapéutica para tratar enfermedades inflamatorias.

Primary chick embryo liver cells were used to explore the regulation of δ-aminolevulinic acid synthase and heme oxygenase, the enzymes that catalyze the rate-limiting reactions of heme anabolism and catabolism, respectively. The general focus of the work was the exploration of the novel observation in which glutethimide and iron synergistically induced both δ-aminolevulinic acid synthase and heme oxygenase, a phenomenon that would not be predicted a priori. The course of events appeared to be: first, that heme synthesis was increased after addition of the glutethimide and that iron potentiated heme synthesis; second, the heme induced heme oxygenase five to ten fold; and third, that heme oxygenase degraded the heme permitting an uncontrolled induction of δ-aminolevulinic acid synthase. This induction of δ-aminolevulinic acid synthase could be prevented by the addition of a metalloporphyrin inhibitor of heme oxygenase. Induced δ-aminolevulinic acid synthase activity could be dramatically reduced by the addition of nanomolar concentrations of a metalloporphyrin, inhibitory for heme oxygenase, and heme. Specific observations related to the synergistic induction of heme oxygenase by glutethimide and iron was that the induction of heme oxygenase activity by glutethimide and iron occurred rapidly, with maximal increases occurring four to six hours after original treatment. Induction of heme oxygenase by glutethimide and iron was shown to be dependent on de novoheme synthesis since 4,6-dioxoheptanoic acid, a potent and specific inhibitor of heme biosynthesis, prevented the activity of heme oxygenase from increasing in the presence of glutethimide and iron. Induction of activity was associated with increases in heme oxygenase mRNA and protein; and, when induction was prevented by 4,6-dioxoheptanoic acid, no increase in either mRNA or immunoreactive protein was observed. δ-Aminolevulinic acid synthase activity was also synergistically increased by glutethimide and iron; this increase occurred 4-6 hours after maximal heme oxygenase activity had been attained. The temporal relationship between the induction of δ-aminolevulinic acid synthase and heme oxygenase suggested that the oxygenase depleted a regulatory heme pool that would normally prevent uncontrolled induction of the synthase. When cultures were exposed to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, induction of δ-aminolevulinic acid synthase, normally produced by glutethimide and iron, was prevented. Addition of tin-mesoporphyrin after δ-aminolevulinic acid synthase induction had already been established promptly halted any further induction. When heme or a combination of heme and tin-mesoporphyrin was added after induction of δ-aminolevulinic acid synthase was established, activity of the synthase was rapidly reduced. Finally, experiments in primary chick embryo liver cells with tin-, zinc- and copper- chelated porphyrins were done to assess their effects on activities of δ-aminolevulinic acid synthase, induced by prior treatment of cells with glutethimide and iron. Nanomolar concentrations of zinc- or tin porphyrins reduced δ-aminolevulinic acid synthase activities, while copper-chelated porphyrins did not. When nanomolar concentrations of heme were added with zinc- or tin-porphyrins, δ-aminolevulinic acid synthase activity was further reduced. Effects of the non-heme metalloporphyrins on δ-aminolevulinic acid synthase were closely correlated with their abilities to inhibit heme oxygenase (r=0.78). The largest decrease of δ-aminolevulinic acid synthase (67%) was obtained with zinc-mesoporphyrin and heme. There was a rapid appearance of the cytosolic, precursor form of δ-aminolevulinic acid synthase in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. Reduction of the half-life of the mRNA from 5.2 hours to 2.2-2.5 hours was observed in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. In summary, the chick embryo liver cell culture model treated with glutethimide and iron may serve as one experimental model for patients suffering from acute porphyrias, in whom uncontrolled induction of hepatic δ-aminolevulinic acid synthase plays a key role in pathogenesis of disease. The synergistic induction of δ-aminolevulinic acid synthase in the presence of glutethimide and iron may serve as an experimental paradigm for this disease. The reduction of δ-aminolevulinic acid synthase by low doses of zinc-mesoporphyrin and heme may help form the experimental foundation for eventual studies in patients suffering from acute porphyrias.

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 (Th2) response following immunization with many vaccine antigens. Although maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. Recent studies have suggested that immunizing with DNA plasmids encoding the vaccine antigen of interest is highly efficacious in a variety of adult animal models. However, similar extensive studies have not been conducted in infants. In this dissertation, we examine both the quantitative and qualitative differences between neonatal and adult humoral and cell-mediated immune responses in the presence or absence of maternal antibody. First, we wished to determine if one-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (HA) by either intramuscular (i.m.) or gene gun (g.g.) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. We found that newborn mice developed stable, long-lived, protective anti-HA-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult i.m. and g.g. DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1-, IgG2a-, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased, but did not change these antibody profiles. We also found that, in contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Further, temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age. To determine if the disparity of polarized IgG responses between neonatal and adult DNA vaccinated mice was due to deficiencies in Th1 promoting cytokines, we addressed the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal DNA immunization. We found that neonatal mice coimmunized with HA and either IL-12 or IFNγ-expressing DNAs developed IgG2a-biased immune responses, regardless of inoculation method, whereas these DNAs had no effect on IgG subtype patterns in adult DNA immunized mice. Consistent with the Th1-promoting effects of these cytokines, we also observed that codelivery of IL-12 or IFNγ DNAs raised T helper responses toward Th1 in mice immunized both as neonates or adults. Thus, codelivery of cytokine DNAs may be effective at tailoring immune responses depending on the required correlates of protection for a given pathogen. Finally, we addressed the effect of maternal antibody on the elicitation of humoral and cell-mediated immune responses. We tested the ability of i.m. and g.g. immunization with DNA expressing influenza HA and/or nucleoprotein (NP) to raise protective humoral and cellular responses in the presence and absence of maternal antibody. We found that neonatal mice born to influenza-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD4+ and CD8+ T cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses, but rather it limited humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. We further observed that protection from influenza virus challenge was dependent on the presence of anti-HA IgG and was independent of the presence T cell responses. Taken together with other published studies, the data presented in this dissertation help better characterize the responses elicited by DNA vaccines at birth. This dissertation presents several novel observations including the temporal development of polarized IgG subtype responses, the ability of codelivered Th1 cytokine DNA to affect both antibody and T cell responses in the neonate, and the ability to generate humoral responses to intracellular, but not plasma membrane proteins, in the presence of maternal antibody. Furthermore, the data provides rationale for further development of DNA vaccines in the neonate.

Dynactin is a multi-subunit complex which was initially identified in 1991 as an activator of cytoplasmic dynein-driven microtubule-based organelle motility in vitro. Although genetic studies also supported the involvement of both complexes in the same functional pathways in yeast, filamentous fungi, and Drosophila, none of these findings yielded significant insights into dynactin's mechanism of action. The full range of cytoplasmic dynein functions in vertebrate cells has also remained poorly understood, due, in large part, to the lack of a specific method of inhibition. The present thesis work was designed to investigate these issues through a study of the 50 kDa subunit of dynactin. As a first step (Chapter 1), I cloned mammalian p50 and characterized its expression at the tissue and subcellular levels. Rat and human cDNA clones revealed p50 to be a novel α-helix-rich protein containing several highly-conserved structural features including one predicted coiled-coil domain. Immunofluorescence staining of p50, as well as other dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase and concentrate near spindle poles thereafter. These findings represented the first evidence for dynactin and cytoplasmic dynein co-localization within cells, and for the presence of dynactin at kinetochores. The second major phase of the thesis (Chapter 2) was focused on investigating dynactin and cytoplasmic dynein function in cultured cells in vivo using a dominant negative inhibition approach based on transient transfections of p50 constructs. Overexpression of wild type human p50 in cultured cells resulted in a dramatic fragmentation and dispersal of the Golgi apparatus. Time-lapse fluorescence microscopy analysis of p50-overexpressing cells revealed that microtubule-based vesicle transport from the endoplasmic reticulum to the Golgi was inhibited. Also, the interphase microtubule organizing center was found to be less well-focused in some but not all transfected cells. Overexpression of p50 also disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still generally bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings provided the first in vivoevidence for the role of dynactin in cytoplasmic dynein function, i.e. mediating the motor's binding to at least one "cargo" organelle, the kinetochore, and probably also to others such as vesicles destined for the Golgi complex. These data also strongly implicated both dynactin and dynein in Golgi organization during interphase, and chromosome alignment and spindle organization during mitosis. Based on the remarkable disruptive phenotypic effects associated with overexpressing of p50, the name of dynamitin was proposed for this polypeptide. In the third and last phase of the thesis (Chapter 3), two issues were addressed: first, the dynamitin-induced mitotic arrest phenotype was studied in greater detail to better understand the exact sites of dynactin and cytoplasmic dynein activity throughout mitosis. Second, a domain analysis of dynamitin was performed to gain insight into its function within the dynactin complex. A time-lapse fluorescence microscopy study of mitosis in living dynamitin-overexpressing COS-7 cells strongly suggested specific defects in interactions of astral microtubules with the cell cortex, and in both spindle pole assembly and maintenance. Analysis of the mitotic arrest phenotype in a second cell line revealed a second arrest point at metaphase, and a clear effect of dynamitin overexpression on spindle axis orientation, again consistent with defects in interactions between microtubules and the cell cortex. Refined analyses of kinetochore and spindle pole components also confirmed specific defects in kinetochore function and spindle pole organization. Taken together, these findings support three main sites of dynactin and cytoplasmic dynein activity during vertebrate mitosis: prometaphase kinetochores, spindle poles, and the cell cortex. Finally, the domain analysis revealed dynamitin to be capable of self-association through at least two separate interaction domains, consistent with models of the mechanism underlying dynamitin-induced dynactin dissociation, and therefore, yielding important new insights into dynactin assembly. This study also indicated that a third region within dynamitin, residues 105 to 154, is essential for dynamitin and dynactin function. An independent study confirmed this finding, implicating this region in binding to ZW10, an upstream kinetochore protein. Dynamitin has therefore been revealed to be the kinetochore-targeting subunit of dynactin, and indirectly, cytoplasmic dynein. Through the body of this thesis work, dynamitin has also emerged as a powerful new tool for studying vertebrate dynactin and cytoplasmic dynein function in vivo and in vitro.

The overall aim of this thesis was to determine how natural killer (NK) cells regulate virus infections in vivo. Anti-viral mechanisms by which NK cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of adult C57BL/6 mice were first studied, revealing different mechanisms of control in different organs. Three days post-infection, MCMV titers in the spleens of perforin-deficient (perforin 0/0) mice were higher than in wild type controls, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers but not in spleen titers. Depletion of IFN-γ in adult C57BL/6 mice by injections with mAbs to IFN-γ resulted in an increase in viral titers in the liver but not in the spleen. Analyses using IFN-γ-receptor-deficient (IFN-γR0/0) mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that even though the donor spleen cells could respond to IFN-γ, the depletion of NK cells in a recipient environment where the host cells could not respond to IFN-γ caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-γ has the ability to induce a variety of cells to produce nitric oxide (NO), and administrating the nitric oxide synthase (NOS) inhibitor Nω-monomethyl-L-arginine (L-NMA) into MCMV-infected adult C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. These data indicate that in adult C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, whereas in the liver the production of IFN-γ by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-1r (Cmv-1-resistant) locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver. The ability of adoptively transferred cells to protect suckling mice from MCMV was another model used to study the mechanisms utilized by NK cells in the regulation of MCMV. Adoptive transfers of 129, C57BL/6 and perforin 0/0 spleen cells or lymphokine-activated killer (LAK) cells into 4 - 6 day old MCMV-infected C57BL/6 suckling mice significantly lowered the splenic MCMV titers in these mice compared to the infected controls. Adoptive transfers of C57BL/6 spleen cells into MCMV-infected 129 suckling mice also decreased the amount of MCMV in the 129 suckling mice, but C57BL/6 spleen cells could not regulate MCMV synthesis when adoptively transferred into 129/IFN-γR0/0 suckling mice. These results suggest that, in the suckling mouse model, the regulation of MCMV by the adoptively transferred NK cells is via an IFN-γ-dependent, perforin-independent, Cmv-1-independent mechanism. The Cmv-1 gene locus resides within the NK gene complex, in close proximity to the Ly49 NK cell receptor family. Analyses were carried out to determine if any of the 4 known Ly49 NK cell receptors (Ly49A, C, D and G2) played a role in the control of MCMV synthesis by NK cells. Studies comparing the expression of the different Ly49 NK cell subsets in the spleen and the peritoneal cavity revealed that there were differences in the distribution of the Ly49 receptors on NK1.1+ cells. Three days post-MCMV infection, the percentage of NK1.1+- Ly49+ NK cells in the spleen and the peritoneal cavity were different than in naive controls. Within the splenic NK1.1+ population, increases in NK1.1+ -Ly49A+ and NK1.1+-Ly49G2+ cells but decreases in NK1.1+-Ly49C+ and NK1.1+-Ly49D+ cells were observed. These changes in the spleen were accompanied by a concomitant decrease in NK1.1+ - Ly49A+ cells and increases in NK1.1+-Ly49C+, NK1.1+-Ly49D+ and NK1.1+-Ly49G2+ cells within the NK1.1+ population in the peritoneal cavity. These data suggest that 3 days post-MCMV infection, there may be movement of NK cells between the different organs. The role of Ly49 NK cell receptors in the regulation of MCMV was tested using adult C57BL/6 mice depleted of single or multiple Ly49 NK cell subsets. These in vivo depletions did not affect the ability of the residual NK cells to regulate MCMV synthesis. LAK cells sorted into the different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice lowered the splenic MCMV titers in these mice. Together, these results indicate that even though there is a redistribution of the Ly49 NK cell subsets during MCMV infection, the presence or absence of anyone of the 4 tested Ly49 NK cell receptors does not affect the regulation of MCMV by NK cells. However, there remain a possibility that one of the undefined Ly49 receptors or an untested NK cell receptor may be important in the control ofMCMV. Most of the cloned NK cell receptors have been shown to bind to MHC class I molecules, and MHC class I antigens have been implicated as modulators of target cell sensitivity to NK cell-mediated lysis. The regulation of virus infections and the fate of NK cells and their natural targets was examined in β2-microglobulin-deficient mice [β2m (-/-)], which have defective MHC class I expression. Infections with either the NK cell-sensitive MCMV or the NK cell-resistant lymphocytic choriomeningitis virus (LCMV) significantly augmented NK cell activity in either C57BL/6 or β2m (-/-) mice. Depletion of NK cells in vivo with antiserum to asialo GM1 markedly enhanced the synthesis of MCMV but had no effect on the synthesis of LCMV in either strain of mouse. Adoptively transferred β2m (-/-) spleen cells lowered splenic MCMV titers in C57BL/6 suckling mice, not unlike adoptively transferred C57BL/6 spleen cells. Analysis of naturally NK cell-sensitive thymocyte targets from these virus-infected β2m (-/-) mice revealed no cell surface expression of class I MHC detectable by conformation-dependent or -independent antibodies, but the virus infections enhanced class I expression on thymocytes from C57BL/6 mice. The sensitivity of C57BL/6 thymocytes to NK cell-mediated lysis was markedly reduced after in vivo poly inosinic:cytidylic (poly I:C) treatment or viral infection; in contrast, the sensitivity of the β2m (-/-) thymocytes was significantly less affected by poly I:C or viral infection. These data indicate that the normal expression of MHC class I antigens on NK cells or their targets is not required for the anti-viral functions of NK cells against an NK-sensitive virus (MCMV) nor do they protect an NK-resistant virus (LCMV) from the anti-viral activity of NK cells. Together, the data presented in this thesis help to further our understanding of the mechanisms utilized by NK cells in the control ofMCMV in both adult and suckling mice, and also help clarify the roles played by Ly49 NK cell receptors and MHC class I molecules in the regulation of MCMV.