The plaque reduction neutralization test (PRNT) is a confirmatory diagnostic assay that is used to confirm a variety of diseases. The performance of PRNT requires the use of infectious wild type viruses, which increases the risk of laboratory acquired infections. For instance, eastern equine encephalitis (EEEV) is a highly virulent pathogen used in PRNT that can result in potentially fatal neurological diseases among humans and equines. Therefore, arboviral PRNT must be performed in Biosafety Level 3 (BSL-3) containment facilities and may require select agent approved scientists, like in the case of EEEV. These stringent requirements restrict the ability of public health laboratories to conduct PRNTs. Chimera viruses, recombinant constructs that have been bio-engineered to express the immunogenic structural proteins from the wild type virus in an attenuated form, can serve as a substitution for infectious viruses when performing PRNT. Since chimera viruses do not require the use of a BSL-3 facility and are not classified as select agents, their use offers advantages over wild type viruses. This study aimed at validating the use of EEE and West Nile chimera viruses as an alternative to the corresponding wild type viruses for diagnostic purposes at the Florida Department of Health (FDOH) Bureau of Public Health Laboratories (BPHL). These evaluations were conducted using human and avian sera. The results illustrate that chimera virus-based PRNT portrays specificity comparable to that of the wild type virus, while a slight reduction in sensitivity was observed when human sera was used. Considering their benefits in increasing safety and reducing regulatory requirements, these chimera viruses are an important alternative to the virulent wild type viruses and could be highly beneficial for diagnostic laboratories.

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.

This thesis is a study of ethics and multiplicity as found within contemporary commercial settings. Drawing on Science and Technology Studies (STS) sensibilities and ethnographic-style research, the thesis proposes that current ethical phenomena should be understood as a user-enacted chimerical object: an object that is multiple in its ontology and as much enacted by what it is, as what it is not. This research is particularly pertinent now because the term 'ethical' has become commonplace in modern Western life, including crucially within commercial activities. In certain uses, doing ethics becomes synonymous with doing business. Despite the increasing prevalence of what is considered 'ethical business', the exploration of how the term is appropriated and enacted remains largely under-examined. Through examination of research material gathered during extensive ethnographic studies in three self-avowedly 'ethical organisations' - an ethical start-up, an ethical confectionery company, and an ethical consultancy - the thesis addresses this research gap. By focusing on the users of ethical business, the investigation questions traditional market assumptions of homogeneity within producing organisations, the supposed linear transfer of ethical knowledge, what we can know about 'users', and the genesis of novel ethical realities. Through this questioning the thesis provides new insights on the ethical object. The thesis additionally builds upon questions of how far we can push the boundaries of what we can know about knowledge, and whether it is possible to bring the mess of investigation back into the reporting. Developing previous applications of constitutive reflexivity, the research symmetrically investigates the appropriateness of my application of STS sensibilities to ethical business as a new research area, and interrogates my thesis as an ethical object in order to address the underlying question(s) of whether 'STS means ethical business?'

A major challenge in cancer treatment is acquired or intrinsic multidrug resistance (MDR) to chemotherapeutics. A notorious mediator of MDR is P-glycoprotein (P-gp, ABCB1), product of the human MDR1 gene, which actively effluxes cytotoxic drugs from cancer cells, resulting in sub-therapeutic intracellular concentrations. Understanding how P-gp interacts with drugs has been severely limited by the lack of high-resolution structures of P-gp. Although numerous efforts to obtain an X-ray crystal structure of P-gp have been attempted, human P-gp has never been crystallized. However, mouse P-gp (87% homologous to human P-gp) has been crystallized, and several structures of mouse P-gp have been recently reported. Despite a high degree of homology, it is currently unknown why mouse P-gp can be crystallized while human P-gp cannot. The studies presented in this thesis describe the creation of novel chimeras of mouse and human P-gp as an approach to investigate whether specific protein domains are responsible for differences in the ability to form crystals between mouse and human P-gp. A range of chimeras, created by protein domain swapping, were expressed in mammalian cells and all were found to retain MDR transport function demonstrating that P-gp can tolerate major structural changes. High-level expression of all chimeras was achieved by baculovirus-mediated heterologous protein expression. Chimeric proteins were purified by a multi-step process including immobilized metal affinity chromatography and size exclusion chromatography. Crystallization screening obtained protein crystals for two of the chimeras, indicating the approach adopted is a successful strategy, and an advance along the path towards a high-resolution structure of human P-gp.

Made available in DSpace on 2017-07-21T19:26:08Z (GMT). No. of bitstreams: 1 Moises Souza Santos.pdf: 6452966 bytes, checksum: 0cca031e2fe3425ee7f63672b4643f3b (MD5) Previous issue date: 2014-02-27 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / This work report on a study of the chimera states in networks composed by logistic maps whose interaction between them may have two types of couplings (nonlocal and power law). The Lyapunov exponents and a derivative of this quantity, the Kolmogorov-Sinai entropy, were used as analysis tool to calculate the average time of “life” or collapse time of chimera for nonlocal case with different sizes of networks. For coupling power law the results show that when it has nonlocal features it is also possible to see chimera states through the network. We also calculate the probable existence of regions of chimeras within the parameter space for both cases. Since the chimera dynamic states occurs when coexist regions that are chaotic and periodic in time, we obtained the region of the parameter space where this behavior occurs. We show the probable relations between the chimera states and the global order parameter that provides information on how the sites of a coupled network are related. / Neste trabalho foi realizado um estudo dos estados quimera em redes compostas por mapas logísticos cuja interação entre os mesmos pode ser de dois tipos de acoplamentos (não-local e lei de potência). Utilizamos como ferramenta de análise os expoentes de Lyapunov e uma quantidade derivada deste, a entropia de Kolmogorov-Sinai, para calcular o tempo médio de “vida” ou tempo de colapso de quimera para o caso não-local em redes de diferentes tamanhos. Para o acoplamento lei de potência mostramos que quando este possui características nâo-locais a rede também pode exibir estados quimera. Além disso calculamos as prováveis regiões de existência de quimeras dentro do espaço dos parâmetros para ambos os casos. Uma vez que a dinâmica de estados quimera ocorre quando coexistem regiões que são periódicas e caóticas no tempo, obtivemos a região, no espaço de parâmetros, onde este comportamento ocorre. Mostramos as relações prováveis que existem entre os estados quimera e o parâmetro de ordem global que fornece informações sobre o quão relacionados estão sítios de uma rede acoplada.

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No. of bitstreams: 2 dissertacao_livia_budziarek_eslabao.pdf: 1788001 bytes, checksum: c14f927ac7e6babdc2bd66cf80275556 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A imunocontracepção é reconhecida como um dos principais novos métodos contraceptivos para o controle e o manejo da fertilidade em diferentes espécies animais. Dentre os potenciais alvos utilizados em vacinas contraceptivas, o hormônio liberador de gonadotrofinas (GnRH) é considerado um dos mais atrativos. O GnRH é um decapeptídeo hipotalâmico que apresenta um papel central na reprodução de mamíferos. Entretanto, devido à sua baixa imunogenicidade, é necessário associar o GnRH com uma molécula carreadora capaz de estimular o sistema imune como, por exemplo, a subunidade B da enterotoxina termolábil de Escherichia coli (LTB). O presente estudo visou avaliar o potencial imunocontraceptivo de duas quimeras LTB/GnRH em camundongos machos da linhagem BALB/c. Os camundongos foram divididos aleatoriamente em oito grupos experimentais. O grupo LTB/GnRH-P recebeu a quimera expressa em P. pastoris. O grupo LTB/GnRH-P/A recebeu a quimera expressa em P. pastoris adsorvida em adjuvante oleoso. O grupo LTB/GnRH-E recebeu a quimera expressa em E. coli. O grupo LTB/GnRH-E/A recebeu a quimera expressa em E. coli adsorvida em adjuvante oleoso. O grupo LTB recebeu a proteína LTB expressa em E. coli. Os grupos Controle e Controle/A não receberam nenhum antígeno vacinal. O grupo Vacina comercial recebeu vacina comercial anti-GnRH utilizada em bovinos. A resposta imune humoral foi avaliada pela técnica de ELISA indireto e o potencial imunocontraceptivo foi avaliado por meio da quantificação de testosterona sérica e das análises histológicas das gônadas dos animais. Os grupos que receberam os antígenos LTB/GnRH (25 μg por vacina) apresentaram níveis de IgG total significativamente superiores quando comparados aos grupos controle, mostrando que ambos os antígenos são imunogênicos. Os grupos LTB/GnRH-P e LTB/GnRH-P/A apresentaram as maiores absorbâncias nos dias 28 (1,11 ± 0,09) e 42 (1,44 ± 0,04), respectivamente. Além disso, o grupo LTB/GnRH-P/A apresentou níveis de testosterona significativamente menores ao controle a partir do dia 28 (192 ng/dl ± 254,55). Os grupos LTB/GnRH-E e LTB/GnRH-E/A também obtiveram os maiores níveis de anticorpos nos dias 28 (0,33 ± 0,005) e 42 (1,44 ± 0,08), respectivamente. O grupo LTB/GnRH-E/A apresentou níveis de testosterona significativamente menores ao controle a partir do dia 28 (<12 ng/dl ± 0). Os resultados da histologia mostraram que ambos os antígenos causam alterações na espermatogênese, sendo que o antígeno expresso em E. coli foi relacionado com as maiores alterações nas gônadas. O presente estudo mostrou quimeras produzidas através da fusão da LTB com uma única molécula de GnRH é capaz de induzir a geração de resposta imune humoral, bloqueio das funções endócrinas relacionadas com a reprodução e alterações teciduais nas gônadas de camundongos machos. / Immunocontraception is recognized as one of the major new contraceptive methods for the control and management of fertility in different animal species. Among the potential targets used in contraceptive vaccines, gonadotropin-releasing hormone (GnRH) is considered one of the most attractive. GnRH is a hypothalamic decapeptide that presents a central role in mammalian reproduction. However, due to its low immunogenicity, it is necessary to associate the GnRH with a carrier molecule capable of stimulating the immune system such as, for example, the B subunit of Escherichia coli heat-labile enterotoxin (LTB). The present study aimed to evaluate the immunocontraceptive potential of two LTB/GnRH chimeras in male BALB/c mice. Mice were randomly divided into eight experimental groups. The LTB/GnRH-P group received the chimera expressed in P. pastoris. The LTB/GnRH-P/A group received the chimera expressed in P. pastoris adsorbed in oil adjuvant. The LTB/GnRH-E group received the chimera expressed in E. coli. The LTB/GnRH-E/A group received the chimera expressed in E. coli adsorbed in oil adjuvant. The Control and Control/A groups did not receive vaccine antigen. The Commercial vaccine group received an anti-GnRH commercial vaccine used in cattle. The humoral immune response was evaluated by indirect ELISA and the immunocontraceptive potential was assessed through the quantification of serum testosterone and the histological analysis of animal’s gonads. The groups that received LTB/GnRH antigens (25 μg per vaccine) presented total IgG levels significantly higher when compared to the control groups, showing that both antigens are immunogenic. The LTB/GnRH-P and LTB/GnRH-P/A groups showed higher absorbances at days 28 (1,11 ± 0,09) and 42 (1,44 ± 0,04), respectively. Furthermore, the LTB/GnRH-P/A group presented testosterone levels significantly lower to the control from day 28 (192 ng/dl ± 254,55). The LTB/GnRH-E and LTB/GnRH-E/A groups also obtained the highest antibody levels at days 28 (0,33 ± 0,005) and 42 (1,44 ± 0,08), respectively. The LTB/GnRH-E/A group presented testosterone levels significantly lower to the control from day 28 (<12 ng/dl ± 0). The results from histology showed that both antigens cause changes in spermatogenesis, wherein the antigen expressed in E. coli was associated with major changes in gonads. The present study showed that chimeras produced through the merge of LTB with a single GnRH molecule is capable to induce the generation of humoral immune response, block of the endocrine functions related to reproduction, and tissue alterations in the gonads of male mice.

Introdução e Objetivos. Fosfatases são enzimas promissoras para aplicação na degradação de organofosforados. Por exemplo, a enzima paraoxonase 1 (PON1), associada à lipoproteína de alta densidade (HDL), hidrolisa lactonas, ésteres aromáticos e compostos organofosforados (OP) neurotóxicos. \"Módulos de ligação a carboidrato\" (CBM) têm diversas aplicações biotecnológicas. Nosso objetivo é a obtenção de proteínas quiméricas contendo fosfatases ligadas a um módulo de ligação de celulose, o que possibilitaria a imobilização dessas enzimas em suportes de celulose. Resultados. Como prova de conceito, uma proteína quimérica contendo uma \"fosfatase ácida\" (appA) de E.coli e CBM familia 2 (CBM2) de uma celulase de Xanthomonas axonopodis pv citri foi montada e produzida em E. coli como uma proteína recombinante solúvel. appA-CBM2 purificada demonstrou ser totalmente funcional exibindo atividade de ligação à celulose microcristalina (Avicel PH101) e atividade de fosfatase sobre p-nitrofenil fosfato. A ligação à Avicel evidenciou um comportamento de saturação descrito por uma \"constante de ligação\" (Kb) de 26 mg e um \"máximo de ligação\" (Bmax) de 4,45 U/&#181;g. Além disso, a ligação de appA-CBM2 em Avicel foi maior em pH 2,5 e diminuiu acima de pH 6,5, como observado anteriormente para CBM2. Finalmente, o efeito de concentração de p-nitrofenil fosfato na atividade catalítica de appA-CBM2 e appA foi idêntico, exibindo um Km de 2,8 mM. Portanto, esses dados mostram que o conceito de uma proteína que combina as propriedades da fosfatase e do domínio de ligação à celulose é possível e funcional. De forma similar, os segmentos de DNA que codificam para o CBM2 e para a PON1 de Homo sapiens, foram fusionados resultando em um segmento que codifica para uma proteína quimérica (PON1-CBM2). PON1 nativa e PON1-CBM2 foram produzidas na forma solúvel e ativa em E.coli cepa Arctic. Embora não tenha sido viável sua purificação, estas enzimas foram caracterizadas. PON1-CBM2 liga-se em Avicel PH101 com um comportamento de saturação, descrito por uma constante de ligação (Kb) de 27 mg, valor idêntico àquele observado para appA-CBM2, o que sugere que o domínio CBM2 é igualmente funcional nestas duas enzimas quiméricas. PON1-CBM2 também exibe atividade paraoxonásica com Km similar àquele observado para PON1 nativa (1,3 mM), sugerindo que o \"domínio\" PON1 encontra-se totalmente funcional na enzima quimérica. Conclusão. Uma estratégia para a construção e expressão heteróloga em E. coli de PON1 e das enzimas quiméricas appA-CBM2 e PON1-CBM2 foi desenvolvida. As enzimas quiméricas mostraram-se totalmente funcionais e conservaram as propriedades de seus \"domínios\" constituintes. / Introduction and Aims. Phosphatases are promising enzymes for application in the degradation of organophosphates, whereas carbohydrate binding module has significant and demonstrated biotechnological applications. The high-density lipoprotein-associated enzyme paraoxonase 1 (PON1) hydrolyzes lactones, aromatic esters, and neurotoxic organophosphorus (OP) compounds. Our aim is to obtain chimeric proteins containing a phosphatase domain linked to a carbohydrate binding module (CBM), which could be immobilized on a cellulose supports. Results. As a proof of concept, a chimeric protein combining an acid phosphatase (appA) from E.coli and a CBM family 2 (CBM2) from Xanthomonas axonopodis pv. citri was assembled and produced in E.coli as a recombinant soluble protein. Purified appA-CBM2 was fully functional, was bound to microcrystalline cellulose and exhibited phosphatase activity upon p-nitrophenyl phosphate. The binding to microcrystalline cellulose Avicel PH101 exhibited saturation with a binding constant (Kb) of 26 and a maximum binding (Bmax) of 4,45 U/&#181;g. In addition, the binding was higher at pH 2.5 and decreased above pH 6.5, as previously observed for CBM2. Finally, effect of p-nitrophenyl phosphate concentration on appA-CBM2 and native appA activities were identical, exhibiting a Km of 2.8 mM. Taken together, these data show that the conceptual design of a protein combining the properties and biotechnological advantages of phosphatases and cellulose binding domains is possible and functional. Similarly, DNA segments coding for CBM2 and for PON1 from Homo sapiens combined resulting in a segment coding for a chimeric protein (PON1-CBM2). Native PON1 and PON1-CBM2 were produced as recombinant protein in E. coli Arctic. Although purification was not accomplished, these enzymes were characterized. PON1-CBM2 binds to microcrystalline cellulose, exhibiting a saturation behavior described by a Kb of 27 mg. PON1 and PON1- CBM2 have the same Km for paraoxon (1.3 mM), indicating that the phosphatase domain was fully functional. Conclusion. An effective strategy for heterologous expression of the native PON1 and chimeric appA-CBM2 and PON1-CBM2 in E. coli was attained. The chimeric enzymes were fully functional and maintained the properties of their original domains

<p>Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.</p><p>IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (<italic>Il1r1</italic>-/-) mice given <italic>Il1r1</italic>-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of <italic>Il1r1</italic>-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given <italic>Il1r1</italic>-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.</p> / Dissertation

The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy is a topical microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal small inhibitory RNA (siRNA) application. To overcome the challenges of using siRNAs to knock down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in both the genital and rectal tracts of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity, provide durable target gene silencing for up to three weeks in vitro, and maintain effectiveness in a hydroxyethyl cellulose (HEC) gel formulation. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in cervicovaginal explants. When applied intravaginally to humanized mice, CD4-AsiCs provided durable protection against transmission of the virus. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent the sexual transmission of HIV.

No description available.