O sistema imune exerce uma contribuição fundamental na proteção contra a infecção pelo plasmódio, o agente causador da malária. Entre os mecanismos envolvidos no controle dos parasitas do sangue destacam-se aqueles mediados por anticorpos. Entretanto, outros mecanismos do sistema imune também parecem exercer papéis fundamentais, e ao mesmo tempo foram pouco estudados. O presente trabalho teve como objetivo descrever alguns dos mecanismos efetores importantes na resposta imune à malária experimental. O projeto subdivide-se em duas partes: a) verificar a relação entre o priming por IFN-g e a imunidade ao parasita durante a fase crônica da infecção sanguínea pelo Plasmodium; e b) descrever separadamente o papel de células dendríticas e macrófagos do baço na fagocitose de eritrócitos infectados durante a infecção em modelo experimental, e o subsequente papel destas células na resposta imune durante esse período. Os resultados obtidos neste projeto deverão contribuir para a compreensão dos mecanismos envolvidos na imunidade contra a malária. / The immune system is considered crucial for the protection against Plasmodium infection. Among the mechanisms involved in the control of blood-stage parasites, the ones mediated by antibodies are widely recognized. However, other mechanisms of immune system can also exert fundamental roles, being less studied. This work had the objective of describing some of the effector mechanisms important in the immune response to blood-stage experimental malaria. This work was subdivided in two main branches: a) to verify the relation between the IFN-g-induced priming and the protective immunity to the parasite during the chronic phase of blood-stage infection; and b) to describe separately the role of dendritic cells and splenic macrophages as phagocytic cells during acute Plasmodium chabaudi infection, and the subsequent role of these cells in the immune responses during this period. The results obtained in this project may contribute for the comprehension of the mechanisms involved in the immunity against malaria.

Schistosomiasis is a water-borne parasitic disease of great public health importance mainly in sub-Saharan African countries. The majority of current control programmes use the antihelminthic drug praziquantel to reduce disease burden in endemic areas. Praziquantel treatment has been reported to accelerate the development of protective immunity against re-infection that otherwise takes years to develop. To date, there is no licensed vaccine for schistosomiasis in humans but an attenuated schistosome parasite vaccine has been tested in animal models. Employing systematic review and meta-analysis approaches, my PhD research has four main objectives relating to attenuated schistosome vaccine and praziquantel treatment: 1) to identify predictors that determine protection levels after treatment with attenuated Schistosoma mansoni vaccines in the mouse model, 2) to quantify the influence of host and schistosome parasite species on attenuated parasite vaccine efficacy, 3) to explore the direction of change (increase/decrease) in schistosome parasite-specific antibody isotypes after praziquantel treatment in humans, 4) to identify predictors of praziquantel efficacy in humans. My analyses revealed three factors that have an influence on the protection levels provided by attenuated schistosome parasite vaccines: increasing numbers of immunizing parasites had a positive effect on the levels of protection whereas increasing the radiation dose and the time to challenge infection had negative effects. Analyses showed that the attenuated schistosome vaccine has the potential to achieve protection levels as high as 79% after a single dose in mice. Alongside this, baboon studies consistently reported protective effects of attenuated schistosome vaccines against re-infection. These results show there is a high potential for an attenuated schistosome parasite vaccine to be effective in humans. A meta-analysis of the influence of praziquantel treatment on the direction of change in schistosome-specific antibody isotypes was conducted. The analysis revealed considerable variability in the antibodies’ direction of change among populations. The results also demonstrated an increase of anti-worm IgA and IgE in the majority of studies. These antibodies have been reported to have a protective effect against re-infection. The combination of age and infection intensity, and the number of days after treatment were identified as influential predictors for some antibody isotypes, but there was no single predictor that consistently affected all antibody isotypes in the same way. Praziquantel efficacy levels in humans were investigated and the analyses revealed that cure rates for schistosomiasis increase with praziquantel dose, and were affected by the identity of the schistosome parasite species (S. mansoni vs. S. haematobium) and the age of the participants (children: 0-19 years old vs. adults: ≥ 20 years old). There has been no clear efficacy level reduction over the treatment years (1979-2013) suggesting that praziquantel is still effective in the treatment of schistosomiasis despite concerns about possible resistance. The development of a schistosome vaccine will benefit from a closer investigation into the mechanisms through which protection is acquired in attenuated schistosome parasite vaccine studies showing high potential efficacy in animal models. Nevertheless, it will take time to develop a schistosome vaccine for human use. The uptake of the vaccine will be made even more challenging by the lack of adequate infrastructure in schistosomiasis endemic areas. In the meantime, close monitoring of praziquantel efficacy levels is necessary to confirm the effectiveness of schistosomiasis control in endemic areas.

Sterile triploid Atlantic salmon represent a solution to the issues of pre-harvest sexual maturation and mature escapees from open aquaculture systems. Although the initial problems of reduced performance and increased deformities in triploids have been thoroughly researched, there is a continued lack of information on their susceptibility and response to disease and routine on-farm treatments compared to diploids. Thus, the main aim of this thesis was to enhance the current understanding of triploid health and immunity through experimental disease challenges and treatments, and aid in determining their robustness and, therefore, suitability for aquaculture. A commercial furunculosis vaccine equally protected diploids and triploids against challenge with Aeromonas salmonicida, and adhesion scores were similar between ploidy (Chapter 2). Interestingly, triploids had lower white blood cell counts but increased cellular activity, e.g. respiratory burst, compared to diploids. Following experimental cohabitation infection with Neoparamoeba perurans, causative agent of Amoebic Gill Disease (AGD), ploidy did not affect the manifestation or severity of AGD-associated gill pathology, or the serum innate immune response (Chapter 3). Hydrogen peroxide, used to treat against parasitic diseases, elicited similar primary and secondary stress responses in both ploidy, but led to differences in the expression of stress (cat, gpx1, gr, hsp70, sod1, sod2) and immune (saa5, crp/sap1a, crp/sap1b, il1β) genes (Chapter 4). Finally, vaccination with different vaccine treatments (4 commercial vaccines, 6 different vaccine combinations and a sham-vaccinated control) showed no ploidy differences in adhesion score or antibody response, although vertebral deformities remained higher in triploids (Chapter 5). Increasing severity of vaccine treatments negatively affected weight, length and thermal growth coefficient in both ploidy. Triploids were heavier than diploids at smolt (+ 14 %) and post smolt (+ 32 %). Overall, this research shows that triploid Atlantic salmon respond as well as diploids to disease and treatment challenges, and supports their application into full-scale commercial aquaculture.

Ants and other social Hymenoptera (social bees and wasps) have a remarkable mating strategy. Social Hymenoptera live in societies where reproduction is monopolized by a fertile caste consisting of males and queens. On the other hand, the logistical tasks of the colony are carried out by a sterile female caste known as workers. Reproductive individuals mate during a single bout early in their life and will never engage in additional reproductive events later on. Males die soon after mating while queens store millions of sperm cells in a specialized organ, the spermatheca. Queens will use this sperm stock to fertilize eggs during the rest of their life that can last up to several decades. With a record of 28.5 years in the black garden ant, ant queens have the longest lifespan recorded to date among the social Hymenoptera. In my thesis, I addressed three aspects of ant queen reproduction. First, I tested the effect of mating on the expression of several genes involved namely in fecundity, longevity and immunity. I found that mating induces an up-regulation of the yolk precursor vitellogenin and of the antimicrobial peptide defensin. Second, I measured the intensity of different immune responses in male and queen genital organs in order to determine which immune pathways are activated to protect sperm. Antimicrobial peptide genes are expressed in the genital tract of both sexes and the queen spermatheca is capable of strongly inhibiting bacterial growth. The immune melanization response is, however, overall inactive in the organs tested probably because its unspecific mode of action and cytotoxic by-products are likely to damage sperm cells. Immunity thus seems to be closely regulated in organs that are in contact with sperm. Third, I determined if activation of the queen immune system had an impact on the survival of sperm stored in the spermatheca. There is no detectable effect in young newly mated queens whereas, in one year old queens, immune activation induces a significant reduction in sperm viability. Life stage thus seems to influence queen ability to preserve sperm viability in the event of an immune challenge. In addition, one year old queens have higher sperm viability than newly mated queens suggesting queens are able to displace dead sperm cells from their spermatheca. Finally, I relied on the well-established sequence of behaviors inherent to the early life of ant queens to try to uncover the largely unknown roles of inotocin, the insect ortholog of the vertebrate hormones oxytocin and vasopressin, in regulating insect behavior. I measured gene expression of the inotocin receptor and found that it is highly expressed during social and reproductive behaviors, which is consistent with previous results in vertebrates. Inotocin might thus also be involved in modulating these behaviors in insects, but further studies are needed to be able to fully understand this complex signaling system. Overall, I show that reproduction and immunity are closely linked in ant queens and that the latter provide promising models for investigating the roles of hormones in insects. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

"Vulvovaginal candidiasis (VVC) is a mucosal infection caused by Candida species and represents one of the most common clinical problems in women of reproductive age (68,71). Annually in the United States there are approximately 13 million cases of VVC, resulting in 10 million gynecologic office visits per year (38). It is estimated that 75% of women will experience at least one episode in their lifetime, with a projected 50% of all women experiencing multiple episodes (23). Candida albicans is a dimorphic commensal organism of the urogenital and gastrointestinal tracts and has been identified as the main pathogenic agent in VVC, accounting for approximately 85-90% of patients with positive cultures (52). Despite extensive research, the invasive mechanism of vaginal yeast infections is not well understood. Traditionally it has been assumed that changes in the host vaginal environment promote the dimorphic transition from blastospore to hyphae, resulting in a shift from asymptomatic colonization to symptomatic vaginitis (28). In contrast to the normal, systemic immune response, which confers an aseptic environment for tissue and organs, immune responses at the mucosal level are designed to prevent tissue invasion and local disease while maintaining an indigenous flora that could be both beneficial and pathogenic (28). Since fungi are eukaryotic, the vital cellular mechanisms that are usually targeted by modern pharmacologic agents, such as DNA replication and protein translation, are either conserved or have a strong homology to their human orthologs. Obtaining a better understanding of natural fungal suppression mechanisms and molecules at the mucosal level may pave the way for the development of more efficacious drugs or preventative regiments. The mechanism by which the human immune system is able to resist fungal invasion at the vaginal mucosa is unknown. Our research was aimed at finding any host factors that might play a role in the suppression of or prevention of a fungal infection at the vaginal mucosa. In order to screen candidate molecules that might be important in this type of vaginal defense, we chose a pathogenic C. albicans strain, SC5314, to test fungal cell viability upon introduction of the candidate molecules. We have identified a host factor that exhibits strong fungicidal activity when organically extracted from both human and bovine serum albumins. Characterization of this factor through organic extractions and acetone separations reveal that this molecule is a non-polar lipid. Serum samples that have been thoroughly stripped of fatty acids and other lipophilic molecules show no apparent fungicidal activity in cell viability assays. Since the factor is extractable from both human and bovine serum albumins, it may be conserved among mammals. Identification and characterization of this molecule may play a pivotal role in understanding host-Candida interactions at the mucosal membrane interface. Due to its human origin, the use of this factor as an antifungal would be extremely advantageous in regards to FDA (Food and Drug Administration) guidelines and ADMET (Adsorption, Distribution, Metabolism, Excretion, Toxicology) properties. "

Foram utilizadas 750 poedeiras comerciais de linhagens brancas com 23 semanas ao início do período experimental, distribuídas num delineamento em blocos completos aleatorizados sendo estes caracterizados pela oncatenação das linhagens com coluna de gaiolas; os tratamentos foram cinco densidades ou taxas de lotação na gaiola (321,43; 375; 450; 562,50 e 750 cm2/ave) com seis repetições, totalizando 150 parcelas experimentais. As dietas experimentais foram à base de milho e farelo de soja, formuladas para suprir as exigências nutricionais das linhagens em todas as fases. O desempenho produtivo e econômico foram avaliados através do peso dos ovos (g), percentagem de postura (%), massa de ovos (g/ave/dia), consumo de ração (g/ave/dia), conversão alimentar por dúzia (kg/dz) e por quilo de ovo (kg/kg). A qualidade de ovos foi avaliada através da gravidade específica (GE), unidades Haugh (UH), percentagens de gema, albúmen e casca, espessura da casca (EC), resistência à quebra (RQ), coloração da gema e índice gema (IG). O bem-estar das aves foi avaliado através de indicadores clínicos, imunológicos e fisiológicos. Para efeito da avaliação dos resultados, foram estabelecidos seis blocos com repetições internas. Os dados foram analisados com auxílio do SAS, sob modelo misto, considerando os efeitos da densidade, período e a interação entre estes, como fixos, além dos efeitos aleatórios de bloco e resíduo. Por se tratar de medidas repetidas longitudinalmente, buscou-se a melhor estrutura de covariância para cada variável. Quando adequado foi aplicado o teste de tukey-kramer para a comparação de médias (p<0,05). O menor consumo de ração, CA/dz de ovos e melhor desempenho econômico, foram obtidos pela densidade 321,4 cm2/ave. Altas densidades de alojamento não influenciaram a qualidade interna de ovos, a % casca e RQ, mas essas variáveis tiveram efeitos dos períodos, com os melhores valores nos primeiros períodos. Altas densidades tiveram efeitos negativos sobre a GE e EC, principalmente no pico de postura das aves. Os ovos avaliados encontravam-se dentro dos valores desejados, mesmo nas maiores densidades, podendo ser classificados como de excelente qualidade. A densidade de alojamento não interferiru (p>0,05) na avaliação clínica das aves, mas o maior espaço proporcionado nas gaiolas do sistema covencional conferiu menor freqüência de lesões nas aves, indicando promoção e melhora ao seu bem-estar geral. Dados de freqüência cardíaca e temperatura da cloaca estavam dentro dos padrões de normalidade para a espécie. Não foram observadas diferenças (p>0,05) das densidades na resposta imunológica das aves, com os títulos dos anticorpos vacinais contra as doenças de Gumboro e Newcastle acima dos níveis do ponto de corte (cutoff). Os tratamentos não modificaram o perfil sanguíneo das aves e não foi possível caracterizar um padrão de corticosterona no plasma e de excreção de metabólitos fecais de glicocorticoides. A técnica de enzimoimunoensaio, empregada utilizando anticorpo primário contra corticosterona e anticorpo secundário anticoelho de cabra, foi capaz de detectar as variações nas concentrações de corticosterona nesses tecidos, contudo, o entendimento do significado desses achados ainda necessita de novas investigações. / 750 commercial laying hens of white strains aged 23 weeks at the beginning of the experimental period were used, distributed in a randomized complete block design, these being characterized by the concatenation of the strains with column of cages; the treatments were five densities or stocking rates in the cage (321,43, 375, 450, 562.50 and 750 cm2/bird) with six replications, amounting to 150 experimental plots. The experimental diets were on the basis of corn and soybean meal, formulated to meet the nutritional requirements of the strains in all the phases. Both the productive and economic performances were evaluated through the weight of the eggs (g), percentage of laying (%), egg mass (g/bird/day), feed intake (g/bird/day), feed conversion per dozen (kg/dozen) and per kg of egg (kg/kg). Egg quality was determined through specific gravity (SG), Haugh units (HU), percentages of yolk, albumen and shell, shell thickness (ST), resistance to breaking (BR), yolk coloration and yolk index (YI). The welfare of the birds was evaluated through clinical, immunological and physiological indicators. For the purpose of evaluation of the results, six blocks with internal replications were established. The data were analyzed with the aid of SAS, under a mixed model, considering the effects of density, period and the interaction between them, as fixed, in addition to the random effects of block and residue. For se tratar de measures longitudinally repeated, the best covariance structure for each variable was sought. When appropriate, the Tukey-Kramer test was used for comparison of means (p<0.05). The lowest feed intake, FI/dozen of eggs and best economic performance, were obtained at the density of 321.4 cm2/bird. High housing densities did not influence the internal quality of the eggs, % shell and BR, but these variables had effects from the periods, with the best values in the early periods. High densities had negative effects on SG and ST, mainly at the laying peak of the birds. The eggs checked lie within the desired values, even at the highest densities and can be classified as of excellent quality. The housing density did not interfere (p>0.05) in the clinical evaluation of the birds, but the greater space provided in the cages of the conventional system conferred a lower frequency of lesions in the birds, indicating promotion and improvement to their general welfare. Data of heart rate and cloacal temperature were within the normal limits for the species. No differences (p>0.05) of the densities in the immune response of the birds, with the titers of the vaccine antibodies against Gumboro and Newcastle diseases above the cutoff levels were found. The treatments did not modify the blood profile of the birds and it was not possible to characterize a standard of plasma corticosterone and excretion of fecal metabolites of glucocorticoids, but the enzyme immunoassay technique, using primary antibody against corticosterone and secondary goat anti-rabbit antibody, was capable of detecting variations in the corticosterone concentrations in these tissues, however, the understanding of the meaning of these findings still requires further investigation.

A própolis é um produto natural elaborado pelas abelhas e vendido comercialmente, muito conhecida pela sua capacidade antimicrobiana, antioxidante e anti-inflamatória. Esse trabalho teve como objetivo caracterizar a influência da própolis vermelha no desempenho, metabolismo e ocorrência de doenças em bezerros leiteiros suplementados diariamente. Foram utilizados 32 bezerros recém-nascidos colostrados e alojados individualmente com acesso a água e concentrado inicial. Os animais foram colocados em blocos de acordo com o peso ao nascer, data de nascimento e sexo, distribuídos em dois tratamentos: 1) Controle e 2) Suplementação diária de 4mL/dia de extrato alcoólico de própolis (30%) no leite integral. O consumo de alimentos e o escore de saúde e fecal foram monitorados diariamente, já as pesagens foram realizadas semanalmente. Amostras de sangue foram colhidas semanalmente, duas horas após o aleitamento da manhã, para realização de hemograma, determinação de hematócrito e das concentrações de glicose, proteína total, &beta;-hidroxibutirato, lactato, ureia, além da capacidade de ligação de ferro. O tratamento não apresentou efeito para os dados de consumo, ganho de peso e desempenho (P>0,05). No entanto, os animais suplementados com própolis vermelha apresentaram significante redução no escore fecal e no número de dias com diarreia (P<0,05). Para os parâmetros sanguíneos, apenas a contagem de eritrócitos totais sofreu efeito do tratamento, apresentando redução em sua concentração nos animais suplementados (P<0,05). O extrato de própolis vermelha brasileira tem grande potencial como alternativa natural para diminuir a incidência de diarreia em bezerros leiteiros, reduzindo a utilização de antibióticos nos sistemas de criação de bezerras. / This study aimed to characterize the influence of propolis or bee glue in the performance, metabolism and diseases of dairy calves. Propolis is a natural product collect by bees and sold commercially, well know for its antibacterial, antioxidant and anti inflammatory effects and the ability to improve the immunity system, both for humans and for animals. For ruminants, there are many positive effects that can contribute positively to animal productivity. Thirty two newborn calves were individually housed, with free access to water and concentrate, and fed 6 L/day of whole milk. The animals were blocked according to their birth weight, birth date and sex, distributed in two treatments: 1) Control and 2) Daily supplementation of 4mL / day of red propolis alcoholic extract (30%) in milk. Food consumption, fecal score and health score of bronchopneumonia were monitored daily, and the weight of the calves were measured every week. Samples of blood were collected weekly, two hours after morning milk feeding, for hemogram and hematocrit determination. Determination of blood glucose, protein, &beta;-hydroxybutyrate, lactate, urea and iron binding capacity have also been done to evaluate effects on metabolism and oxidative stress. Starter feed intake, daily weight gain and body measurements were not affected by the red propolis supplementation (P>0.05). Blood parameters were also not affected (P>0.05), exception made for the red blood count, which were lower in animals supplemented with ethanolic red propolis extract (P<0.05). Fecal score and days with diarrhea were significantly affected by the treatment (P<0.05). Fecal score and days with diarrhea were inferior in animals supplemented with red propolis extract. Propolis has a big potential as a natural alternative to improve calves health, reducing the incidence of diarrhea and as a consequence, the use of antibiotics in calf rearing systems.

The inferior turbinates are important anatomical nasal structures that provide warmth, humidification, and filtration of the inspired air to provide optimal conditioning for lung function. Besides these well-established basic functions, the turbinates are also important in immune function as the interface between the airway and the environment. Proper functioning of the inferior turbinates relies on an intact autonomic system, which maintains homeostasis within the nasal cavity. The autonomic nervous system innervates the submucosal glands and the vasculature within the inferior turbinate, resulting in control of major turbinate functions: nasal secretions, nasal patency, and amount of warmth and humidification provided. This thesis will summarize the normal and abnormal autonomic processes that contribute to the inferior turbinate as an autonomic organ.

Several successful pathogens evade host defenses resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory oral bone loss and systemic inflammation manifested as atherosclerosis. The pathogenic mechanisms contributing to P. gingivalis evasion of host immunity and chronic inflammation are not well defined. P. gingivalis evades host immunity at Toll-like receptor (TLR)-4 through expression of an atypical lipopolysaccharide (LPS) that contains lipid A species that exhibit TLR4 agonist or antagonist activity or fail to activate TLR4. By utilizing a series of P. gingivalis lipid A mutants we demonstrated that expression of antagonist lipid A structures resulted in weak induction of proinflammatory mediators. Moreover, expression of antagonist lipid A failed to activate the inflammasome, which correlated with increased bacterial survival in macrophages. Oral infection of atherosclerotic prone apolipoprotein E (ApoE) deficient mice with the antagonist lipid A strain resulted in vascular inflammation characterized by macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain expressing exclusively agonist lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11 dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE deficient mice infected with the agonist lipid A strain exhibited diminished vascular inflammation. Notably, the ability of P. gingivalis to induce local inflammatory oral bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. We next investigated the role of TLRs and lipid A on bacterial trafficking by the autophagic pathway. Originally characterized as a cell autonomous pathway for recycling damaged organelles and proteins, autophagy is now recognized to play a critical role in innate defense and release of the proinflammatory cytokine interleukin (IL)-1β. We demonstrated that P. gingivalis suppresses the autophagic pathway in macrophages for pathogen survival and intercepts autophagy-mediated IL-1β release. P. gingivalis-mediated suppression of autophagy was independent of lipid A expression but partially dependent on TLR2 signaling. Collectively, our results indicate that P. gingivalis evasion of innate immunity plays a role in chronic inflammation.

Immunity, behavior, and circadian regulation are important ways that animals maintain homeostasis. Defects in these physiologies often lead to disease or even death, yet many questions remain about how these physiologies are related. I explored the interactions between innate immunity, behavior, and circadian regulation by using Drosophila melanogaster, a convenient, genetically tractable model organism with both functionally and molecularly conserved innate immune and circadian clock systems. In the first chapter, I show that feeding, a circadian-regulated behavior, increases immunity to a sepsis-like infection. In the second, I present evidence suggesting that aging-related changes in immunity may be linked to circadian defects. Finally, I use a novel automated method to demonstrate that reduced grooming is a conserved sickness behavior in Drosophila. The feeding project ultimately showed that mutating TORC2 components could increase the host’s ability to kill and clear a bacterial infection, as well as survive the pathogenic effects of infection. Therefore we have identified a possible drug target to create host-based therapies for sepsis patients. We also have established Drosophila as a model system for studying a conserved sickness behavior: reduced grooming. This experimental paradigm will allow researchers to isolate mutants that do not show reduced grooming, and investigate whether this sickness behavior is adaptive or not.