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The investigations have given rise to the following findings: 1. CsA is an effective immunosuppressant of both the induction and elicitation phases of tuberculin-like and contact delayed-type hypersensitivity (DTH) and of the induction phase of Jones-Mote hypersensitivity. 2. The effective suppression of tuberculin-like DTH responses in the guinea pig is not dependent upon cyclophosphamide-sensitive suppressor cells. 3. Topically applied CsA inhibits the elicitation of contact dermatitis in experimental animals. 4. The kinetics of percutaneous CsA absorption have been determined, as has the extent to which this mode of drug delivery obviates systemic toxicity. 5. The investigation of CsA-induced DTH enhancement indicates that the phenomenon is restricted to cellular as opposed to humoral responses, develops some days after drug withdrawal and can be reversed by the administration of putative suppressor cells from immunised but untreated animals. 6. CsA is able to effectively inhibit T cell dependent hyper-eosinophilia. It would appear that the effects of CsA on DTH responses and on T dependent eosinophilia occur primarily by the inhibition of T helper cell function. Enhancement phenomena seem to arise from drug impaired development of antigen-specific suppressor cells which, following drug withdrawal, fail to develop, in contrast to the maturation of T effector cells. Although the phenomenon of enhancement may limit the potential of CsA in the control of diseases in which DTH responses are a component, topical application is effective and may well be suitable for use over a prolonged period without systemic toxicity.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to a-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and realtime quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/ macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

In renal transplantation, immunosuppressants are prescribed to patients to prevent graft loss. Although the extent of adherence required for such treatment to prevent graft loss has not been determined, it is thought to be high. Despite this, research suggests adherence rates for renal transplant recipients to be only between 50% and 95%. Considering the impact of graft loss on the renal patient, the national healthcare budget and on the limited resource of donor organs, it is important to identify and understand factors that contribute to nonadherence, and thereafter to address those that are most influential. This thesis seeks to understand adherence of renal transplant recipients, and to identify the cognitive and behavioural factors influencing this behaviour. To achieve this, three main activities were performed, a literature review, an interview study and a questionnaire study, the methods and findings of which are presented following an overview of two social cognition models, used in two of the activities, and renal disease. The first activity, a comprehensive literature review, identified 55 research articles that explored factors influencing adherence of renal transplant recipients to immunosuppressant drug therapy. It included original research studies published between 1980 and 2009, and was updated in 2011. The findings were categorised into the five dimensional framework suggested by the World Health Organisation: patient- related factors; socio-economic factors; condition-related factors; therapy-related factors; and healthcare team and system-related factors. Secondly, a semi-structured interview study with 27 renal transplant recipients was conducted. The study explored their attitude towards and behaviours related to taking immunosuppressants. The interview schedule was informed by the health belief model, and framework analysis of the data identified five key themes. These were: satisfaction with renal replacement therapy; the importance of taking immunosuppressants; perception of side effects and risks; responding to side effects and risks; and 'compliance is routine'. Finally, a questionnaire was developed using the theory of planned behaviour and the findings of the previous two activities. Its purpose was to determine the predictors of renal transplant recipients' self-reported adherence and to explain their adherence. A logistic regression model of 528 survey responses suggested respondents were more likely to be highly adherent if they, in descending order of influence: had well- established habits; were unemployed; had a better prospective memory; were a shorter time post-transplantation; had higher levels of anticipated affect; and lower levels of perceived behavioural control. The thesis concludes with discussing the findings of the studies, their strengths and limitations, and their implications for practice and future research. The findings of this thesis suggest unintentional nonadherence to prevail and encourage the development of interventions which promote habit formation and maintenance.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-20T12:35:56Z No. of bitstreams: 1 arquivototal.pdf: 2158020 bytes, checksum: 21163425bf778a5e7dcea450996ed689 (MD5) / Made available in DSpace on 2017-02-20T12:35:56Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2158020 bytes, checksum: 21163425bf778a5e7dcea450996ed689 (MD5) Previous issue date: 2016-11-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Pseudomonas aeruginosa is an opportunistic pathogen ubiquitous often associated with individuals with pathologies that cause immunodeficiency, such as cystic fibrosis. This pathogen synthetizes a typical greenish-blue pigment, the pyocyanin, which represents its major virulence factor, inducing the generation of reactive oxygen species by host cells, in addition to inflammation in several tissues and neutrophil apoptosis. However, the protocols used to study these effects are distinct from each other and the pyocyanin mechanism of action in acute inflammation processes is unknown. The aim of this work was to understand the interactions between pyocyanin and acute inflammatory processes, evaluating in vitro effect of pyocyanin on cell viability and nitric oxide, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α production by lipopolysaccharide-activated mice peritoneal macrophages. In addition, the in vivo effect of pyocyanin on zymosan-induced peritonitis in mice was evaluated. Our results show that pyocyanin 50 or 100 μM induced cell death about 90 and 95 % (p < 0.0001), respectively, while pyocyanin 1, 5, or 10 μM was not able to affect cell viability. The pigment at 5 or 10 μM reduced nitric oxide production about 26 % (p < 0.05) and 51 % (p < 0.0001), respectively, in addition to reducing IL-1β (38 %, p < 0.001) and TNF-α (48 %, p < 0.001) levels in macrophages treated with pyocyanin 5 μM. In the in vivo model, pyocyanin 5 mg/kg has not affect leukocyte migration to the inflammation site. The pyocyanin-induced reduction of nitric oxide, IL-1β, and TNF-α levels may be a pathogen-friendly escape mechanism, reducing the host's immune response at the concentrations evaluated in this work. This effect seems to be independent of interference in cell migration. / A Pseudomonas aeruginosa é um bacilo ubíquo oportunista frequentemente associado à indivíduos portadores de patologias que causam imunodeficiência, como a fibrose cística. Essa bactéria produz um pigmento típico, a piocianina, que representa o seu fator de virulência majoritário, induzindo a geração de espécies reativas de oxigênio por células do hospedeiro, além de induzir a inflamação em vários tecidos e a apoptose em neutrófilos. No entanto, os protocolos utilizados para a observação desses efeitos são distintos entre si e não se conhece o mecanismo de ação da piocianina em processos de inflamação aguda. O objetivo desse trabalho foi compreender as interações entre a piocianina e os processos inflamatórios agudos, avaliando in vitro o efeito da piocianina na viabilidade celular e na produção de óxido nítrico, interleucina (IL)-1β e fator de necrose tumoral (TNF)-α por macrófagos peritoneais de camundongo ativados por lipopolissacarídeo. Em adição, foi avaliado in vivo o efeito da piocianina no modelo de peritonite induzida por zimosan em camundongo. A piocianina nas concentrações de 50 ou 100 μM induziu a morte celular em 90 e 95% (p < 0,0001), respectivamente, enquanto a piocianina nas concentrações de 1, 5 ou 10 μM não foi capaz de afetar a viabilidade celular. O pigmento nas concentrações de 5 ou 10 μM reduziu a produção do óxido nítrico em 26 % (p < 0.05) e 51 % (p < 0.0001), respectivamente, além de diminuir os níveis da IL-1β (38 %, p < 0,001) e do TNF-α (48 %, p < 0,001) em macrófagos tratados com a piocianina na concentração de 5 μM. No modelo in vivo, a piocianina na dose de 5 mg/kg não interferiu na migração dos leucócitos para o sítio da inflamação. A redução nos níveis do óxido nítrico, da IL-1β e do TNF-α induzida pela piocianina pode ser um mecanismo de fuga favorável ao patógeno, capaz de reduzir a resposta inflamatória do hospedeiro nas concentrações avaliadas nesse trabalho, e esse efeito parece ser independente da interferência na migração celular.

En transplantation d'organes solides ou en greffe de moelle osseuse, la ciclosporine et letacrolimus ont prouvé leur efficacité. Ils sont cependant de maniement délicat du fait d'unintervalle thérapeutique étroit et d'une grande variabilité pharmacocinétique inter et intraindividuelle.Un suivi thérapeutique et une adaptation des posologies de ces médicaments sontnécessaires pour diminuer le risque de rejet et limiter leur toxicité.Un modèle PBPK est construit à partir de considérations anatomiques, physiologiques etbiochimiques. Il permet d'apporter des informations sur les cinétiques tissulaires et sur lesrépercussions des altérations physiologiques ou pathologiques.Les modalités optimales d'administration de la ciclosporine en greffe de moelle osseusepédiatrique, ainsi que les zones thérapeutiques à atteindre, font l'objet de débats. Un modèlePBPK-PD pour la ciclosporine construit à partir de données chez le rat puis extrapolé etvalidé chez l'homme a permis d'estimer l'exposition à la ciclosporine dans les organes ciblesde la GVH, de comparer les modalités d'administration en perfusion intraveineuse, et dedéfinir des concentrations cibles en fonction des indications. L'adaptation posologique du tacrolimus en transplantation hépatique par la méthodeBayésienne reste relativement imprécise dans la période initiale après la greffe, parce que lesfacteurs de variabilité sont imparfaitement connus. Un modèle PBPK a été construit et évaluéafin de rechercher les covariables pertinentes par une approche bottom-up : la fonctionhépatique, l'hématocrite, le génotype du cytochrome P450 3A5 du donneur, la fraction libre etcertaines comédications ont été retrouvées. / In solid organ or bone marrow transplantation, cyclosporine and tacrolimus have proven theireffectiveness. However, their handling remains difficult because of a narrow therapeuticwindow and high inter- and intra-individual pharmacokinetic variabilities. Therapeutic drugmonitoring and dose adjustments of these drugs are necessary to reduce the risk of rejectionand minimize their toxicity.A PBPK model is built from anatomical, physiological and biochemical data. It can provideinformation on the kinetics in tissues and on the effects of physiological or pathologicalalterations.How to best administer cyclosporine in pediatric bone marrow transplantation, as well astherapeutic ranges to achieve, are discussed. A PBPK-PD model for cyclosporin built fromdata in the rat and then extrapolated and validated in humans was used to estimate exposure tocyclosporine in the target organs of GVHD, to compare schedules of administration byintravenous infusion, and to define target blood concentration based on therapeuticindications.Dose adjustment of tacrolimus in liver transplant patients by the Bayesian method is relativelyinaccurate in the initial period after transplantation because factors of variability areincompletly understood. A PBPK model was constructed and evaluated in order to findrelevant covariates by a bottom-up approach. Liver function, hematocrit, cytochrome P4503A5 genotype of the donor, the unbound fraction and some comedications were found. Newdosing regimen recommendations have been developed from this model.

To sustain the health and viability of renal transplants, adherence to immunosuppressant therapy (IST) medications is critical. Studies continue to identify decreased adherence rates as time from transplant increases (Chisholm-Burns, Kwong, Mulloy & Spivey, 2008; Chisholm, Lance, Mulloy, 2005; Chisholm, Mulloy, & DiPiro, 2005; Nivens & Thomas, 2009). While previous research has explored the effect of variables known to influence IST adherence in adult renal transplant recipients, limited studies have explored these variables in a population of renal transplant recipients with longer time posttransplant intervals. The purpose of this study was to examine demographic variables, time posttransplant, immunosuppressive agents, health beliefs, social support, and symptom experience and test their relationship to adherence in a population of long-term renal transplant recipients. A cross-sectional correlational design was used to collect data from a convenience sample of 98 adult renal transplant recipients who were three or more years from transplant. Participants completed five instruments: 1) demographic survey, 2) the Beliefs About Medicines Questionnaire (BMQ), 3) the Medical Outcomes Study (MOS) Modified Social Support Survey (MSSS), 4) the Basel Assessment of Adherence with Immunosuppressive Medication Scales (BAASIS), and 5) the Modified Transplant Symptom Occurrence and Symptom Distress Scale- 59R (MTSOSD-59R). A composite adherence score (CAS) consisting of a self-report measure of adherence (BAASIS), nontherapeutic serum drug assay, and collateral report of adherence as provided by two transplant clinic professionals was used to determine final adherence group classification (adherent/nonadherent). Analysis of the relationship between all independent variables and adherence was conducted using Spearman’s rho correlation coefficient. Mean scores for medication complexity, health beliefs, social support, and symptom experience were 4 compared between age, gender, and time posttransplant groups using independent-samples t tests. A logistic regression prediction of probability was conducted to determine which of the variables that demonstrated a significant relationship to adherence were most predictive of adherence. Of the total sample population (N = 98), 39.8% (n = 39) were classified as adherent and 60.2% (n = 59) were nonadherent. Results demonstrated no significant relationship between age (continuous variable), time posttransplant, immunosuppressant medications (measured by a medication complexity index), health beliefs, symptom experience, and adherence. Weak, but significant relationships between age groups (r = -.213, p=.035), tangible social support (r = .215, p =.017), emotional informational social support (r = .274, p = .003), positive social interaction support (r = .199, p = .025), total overall social support (r = .274, p =.003) and composite adherence group classification were found. Older participants ( > 55 yrs) were significantly less adherent than younger ( < 54 yrs) participants. Mean scores for emotional / informational (EMI), positive social interaction (POS), and total social support (MSSS) were significantly lower in nonadherent participants. Regression results indicated the overall model of two predictors (age grouped [ < 54 yrs; > 55 yrs] and EMI social support subscale) was statistically reliable in distinguishing between adherent and nonadherent participants (-2 Log Likelihood 116.244; Goodness-of-Fit x 2 (2) = 13.664, p = .001), correctly classifying 69.1% of the cases. Findings from this study contribute to the body of research exploring predictors of immunosuppressant adherence in long-term renal transplant recipients. Data suggest both younger age (< 55) and categories of social support predict adherence in long-term renal transplant recipients. Healthcare providers caring for renal transplant recipients long-term 5 should consider annually assessing older participants for adherence as well as for changes in social networks.

Les produits naturels (PNs) englobent une énorme diversité chimique qui a conduit à la découverte de médicaments révolutionnaires contre le cancer, contre les maladies infectieuses et contre d'autres maladies. La majorité des médicaments actuellement approuvés sont des dérivés de PNs, où nombre d’entre eux engagent des cibles considérées comme non thérapeutiques. Malgré ces avantages, les PNs posent des problèmes au niveau de l’isolement, de la déréplication, du réapprovisionnement et de la traçabilité chimique. Compte tenu du besoin urgent de découvrir de nouvelles molécules bioactives contre de nouvelles cibles pour tous les types de maladies, des stratégies innovantes sont nécessaires pour revigorer la découverte de médicaments à partir des PNs. Nous avons développé une plateforme utilisant Saccharomyces cerevisiae pour la production hétérologue de molécules similaire aux PNs, appelée « produits naturels synthétiques » (PNSs). Nous avons synthétisé une vaste bibliothèque de gènes impliqués dans la biosynthèse de PNs (GBSs) provenant de plantes, de champignons et de bactéries, pour lesquels leur contenu en GC et leurs codons ont été optimisés pour l’expression dans S. cerevisiae. Ces gènes sont assemblés en chromosomes artificiels de levure pour générer de vastes bibliothèques combinatoires de BSG pour la production de molécules similaires aux PNs. Les bibliothèques de PNSs peuvent être directement criblées contre des microorganismes ou des cibles spécifiques dans des essais à haut débit. J'ai effectué le criblage de bibliothèques de PNSs contre une variété de cibles bactériennes et humaines. L'un de ces criblages a conduit à la découverte de PNSs ayant une activité antimicrobienne contre un groupe de pathogènes cliniquement pertinents. Récemment, certaines équipes scientifiques, dont la nôtre, ont découvert que l'hyperactivation de la protéase mitochondriale humaine CLPP par les composés anticancéreux ONC201 et ONC212, qui sont présentement en phase préclinique, provoque la mort cellulaire par protéolyse mitochondriale incontrôlée. Cependant, j'ai trouvé que ONC201/212 activent également la version bactérienne de ClpP et ils pourraient donc perturber le microbiome. J'ai donc développé des essais génétiques de substitution dans la levure pour les protéases ClpP afin de cribler pour des activateurs plus spécifiques. Ensuite, j'ai adapté mon approche dans la levure pour le criblage d’inhibiteurs de la protéase principale (Mpro) et de l'endoribonucléase (NendoU) de SRAS-CoV-2, afin de répondre au besoin pour des thérapies antivirales efficaces afin de traiter les personnes atteintes de la forme grave de la COVID-19. Enfin, une autre variante de mon approche dans la levure a également été développée pour le criblage de stabilisateurs de l'interaction entre FKBP12 et calcineurine dans le but d'identifier de nouveaux immunosuppresseurs qui présentent moins d'effets secondaires. Le criblage de ces différents essais m’a permis d’identifier des candidats potentiels pour chaque cible. Bien que les tests faits dans la levure soient utilisés dans le contexte de criblages traditionnels, l’utilisation de la plateforme PNS permet d’explorer un espace chimique inaccessible auparavant afin de favoriser la découverte de médicaments, le tout de manières économique, modulable et durable. / Natural products (NPs) encompass enormous chemical diversity, leading to revolutionary medicines in cancer, infectious disease, and other indications. The majority of currently approved drugs are derived from NPs, with many of them engage targets otherwise viewed as undruggable. Despite these advantages, NPs pose problems in isolation, dereplication, resupply and chemical tractability. Given the pressing need to discover bioactive chemical matter against new targets in all disease areas, innovative strategies are required to reinvigorate NP-based drug discovery. We have developed a Saccharomyces cerevisiae platform for heterologous production of NP-like chemical matter, termed Synthetic Natural Products (SynNPs). We synthesized an extensive library of codon- and GC-content optimized NP biosynthetic genes (BSGs) from plants, fungi and bacteria. These genes are then assembled into programmable yeast artificial chromosomes (YAC) to generate vast combinatorial BSG libraries that produce NP-like molecules. SynNP libraries can be directly screened in high-throughput in either cell- or target-based assays. I constructed and screened SynNP libraries in yeast-based surrogate genetic assays against a variety of bacterial and human targets. One of these screens led to the discovery of SynNPs with antimicrobial activity against a panel of clinically relevant pathogens. Recently, we and others discovered that hyperactivation of the human mitochondrial caseinolytic protease proteolytic subunit (CLPP) by the preclinical anti-cancer compounds ONC201 and ONC212 causes cell death by rampant mitochondrial proteolysis. However, I found that ONC201/212 also activates bacterial ClpP and could therefore disrupt the microbiome. I thus developed yeast-based surrogate genetic assays for ClpP proteases to screen for more specific activators. Then, I adapted my yeast-based approach to screen for inhibitors of SARS-CoV-2 main protease (Mpro) and endoribonuclease (NendoU) to address the need for efficacious antiviral therapies to mitigate the COVID-19 pandemic. Finally, I developed another variant of my yeast-based approach to screen for stabilizers of the interaction between FKBP12 and calcineurin to identify novel candidate immunosuppressants. Screens with these various assay formats allowed me to identify candidate hits for each target. In summary, the SynNP platform allows the exploration of new-to-nature NP-like chemical space for drug discovery in a cost-effective, scalable and sustainable manner, and yeast-based surrogate genetic assays can be used to screen both existing chemical libraries and SynNP libraries.