Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-11-23T11:17:22Z No. of bitstreams: 0 / Made available in DSpace on 2017-11-23T11:17:22Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP / FAPESP:10/10935-9

Cette thèse décrit la synthèse de nanoparticules de polymères à empreintes moléculaires (MIP, de l’anglais molecularly imprinted polymer) pour la reconnaissance de protéines, par une approche de synthèse en phase solide. Les polymères à empreintes moléculaires sont des récepteurs biomimétiques synthétisés sur mesure par un processus de nanomoulage du polymère autour de la molécule unique. Ils possèdent ainsi des cavités de reconnaissance spécifiques pour leur molécule cible. La technique de l'impression moléculaire pour les petites molécules cibles est bien établie, alors que l'impression de protéines reste encore un défi en raison de la flexibilité et complexité de leur structure native et de leurs nombreux sites fonctionnels, mais aussi en raison de leur faible stabilité dans des conditions inhabituelles. Par conséquent, une approche de synthèse en phase solide a été développée ici où la protéine est immobilisée sur un support avant la synthèse de nanoparticules hydrosolubles de MIP par polymérisation radicalaire. Les MIPs obtenus ont des affinités comparables à celles des anticorps, et des réactivités croisées faibles. Ils possèdent des avantages tels qu'une stabilité meilleure, un coût plus faible et peuvent potentiellement être régénérés et réutilisés, devenant ainsi des alternatives prometteuses aux anticorps naturels. Nous avons fabriqué des MIPs contre des protéases à sérine, telles la trypsine et la kallikréine, mais aussi contre un épitope peptidique de la protéine gp41 du VIH. Des nanogels de MIP thermosensibles ont été synthétisés dans un réacteur sous la forme d’une colonne thermostatée ou une boîte de Pétri, par polymérisation radicalaire initiée par voie thermique ou photochimique. Un simple changement de la température permet de libérer les MIPs de la protéine immobilisée. Ces MIPs sont hydrosolubles en fonction de la température et ont un diamètre inférieur à 100 nm. Leur affinité pour leur cible est élevée, avec un Kd du nano ou picomolaire. Ces 'anticorps synthétiques' ont été appliqués dans des tests d'adsorption sur microbalance à cristal de quartz, mais également comme 'chaperons synthétiques'. Des études préliminaires de la protection des protéines d'une dénaturation thermique ou par un pH défavorable ont été effectuées. L'utilisation d'un iniferter pour initier la photopolymérisation vivante du MIP a permis de synthétiser des nanogels de type core-shell. En introduisant des marqueurs fluorescents dans les MIPs, les tests d’immunoessai dans des fluides biologiques ont été démontrés, ce qui indique le grand potentiel de ces MIPs dans le diagnostic clinique. En conclusion, nous avons développé une nouvelle approche de synthèse de nanoparticules de MIP hydrosoluble ayant une haute affinité pour une protéine, utilisables à la place des anticorps dans des applications dans le monde réel tel que la détection de protéines biomarqueurs dans des échantillons complexes, et potentiellement comme principe actif in vivo. / This thesis describes the synthesis, by a solid-phase synthesis approach, of nanoparticles of molecularly imprinted polymers (MIPs) for the recognition of proteins. Molecularly imprinted polymers are biomimetic receptors synthesized by a nanomolding process of the polymer around single molecules. They therefore possess specific recognition cavities for their target molecule. The technique of molecular imprinting for small target molecules is well established, while protein imprinting remains a challenge due to the flexibility and complexity of their native structure and functional sites, but also because of their low stability under unusual conditions. Therefore, a solid-phase synthesis approach has been developed where the protein is immobilized on a support before the synthesis of water-soluble MIP nanogel particles by radical polymerization. The MIPs obtained have affinities comparable to those of antibodies, and low cross-reactivities. They have advantages such as better stability, lower cost, and can potentially be regenerated and reused, thus becoming promising alternatives to real antibodies. We have synthesized MIPs against serine proteases such as trypsin, and kallikrein, but also against a peptide epitope of the HIV gp41 protein. Thermosensitive MIP nanogels were synthesized in a thermostated column-type reactor or a petri dish, by thermally or photo-initiated radical polymerization. Their thermosensitivity allows the MIPs to be released from the immobilized protein by a simple temperature change. They are water-soluble as a function of temperature and have a diameter of less than 100 nm. Their affinity for their target is strong, with a Kd in the nano or picomolar range. These 'synthetic antibodies' have been applied in binding assays with quartz crystal microbalance, but also as 'synthetic chaperones'. Preliminary studies of the protection of proteins from thermal denaturation or from denaturation by an unfavorable pH have been carried out. The use of an iniferter to initiate the living photopolymerization of MIP made it possible to synthesize nanogels of core-shell type. By introducing fluorescent markers into MIPs, immunoassay applications in biological fluids have been demonstrated, indicating the great potential of these MIPs in clinical diagnostics. In conclusion, we have developed a novel approach to the synthesis of soluble MIP nanoparticles having high affinity for a protein, usable in place of antibodies in real world applications such as the detection of biomarker proteins in complex samples, and potentially as an active principle in vivo.

Age stratified seroepidemiological studies play a crucial role in the design and assessment of vaccination strategies. An existing multiplex bead immunoassay for measles, mumps, rubella and varicella zoster virus antibodies together with a newly developed multiplex bead immunoassay for herpes simplex virus type 1 and type 2 antibodies were used to investigate the age-related seroepidemiology of these viruses in England during 2012.To develop the HSV-1 and HSV-2 antibody assay, attempts were made to produce full length of HSV-1 and HSV-2 glycoprotein G using a baculovirus vector expression system. While HSV-1 gG protein was produced, the proteins were extensively aggregated. Native glycoprotein G molecules undergo partial removal of HSV-1 signal sequence and HSV-1 short membrane anchor sequence during post translational modification. It is possible that such post translational modification is not performed when protein is processed in insect cell culture. Attempts to produce an HSV-2 glycoprotein G were not successful. It is possible that the high GC-content of HSV-2 glycoprotein G led to poor fidelity of copying the PCR amplification sequence. Commercially available truncated HSV-1 gG and HSV-2 gG were therefore used to develop a duplex microbead immunoassay for the simultaneous detection of specific HSV antibodies in human sera. The resultant assays performed with low sensitivity and specificity (HSV-1 of 89% and 66%, respectively and for HSV-2 of 79% and 85%, respectively) compared to the reference HerpeSelect ELISA.The MMRV multiplex bead immunoassay proved rapid, and required minimal sample volume to semi-quantify MMRV specific antibodies. The seroepidemiology of MMR results was compared with previous seroepidemiological studies performed in 1996 in England. The comparison showed an increase in the proportion of individuals who were positive for mumps and measles antibodies in the 2012 survey. The proportion of individuals positive for rubella was essentially unchanged. The increase in the proportion of individuals positive for mumps and measles antibodies in 2012 show the effectiveness of the change in MMR vaccination policy for England from 1996 onward. For VZV, the proportion of individuals who were positive for varicella antibodies between the 1996 and 2012 serological surveys were essentially unchanged. The comparison showed that most young children are susceptible to VZV. At this level of immunity, it can be expected that varicella will continue to produce epidemics of infection in the population, unless varicella vaccination is implemented as a part of routine childhood vaccination.

Reproductive biology is a necessary element for the management of elasmobranch fisheries. Traditionally, characterization of elasmobranch reproduction has involved lethal sampling to examine gross reproductive structures and development of embryos. However, this method is counterproductive to the conservation of shark populations. One non-lethal alternative is the measurement of serum hormones, which often vary according to reproductive events. Radioimmunoassay (RIA) has been used to measure hormone concentrations in reproductive endocrinology, but can be problematic for researchers. Alternatively, chemiluminescence immunoassays (CLIA) are routinely used for measuring circulating hormone concentrations in low-volume, non-extracted human serum samples. However these assays have not been previously examined for use with elasmobranch blood. In the first component of this study, I examined whether CLIA was a suitable alternative for detecting seasonal profiles of these hormones in the bonnethead, Sphyrna tiburo. This was accomplished by collecting serum from sexually mature male (n = 35) and female (n = 32) bonnetheads , measuring reproductive organs for maturity and reproductive stage, and measuring concentrations of testosterone (T) in males, and 17β-estradiol (E2) and progesterone (P4) in females using RIA and CLIA. CLIA was successfully validated for use with shark serum by assessing parallelism and spike recovery. CLIA-derived measurements were significantly correlated with those obtained with RIA (r = 0.809, 0.773, and 0.908 for T, E2, and P4, respectively; p

Foram testadas, com o teste não treponêmico VDRL e com o teste treponêmico imunoenzimático de competição, 23.531 amostras de soros, coletados em todas as regiões do Brasil, com o objetivo de verificar o comportamento do teste imunoenzimático treponêmico na triagem de amostras. A prevalência obtida foi de 0,63% com o VDRL e de 0,84% para o teste imunoenzimático. A análise dos dados foi feita comparando-se os resultados dos dois testes com os resultados do teste treponêmico de imunofluorescência indireta (FTA-ABS), considerado como teste de referência. No total, 1120 amostras foram submetidas ao teste FTA-ABS, incluindo todas as que foram reagentes em qualquer um dos testes de triagem e 872 amostras negativas. Amostras com resultados discordantes entre os testes foram submetidas a um teste imunoenzimático do tipo Western blot. Nas amostras por nós estudadas, o teste imunoenzimático apresentou sensibilidade de 89,95% e especificidade de 99,78%, muito superior aos 55,11% de sensibilidade e 97,43% de especificidade que encontramos para o VDRL. Os resultados dos testes detectaram positividade em amostras diferentes portanto, recomendamos utilizar a associação dos dois testes, como método de triagem, quando se trata de populações de baixa prevalência. Resultados preliminares do Western blot sugerem a participação doas proteínas de 43 kD, 17 kD e 15,5 kD na reação de ELISA treponêmico competitivo. / The VDRL, a non treponemal test, and a treponemal competitive ELISA were used to test 23,531 serum samples, collected from conscript men throughout Brazil, with the objective of assessing the performance of the competitive ELISA on the screening of serum samples. The VDRL showed a prevalence of 0.63% contrasting with a 0.84% prevalence showed by the competitive ELISA. The results obtained with the two tests were then compared to those obtained by fluorescent treponemal antibody absorption (FTA-ABS) test which is considered the gold standard method for detection of antibodies for syphilis. A total number of 1,120 samples, which included all that were reagent in at least one of the screening test plus 872 that were negative in both tests, were submitted to the FTA_ABS test. In addition, some of the samples that presented discrepant results between the two tests studied were also submitted to the Western blot test. The results of the screening tests showed an 89.95% sensitivity and a 99.78% specificity for the competitive ELISA, which are much higher than the 55.11% sensitivity and 97.43% specificity presented by the VDRL. Also, the tests detected positivity in different samples. In conclusion, we recommended the use in tandem of both tests as screening for syphilis antibodies in low prevalence populations. In addition, the results of the Western blot seemed to suggest the positivity of the ELISA becoming non reactive after treatment of the patient and that the 43 kD, 17 kD and 15 kD proteins are the main proteins involved in the ELISA competitive reaction.

This thesis discusses the technique of flow cytometry for multiplex analysis and its use in conjunction with imunochemical methods. As part of this work was carried out clinical studies dealing with secondary prevention of myocardial infarction and atherosclerosis in 186 pacientů. In this time represents myocardial infarction worldwide civilizational problem. A number of possible parameters for monitoring atherosclerosis in the world is still an unresolved issue. In the practical part of this work we performed an analysis using Luminex xMAP technology for new parameters (adiponectin, resistin, osteopontin) to predict atherosclerotic disease associated with myocardial infarcion. Also we wanted to see how these parameters are changed in patients after increasing the dose of therapeutic drugs.

Hormones influence life of all animals. Not only they affect physiological changes in organisms, but also impact their behaviour. This work focuses at two main groups of steroid hormones: glucocorticoids and androgens. Glucocortiods are activated in response to stress. Their levels can be measured using non-invasive methods, which have a range of advantages. The main advantage is the feedback-free sample collection for enzyme immunoassay. As the measurement involves metabolites of the hormones rather than the hormones themselves, prior validation of the method is, however, necessary. This work reports on a study aiming to validate non-invasive measurement on the Madagascar Ground Gecko (Paroedura Picta). The validation was based on ACTH challenge test: Synacthen Depot was injected, which should lead to increased blood level of glucocorticoids. The validation, however, was not successful. The measurement did not discover significant increase in the levels of the metabolites of glucocorticoids. In addition, the work focuses on behavioural effects of testosterone, the primary androgen. Hormonal manipulations have been carried out on several male and female specimens. The results have discovered differences in sexual behaviour between control groups. On the other hand, the hormonal manipulations had no...

To diagnose patients suffering from blood clotting disorders latex immununoassays (LIA) can be used. A time consuming manual tangential flow filtration (TFF) process suggests the implementation of an automatic TFF system to improve the efficiency, profitability, and expandability of the production facility of LIA at Nordic Biomarker. Tests were made of the automatic TFF system's ability to perform the desired steps of concentration, dilution and diafiltration, both with purified water and mimicked product. The mimicked product of micro particles (MP) mixed with monoclonal antibodies (mAb) was also used to further test the system's pressure control, safety alarms and stops, and to determine a permeate flux by a critical flux experiment. The results imply a functional TFF system able to automatically concentrate the process fluid and maintain a stable volume during diafiltration, although an additional permeate pump was ordered to be able to attain a fully functional performance of the automatic TFF process. The final part of the implementation was to initiate a validation draft including a risk assessment, OQ plan and PQ plan that resulted in a plan of the main tests to be performed. To conclude, the essential part of the implementation of a high quality and efficient automatic TFF process was conducted to facilitate future expansion of the production of LIA.

Immunoassays are analytical methods that use the highly specific binding of antibodies in order to detect and quantify an analyte. The technique has become a staple in modern biopharmaceutical research and diagnostics, however the measurement of biomarkers like dysregulated cytokines require ultra-sensitive immunoassays that can detect molecules at sub pg/mL concentrations. One such method is the Exazym® signal amplification. Based on a method called Binding Oligo Ladder Detection (BOLD), it is a set of add-on reagents where a primer is conjugated to a detection antibody which is then combined with a template, polymerase and modified DNA nucleotides to generate a oligonucleotide ladder that is detected with a secondary detection antibody; this amplifies the signal by a factor of 10-100 in an existing immunoassay.  By applying this method to the Gyrolab® microfluidic immunoassay system, a sensitivity increase of 880x-1800x was achieved between a pre-synthesised BOLD product and the polymerised BOLD product. Several key factors for successful polymerisation in the microfluidic system were identified: adding the template separately before the polymerase and using a buffer with low ionic strength for the secondary detection antibody. Applying the BOLD amplification to an existing Gyrolab TNF-α assay only resulted in similar sensitivity as previous methods however. This report demonstrates that BOLD amplification can be successfully performed in a flow-through format on miniaturized affinity columns in the Gyrolab system to increase the sensitivity by orders of magnitude, where both the immunoassay and the amplification steps are automated in the system. However, further optimisation is needed for application in biomarker assays.

Borrelia burgdorferi sensu lato (s.l.) and tick-borne encephalitis (TBE) are two of the most common tick-borne diseases in Sweden. Varicella-zoster-virus (VZV) is a contagious, airborne herpesvirus with nearly 100% prevalence in individuals over 30 years old. At the Laboratorymedicine, County Hospital Ryhov, Jönköping, IgG-antibody analysis against B. burgdorferi s.l., TBE and VZV is performed on serum. The aim of the study was to compare ethylenediaminetetraacetic-acid-plasma and lithium-heparin-plasma with serum for thesea nalyses. The goal was to investigate whether it was possible to switch the sample material from serum to plasma for these analyses. Samples from 500 blood donors were collected for analysis in serum, ethylenediaminetetraacetic-acid-plasma and lithium-heparin-plasma. The qualitative data from the study indicated that there was no statistically significant difference in the interpretations (positive and negative) between the sample materials. The quantitative analysis of antibody levels showed a statistically significant difference between the sample materials. Due to the study´s limited sample size, no general conclusions could be drawn about whether plasma could replace serum. Further studies on the subject could provide increased understanding of the relationship between serum and plasma for analysis of Borrelia, TBE and VZV and how results are affected in the different sample materials. / Borrelia burgdorferi sensu lato (s.l.) och tick-borne encefalit (TBE) är två av de vanligaste fästingöverförda sjukdomarna i Sverige. Varicella-zoster-virus (VZV) är ett smittsamt, luftburet herpesvirus med nära 100% prevalens hos personer över 30 år. På Laboratoriemedicin, Länssjukhuset Ryhov, Jönköping utförs analys av IgG-antikroppar mot B. burgdorferi s.l. TBE och VZV på serum. Syftet med studien var att jämföra etylendiamintetraättiksyra-plasma och litiumheparinplasma mot serum för dessa analyser. Målet var att undersöka om det var möjligt att byta provmaterial från serum till plasma för nämnda analyser. Prover från 500 blodgivare samlades in för analys i serum, etylendiamintetraättiksyra- och litiumheparin-plasma. Resultatet från studiens kvalitativa data indikerade att ingen statistisk signifikant skillnad förelåg vad det gäller tolkningarna (positiv och negativ) mellan provmaterialen. Kvantitativa analysen av antikroppsnivåerna visade en statistisk signifikant skillnad mellan provmaterialen. På grund av studiens begränsadeprovstorlek kunde inga generella slutsatser dras kring huruvida plasma kunde ersätta serum. Vidare studier kring ämnet kan ge ökad förståelse kring sambandet mellan serum och plasma för analys av Borrelia, TBE och VZV samt hur resultaten påverkas i de olika provmaterialen.