O veneno de serpentes do gênero Bothrops (responsáveis por 80% dos envenenamentos no Brasil) é composto por metalo e serinoproteases, disintegrinas, fosfolipases, entre outros, e pode causar edema, hemorragia, necrose, e manifestações sistêmicas, como coagulação intravascular, choque, falência renal e hemorragia. O veneno da B. pirajai ativa o sistema complemento (C), sugerindo uma contribuição para o agravamento dos sintomas. Considerando a importância do C no processo inflamatório e o papel das metaloproteinases nos envenenamentos, verificou-se que o tratamento do sangue total humano (onde células e mediadores plasmáticos interagem entre si) com ou sem a compstatina (inibidor de C3 do C), e com a metaloproteinase de classe PI do veneno de B. pirajai, levou a diferenças bastante significativas na expressão dos marcadores analisados nos leucócitos, na geração de anafilatoxinas e TCC, e na quantificação de citocinas e quimiocinas no plasma, sugerindo que a inibição do C reduz o processo inflamatório, podendo ser uma terapia efetiva para envenenamentos botrópicos. / The snake venom of Bothrops genus (responsible for 80% of envenomation in Brazil) is composed by metallo and serine proteases, disintegrins, phospholipase, among others, and can cause edema, hemorrhage, necrosis, and systemic manifestations, such as intravascular coagulation, shock, renal failure and systemic hemorrhage. The venom of B. pirajai is able to activate the complement system (C), suggesting a contribution to the worsening of symptoms. Considering the importance of C in the inflammatory process and the role of metalloproteinases in envenomation, it was found that treatment of human whole blood (where cells and plasma mediators interact) with or without compstatin (C3 inhibitor), and with a class PI metalloproteinase from B. pirajai\'s venom led to highly significant differences in the expression of the markers analyzed in leukocytes, in generation of anaphylatoxins and TCC, and quantification of cytokines and chemokines in plasma, suggesting that inhibition of C reduces the inflammatory process and may be an effective therapy for bothropic envenomations.

The aim of cancer immunotherapy is to eradicate tumours by inducing a tumour-specific immune response. This thesis focuses on how antibodies and peptides can improve antigen presentation and the subsequent tumour-specific T cell response. Tumour recognition by the immune system can be promoted through delivery of antigen in the form of a vaccine. One example is the development of a therapeutic peptide vaccine containing both CD4+ and CD8+ T cell epitopes. So far, peptide vaccinations have shown limited success in clinical trials and further improvements are needed, such as choice of adjuvant and T cell epitopes, as well as targeted delivery of peptides and adjuvants to the same DC. In paper I, we describe the development of a peptide-peptide conjugate (with a tumour T cell epitope) that, via immune complex formation and FcγR binding, enhance antigen uptake and activation of DCs. The conjugate consists of three tetanus toxin-derived linear B cell epitopes (MTTE) that were identified based on specific IgG antibodies in human serum. Three MTTE peptide sequences were conjugated to a synthetic long peptide (SLP) that consists of a T cell epitope derived from the desired target tumour. In paper II, the conjugate was evaluated in a modified Chandler loop model containing human blood, mimicking blood in circulation. The conjugate was internalised by human monocytes in an antibody-dependent manner. A conjugate containing the model CMV-derived T cell epitope pp65NLV generated recall T cell responses dependent on MTTE-specific antibodies and the covalent conjugation of the three MTTE with the SLP. In paper III, a CD40-specific antibody was characterised for local treatment of solid tumours. The antibody eradicated bladder tumours in mice and induced T cell-mediated immunological memory against the tumour. In paper IV, we characterised the Chandler loop model (used in paper II) for its potential use in predicting cytokine release syndrome (CRS) in response to monoclonal antibodies (mAbs). Superagonistic antibodies (e.g., OKT3) induced rapid cytokine release whereas no cytokine release was induced by antibodies (e.g., cetuximab) associated with low incidence of CRS in the clinic. In conclusion, this thesis work demonstrates proof-of-concept of improved strategies for antibody- and peptides-based cancer immunotherapies and their potential use in multiple cancer indications.

Magister Scientiae - MSc / The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P<0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised. / South Africa

O Hemocentro São Paulo é responsável por manter um banco de dados com informações sobre cada doação ou tentativa de doação de sangue. No entanto, os dados desse banco de dados não possuem a qualidade requerida pelas ferramentas/técnicas de análise. Por essa razão, fica difícil utilizar tais dados para estabelecer relações sistemáticas entre as variáveis armazenadas. A principal contribuição desta tese é a descrição da proveniência para atributos selecionados usando critérios de classificação definidos por especialistas. Este trabalho mostra que é possível fazer investigações detalhadas usando a descrição dos dados sem a necessidade de alterar a estrutura do banco de dados. Durante o período de 1996 a 2006, 1.469.505 doadores foram responsáveis por mais de 2.8 milhões de doações. Após a descrição da proveniência, foram obtidos 252.301 doadores do sexo masculino e 133.056 doadores do sexo feminino e que atenderam aos critérios de inclusão usados nesta tese. Dos 385.357 doadores incluídos na análise, 21.954 (5,7%) tiveram suas doações adiadas devido a seus baixos níveis de hematócrito, 3.850 (1,5%) eram do sexo masculino e 18.104 (13,6%) do sexo feminino. Os resultados obtidos demonstram que, embora os intervalos de espera entre as doações de sangue sejam grandes entre os doadores do sexo feminino e masculino, as mulheres são recusadas mais cedo, por risco de desenvolver anemia, do que os homens. Aproximadamente 12,84% das mulheres e 1,21% dos homens desenvolveriam hematócrito baixo antes da sétima doação. Os dados sugerem que indivíduos com baixo nível de hematócrito devem esperar mais tempo antes de executarem a próxima doação. Portanto, é importante compreender se existe uma ligação entre a doação de sangue e a diminuição no nível de hematócrito, a fim de evitar resultados indesejáveis para os doadores de sangue. O modelo de proveniência apresentado nesta tese não foi definido de acordo com os modelos de proveniência genéricos já implementados. Esta tese apresenta um modelo de proveniência que foi capaz de acrescentar informações semânticas para adquirir conhecimento de um experimento in silico. Um dos principais objetivos foi desenvolver uma abordagem baseada em declarações, tentando responder a importantes questionamentos biológicos. O modelo descrito combina ricas informações em cada processo usando declarações, e se baseia no conhecimento de especialistas. Esta tese também utilizou estatística descritiva e Análise de Sobrevivência. Finalmente, com a validação do modelo em um domínio conhecido, é pretendido expandir esse método para outros sistemas de informação voltados para hemoterapia. / The São Paulo Blood Center is responsible to maintain a database with information on each donation. However, this database does not have the quality required by techniques of analysis. For this reason, it is difficult to use it directly to establish systematic relationships between the variables. The main contribution of this paper is a provenance description of attributes selected using classification criteria defined by specialists. We show that it is possible to make detailed investigations using the data description without the need to change the structure of the database. During 1996 2006, 1,469,505 donors were responsible for more than 2.8 million of donation. After the provenance description, we obtained 252,301 male and 133,056 female that met our inclusion criteria. Of the 385,357 donors included in the analysis, 21,954(5.7%) were deferred due to low hematocrit, 3,850(1.5%) were males and 18,104(13.6%) were females. Our results show that, although the intervals between donations for female and male donors are wider, women presented anemia earlier than men. Approximately 12,84% of the females and 1,21% of the males would develop low hematocrit before the 7th donation. Our data suggest that individuals with low hematocrit level should wait longer before the next donation. Therefore, it is important to understand if there is a connection between blood donation and decrease in hematocrit level in order to prevent undesirable outcomes to blood donors. The provenance model presented here was not defined according to the generic provenance models already implemented. This thesis presents a provenance model that is able to add semantic information to acquire knowledge of an in silico experiment. One of the main purposes is to develop an approach based on declarations in order to answer biological questions. The provenance model described in this paper combines rich information for each process using the declarations, each having expert knowledge as a basis. To evaluate this provenance model we use descriptive statistics and Survival Analysis. Finally, with the validation of the model in a known domain, we intent to apply and validate this provenance model to other hemotherapy information systems.

Introduction: Post-mortem whole blood samples differ greatly in quality, lipemia is one cause of concern in toxicological analyses. Around 4 % of all samples sent to RMV are given a notation of lipemic content. The aim of the thesis was to study the effects of lipemia on the quantification of 14 benzodiazepines and 5 similar sedative and antianxiety drugs as well as evaluate the pre-analytical process aiming to reduce the effects of lipemia.  Methods: Blood samples were simulated with bovine blood, analyte spiking, and lipid spiking with either the nutrition emulsion Intralipid or with a mixture of post-mortem lipids from authentic samples. The outset was the by RMV currently used LLE method followed by UPLC- MS/MS and the extraction method was altered and evaluated. Matrix effects were also studied.  Results: Lipemia were found to be a great interference when quantifying benzodiazepines. For most analytes, internal standard could compensate for the loss of analyte but there was a problem with analytes not having their own IS. The 7-amino-compounds were greatly affected by lipemia and propiomazine and dihydropropiomazine showed extreme losses. Equilibration of IS did not result in similar loss as analyte. Dilution of sample reduced losses caused by lipemic content. SPE resulted in extracts free from lipids and high yields but there were analyte losses similar to LLE. No matrix effects from the lipids were found. Samples spiked with Intralipid gave poorer analyte yields than those spiked with post-mortem lipids.  Conclusion: Dilution is the most successful method to reduce pre-analytical matrix effects as long as the concentration is not so low that it risks getting lower than the analytical limits when doing so. Not homogenising samples before sampling is giving incorrect results. SPE could, if optimised for the analyte retention and elution, remove lipids from samples and obtain accurate analyte concentrations. Pooling lipids from post-mortem samples is a possible method for simulating lipemic whole blood. Intralipid and the PM-mix gave the same indications, but to different extents. Further studies where the ability to mimic authentic lipids are needed for both Intralipid and PM-mix.

Hematology-instruments that analyze blood cells can at specific casesbe followed by manual counting with a hemocytometer. The plastic hemocytometer which has a fixed coverslip and is assigned for disposable use, appears to reduce the problems arising from the use of the traditional glass hemocytometer, for instance coverslip-application difficulty and dust particles. Capillary whole blood sampling may be useful for patients at the emergency department and in addition, a smaller volume of blood is taken compared to venous blood sampling. This study will focus on the platelet- and leukocyte count (PLT and WBC) in venous and capillary blood, in order to compare the plastic- and glass hemocytometers with Sysmex XN-2000 (Sysmex Corporation, Kobe, Japan), and verify if capillary whole blood sampling can be used for the measurement of these hematological parameters. 30 subjects donated both capillary- and venous blood samples and these blood samples were analyzed with glass- and plastic hemocytometers with Sysmex XN-2000 as the reference method. The study results showed high correlation between the reference method and plastic hemocytometer at low PLT but were not suitable for WBC-determination. The capillary whole blood showed high correlation to the reference method at WBC-determination, but was unusable at PLT-determination. Glass hemocytometer was closer to the reference method results than plastic hemocytometer was.

(1) Background: To date, the response of patients with rheumatoid arthritis (RA) to the various biologic DMARD available cannot be predicted due to a lack of reliable biomarkers. Based on our preliminary work on tmTNF reverse signaling, we developed a whole-blood assay measuring tmTNF crosslinking-induced IL-10 production to predict the response to TNF inhibitor (TNFi) therapy. (2) Methods: This prospective study included patients with active RA. Depending on the clinical judgment of the attending rheumatologist, either therapy with a TNF or JAK inhibitor was initiated. Clinical parameters and blood samples were obtained at baseline and after 8 weeks of therapy. The blood samples were collected using a newly developed whole-blood assay based on the principle of tmTNF reverse signalling. Subsequently, IL-10 was measured via enzyme-linked immunosorbent assay (ELISA) technique. (3) Results: 63 patients with RA were enrolled. In fifteen patients, TNFi therapy was initiated, while eight patients started a JAKi treatment. The cross-sectional analysis of all patients showed a positive correlation between tmTNF crosslinking-induced IL-10 and parameters of disease activity (CRP [r = 0.4091, p = 0.0009], DAS28 [r = 0.3303, p = 0.0082]) at baseline. In the TNFi treatment study, IL-10 was found to be significantly higher in EULAR responders than in non-responders (p = 0.0033). After initiation of JAKi treatment, in contrast, IL-10 induction was not linked to response. Longitudinal analysis of the TNFi-treated patients revealed IL-10 to decrease in responders (p = 0.04), but not in non-responders after 8 weeks of therapy. Of importance, the IL-10 production at baseline correlated inversely with TNFi response determined by DDAS28 in patients with TNFi treatment (r = 0.5299, p = 0.0422) while no such link was observed under JAKi therapy (p = 0.22). Receiver operation characteristics (ROC) analysis demonstrated a high performance of tmTNF/crosslinking-induced IL-10 in predicting a TNFi therapy response according to the EULAR criteria (AUC = 0.9286, 95% Confidence interval 0.7825–1.000, p = 0.0055). (4) Conclusions: In this pilot investigation, we demonstrated the feasibility of a whole-blood assay measuring tmTNFinduced IL-10 to predict clinical response to TNF inhibitor treatment. This approach might support rheumatologists in their decision for an individually tailored RA therapy.

Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by severe and debilitating fatigue and although its etiology is still unknown, recent studies have found considerable evidence that mitochondrial dysfunction and oxidative stress might be responsible for the underlying energy deficit in these patients. Adenine and pyridine nucleotides could be used as potential biomarkers for energy related disorders such as chronic fatigue syndrome because of their various functions in the energy and redox pathways. The first part of this study focussed on developing a liquid chromatography electrosprayionisation tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of these nucleotides in blood samples. Due to the instability of nucleotides in biological matrices it was also necessary to find a suitable extraction method that would be able to stop enzymatic activity via protein precipitation. Out of the four extraction methods investigated during this study, deproteinisation of whole blood samples with perchloric acid produced the highest nucleotide abundances. Although nucleotide standards were found to be stable in perchloric acid, nucleotide levels in blood samples were not stabilised by addition of perchloric acid. The second part of this study consisted of measuring the nucleotide levels in blood samples of controls and possible CFS patients in order to test the proof of concept of the new LCESI- MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and problems with nucleotide instability, it was still possible to distinguish between the two groups based on the results obtained with the new LC-ESI-MS/MS method. The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for nucleotide quantification in whole blood samples, thus the aim of this study was achieved. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

O agregado proteico P-MAPA apresentou potencial imunomodulatório em diversos estudos, mas a sua ação sobre o sistema complemento e receptores do tipo Toll (TLRs) não é, ainda, conhecida. Neste estudo o P-MAPA promoveu ativação das vias clássica e alternativa do sistema complemento e produção de C3a e C5a. Utilizando um modelo ex vivo de sangue total humano, o composto promoveu aumento da expressão de CD11b e CD14, diminuição da expressão de C5aR, TLR2 e TLR4, em leucócitos de sangue periférico e também quando combinado com LPS, porém não promoveu alterações na expressão de C3aR. O P-MAPA induziu redução de IFN-g no plasma, aumento da produção de TNF-α, IL-8, IL-12 e peróxinitrito, mas não induziu produção de superóxido, IL-6, IL-1β, TGF-β ou IL-10. Por meio de testes in vivo, foi possível determinar a dose letal do P-MAPA. Em conjunto, os dados obtidos mostram que o P-MAPA apresenta ação pró-inflamatória em modelo ex vivo de sangue total humano e que o tratamento combinado com LPS leva a uma amplificação dos seus efeitos. / P-MAPA, a protein aggregate has been described as a promising immunomodulator, however, its role on the complement system and Toll-like receptors (TLRs) is unknown. In the study, P-MAPA has promoted activation of the complement\'s classical and alternative pathways and the production of C3a and C5a. Using an ex vivo model of human whole blood, the compound promoted increase of CD11b and CD14 expression, decrease of C5aR, TLR2 and TLR4, in peripheral blood leucocytes and when combined with LPS, but did not change C3aR expression. P-MAPA promoted reduction of IFN-g in plasma, increased production of TNF-α, IL-8, IL-12 and peroxynitrite, but did not induce the production of superoxide, IL-6, IL-1β, TGF-β or IL-10. Through in vivo tests, we were able to determine a lethal dose for P-MAPA. Altogether, our data indicate that P-MAPA has proinflammatory action in ex vivo model of human whole blood and that the treatment combined with LPS leads to amplification of its effects.

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.