<p>In this thesis some multivariate spectroscopic methods for the analysis of solutions are proposed. Spectroscopy and multivariate data analysis form a powerful combination for obtaining both quantitative and qualitative information and it is shown how spectroscopic techniques in combination with chemometric data evaluation can be used to obtain rapid, simple and efficient analytical methods. These spectroscopic methods consisting of spectroscopic analysis, a high level of automation and chemometric data evaluation can lead to analytical methods with a high analytical capacity, and for these methods, the term high-capacity analysis (HCA) is suggested. It is further shown how chemometric evaluation of the multivariate data in chromatographic analyses decreases the need for baseline separation. </p><p>The thesis is based on six papers and the chemometric tools used are experimental design, principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), partial least squares regression (PLS) and parallel factor analysis (PARAFAC). The analytical techniques utilised are scanning ultraviolet-visible (UV-Vis) spectroscopy, diode array detection (DAD) used in non-column chromatographic diode array UV spectroscopy, high-performance liquid chromatography with diode array detection (HPLC-DAD) and fluorescence spectroscopy. The methods proposed are exemplified in the analysis of pharmaceutical solutions and serum proteins.</p><p>In Paper I a method is proposed for the determination of the content and identity of the active compound in pharmaceutical solutions by means of UV-Vis spectroscopy, orthogonal signal correction and multivariate calibration with PLS and SIMCA classification. Paper II proposes a new method for the rapid determination of pharmaceutical solutions by the use of non-column chromatographic diode array UV spectroscopy, i.e. a conventional HPLC-DAD system without any chromatographic column connected. In Paper III an investigation is made of the ability of a control sample, of known content and identity to diagnose and correct errors in multivariate predictions something that together with use of multivariate residuals can make it possible to use the same calibration model over time. In Paper IV a method is proposed for simultaneous determination of serum proteins with fluorescence spectroscopy and multivariate calibration. Paper V proposes a method for the determination of chromatographic peak purity by means of PCA of HPLC-DAD data. In Paper VI PARAFAC is applied for the decomposition of DAD data of some partially separated peaks into the pure chromatographic, spectral and concentration profiles. </p>

Les immunoglobulines intraveineuses (IVIg) constituent une préparation polyclonale d’IgG isolée et regroupée à partir du plasma sanguin de multiples donneurs. Initialement utilisé comme traitement de remplacement chez les patients souffrant d’immunodéficience primaire ou secondaire, les IVIg sont maintenant largement utilisées dans le traitement de plusieurs conditions auto-immunes, allergiques ou inflammatoires à une dose élevée, dite immunomodulatrice. Différents mécanismes d’action ont été postulés au fil des années pour expliquer l’effet thérapeutique des IVIg dans les maladies auto-immunes et inflammatoires. Entre autre, un nombre grandissant de données issues de modèles expérimentaux chez l’animal et l’humain suggère que les IVIg induisent l’expansion et augmentent l’action suppressive des cellules T régulatrices (Tregs), par un mécanisme qui demeure encore inconnu. Également, les patients atteints de maladies auto-immunes ou inflammatoires présentent souvent un nombre abaissé de Tregs par rapport aux individus sains. Ainsi, une meilleure compréhension des mécanismes par lesquels les IVIg modulent les cellules T régulatrices est requise afin de permettre un usage plus rationnel de ce produit sanguin en tant qu’alternative thérapeutique dans le traitement des maladies auto-immunes et inflammatoires. Par le biais d’un modèle expérimental d’allergie respiratoire induite par un allergène, nous avons démontré que les IVIg diminuaient significativement l’inflammation au niveau des voies aériennes ce, en association avec une différenciation des Tregs à partir des cellules T non régulatrices du tissu pulmonaire. Nous avons également démontré qu’au sein de notre modèle expérimental, l’effet anti-inflammatoire des IVIg était dépendant des cellules dendritiques CD11c+ (CDs) pulmonaires, puisque cet effet pouvait être complètement reproduit par le transfert adoptif de CDs provenant de souris préalablement traitées par les IVIg. À cet effet, il est déjà établi que les IVIg peuvent moduler l’activation et les propriétés des CDs pour favoriser la tolérance immunitaire et que ces cellules seraient cruciales pour l’induction périphérique des Tregs. C’est pourquoi, nous avons cherché à mieux comprendre comment les IVIg exercent leur effet sur ces cellules. Pour la première fois, nous avons démontré que la fraction d’IgG riche en acide sialique (SA-IVIg) (constituant 2-5% de l’ensemble des IgG des donneurs) interagit avec un récepteur dendritique inhibiteur de type lectine C (DCIR) et active une cascade de signalement intracellulaire initiée par la phosphorylation du motif ITIM qui est responsable des changements observés en faveur de la tolérance immunitaire auprès des cellules dendritiques et des Tregs. L’activité anti-inflammatoire de la composante SA-IVIg a déjà été décrite dans des études antérieures, mais encore une fois le mécanisme par lequel ce traitement modifie la fonction des CDs n’a pas été établi. Nous avons finalement démontré que le récepteur DCIR facilite l’internalisation des molécules d’IgG liées au récepteur et que cette étape est cruciale pour permettre l’induction périphérique des Tregs. En tant que produit sanguin, les IVIg constitue un traitement précieux qui existe en quantité limitée. La caractérisation des mécanismes d’action des IVIg permettra une meilleure utilisation de ce traitement dans un vaste éventail de pathologies auto-immunes et inflammatoires. / Intravenous immunoglobulin (IVIg) is a therapeutic preparation of normal human polyclonal IgG derived from pooled plasma from a large number of healthy donors. Initially used as replacement therapy for patients with primary and secondary immune deficiencies, IVIg is now also widely used for the treatment of a variety of autoimmune, allergic and systemic inflammatory disorders, at high immunomodulatory doses. The beneficial effect of IVIg in autoimmune and inflammatory diseases has been attributed to different mechanisms. Increasing evidence shows that IVIg induces expansion and enhances the suppressive function of regulatory T cells (Tregs) in different experimental animal models and human subjects, through an unknown mechanism. Human inflammatory and autoimmune diseases are known to be associated with Treg deficiency. Therefore, a more precise understanding of the mechanisms by which IVIg modulate Treg populations seems to be needed for more rational use of this compound as an alternative therapy in context of various inflammatory and autoimmune disorders. Using a robust antigen-driven model of allergic airway disease, we have demonstrated that IVIg markedly attenuates airway inflammation and this effect is associated with the induction of Tregs from non-regulatory T cells in pulmonary tissues. We have also demonstrated that the antiinflammatory actions of IVIg, in our model are dependent on a population of pulmonary CD11c+ dendritic cells (DCs), as the action of IVIg could be completely replicated by adoptive transfer of CD11c+ DCs from IVIg-treated mice. we have shown that tolerogenic DCs involve in the peripheral induction of Tregs. Given the requirement of DCs in the induction of Tregs, we explored the mechanism by which IVIg interacts and modulate these cells and for the first time demonstrated that the purified sialylated fraction of human IgG (SA-IVIg) (that consists 2-5% of whole IgG) interacts with an inhibitory C-type lectin receptor on dendritic (DCIR) and this interaction triggers an ITIM intracellular signaling cascade. This subsequently results in rendering tolerogenic activities to DCs and peripheral induction of Tregs. The anti-inflammatory activity of SA-IVIg has been shown in previous studies, but the mechanism by which it modulates DCs functions is not well understood. We also demonstrated that DCIR facilitates the internalization of IgG molecules into DC and this internalization appears to be a crucial step for induction of Tregs. IVIg is a costly therapeutic compound. Characterization of the mechanism of action of IVIg can lead to a better application of this plasma based therapy in a wide range of autoimmune and inflammatory diseases.

A via de Reparo por Excisão de Nucleotídeos (NER) é responsável pelo reparo das lesões causadas pela luz ultravioleta (UV) e de outras lesões capazes de distorcer a dupla hélice, bloqueando a replicação e a transcrição. Os pacientes que apresentam as síndromes recessivas raras Xeroderma Pigmentosum (XP), tricotiodistrofia (TTD) e síndrome de Cockayne (CS) possuem mutações em algum dos 11 genes relacionados ao NER e à transcrição basal. Mutações na proteína XPD levam ao surgimento de diferentes fenótipos: XP, TTD, XP/CS ou COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), uma forma rara de CS. Os pacientes XP apresentam alta incidência de câncer de pele, o que não ocorre com os pacientes TTD e CS, além de poderem apresentar perda neuronal progressiva, enquanto todos os CS e TTD apresentam uma diminuição na mielinização do cérebro. As neuropatologias são provavelmente associadas a problemas no reparo de danos endógenos no DNA das células nervosas. Diversos trabalhos mostraram o envolvimento do NER no reparo desses danos, os quais pensava-se serem reparados apenas por outro mecanismo, o Reparo por Excisão de Base. Neste trabalho mostramos que fibroblastos de pacientes XP-D, XP-D/CS e TTD, portadores de mutações em XPD, são sensíveis ao estresse oxidativo induzido pelo tratamento com azul de metileno fotoativado, apresentando bloqueio prolongado no ciclo celular e permanência da sinalização de danos ao DNA. A complementação das diferentes linhagens com o gene XPD/ERCC2 foi capaz de restaurar a sobrevivência celular. Foram detectadas diferenças importantes na capacidade de reparo/retomada da transcrição após danos gerados por estresse oxidativo em DNA plasmidial, além da ativação de vias diferentes de morte celular: fibroblastos XP-D apresentam maior capacidade de reparo e apresentam morte por apoptose após estresse oxidativo, enquanto os fibroblastos XP-D/CS e TTD apresentam menor capacidade de reparo ativação de mais de uma via de morte celular (apoptose e necrose), diferenças que podem estar ligadas ao fenótipo dos pacientes. Mutações no gene codificante para a DNA polimerase n, POLH, estão associadas à forma variante de XP (XP-V). Pol n é uma polimerase especializada na síntese translesão (TLS) de fotoprodutos, além de estar implicada na TLS de outros tipos de lesões como bases oxidadas, e em vias não relacionadas à TLS como a hipermutação somática e à replicação de regiões de DNA com arquiteturas não-canônicas. Neste trabalho mostramos que os fibroblastos de pacientes XP-V apresentam sensibilidade ao estresse oxidativo. Mostramos uma indução da proteína pol n em fibroblastos primários após danos genotóxicos, associada ao aumento da capacidade de lidar com a parada na forquilha de replicação, possibilitando a continuidade da replicação do DNA e ao aumento da sobrevivência celular. Mostramos uma diferença na estabilidade genômica nos genes das imunoglobulinas dos pacientes XP-V idosos em comparação com os pacientes jovens e controles de idade pareada, mostrando que a ausência dessa polimerase pode estar ligada ao aumento da instabilidade genômica nesses genes / The Nucleotide Excision Repair (NER) pathway is responsible for the repair of UV photoproducts and other bulky lesions that block both replication and transcription. Patients with the rare recessive disorders Xeroderma Pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne Syndrome (CS) carry mutations in one of the 11 NER genes, linked to repair and basal transcription. Mutations in XPD lead to different phenotypes: XP, TTD, XP/CS or COFS (Cerebro-Oculo-Facio-Skeletal Syndrome), a rare form of CS. XP patients have high incidence of skin cancer, which does not occur in TTD or CS patients, although ther may present neurodegeneration, while all CS and TTD patients have neurodevelopmental symptoms linked to dysmielynation. The pathology of these neurological diseases is probably associated with deficient repair of DNA lesions in nervous cells, generated by endogenous processes. Many groups including ours have demonstrated the involvement of NER in the repair of these lesions, previously thought to be exclusively repaired by Base Excision Repair. In this work we show high sensitivity of both primary and transformed XP-D, XP-D/CS and TTD human fibroblasts in response to oxidative stress generated by photoactivated methylene blue, with prolonged cell cycle arrest and DNA damage signaling. The complementation of the three different cell lines with the XPD/ERCC2 gene was able to restore cell survival. We detected important differences in repair capacity/transcription resumption after damage generated by oxidative stress in plasmid DNA, besides the activation of different cell death pathways: XP-D cells have higher repair capacity and die by apoptosis, while XP-D/CS and TTD cells have little repair capacity and activate more than one death pathway (apoptosis and necrosis). We believe these differences can be related to the patients\' phenotypes. Mutations in DNA polymerase n coding gene, POLH, are associated with the variant form of XP (XP-V). Pol n is a translesion synthesis (TLS) polymerase specialized in the TLS past CPD photoproducts, besides other lesions like oxidized bases, and in other processes like somatic hypermutation and DNA replication in structured regions. In this work we show XP-V human fibroblasts are sensitive to oxidative stress. We detected an induction of pol n after genotoxic stress in primary cells, associated with increased ability to deal with the stalled replication fork, and consequently to DNA replication restart and cell survival. In addition, we detected a difference in genomic stability in immunoglobulin genes in aged XP-V patients in comparison to both young patients and age-matched controls, showing the absence of this polymerase may be linked to increased genomic instability in these genes

INTRODUÇÃO: A endomiocardiofibrose (EMF) é uma doença típica de países tropicais, na qual ocorre a deposição de uma capa fibrosa na região endomiocárdica com graves consequências clínicas. A patogenia da EMF ainda não foi elucidada, mas uma das principais hipóteses etiológicas sugere que a EMF seja consequência de um processo inflamatório crônico com envolvimento de respostas imunes Th2 pós-infestação helmíntica, mediada por eosinófilos nas fases iniciais da doença. Neste estudo avaliamos o perfil da resposta imune e inflamatória, assim como o perfil de proteínas expressas no endomiocárdio afetado, para compreender melhor os mecanismos envolvidos na patogênese da EMF. MÉTODOS: Foram coletadas amostras de plasma e soro de pacientes com diagnóstico de EMF e de indivíduos saudáveis, para dosagens de 6 citocinas plasmáticas do perfil Th1/Th2, Proteína C Reativa ultrassensível (PCRus), IgE total e IgE específico contra aero-alérgenos prevalentes e alérgenos de helmintos. A análise proteômica do endomiocárdio afetado de quatro pacientes com EMF submetidos à ressecção cirúrgica da fibrose, levou à identificação por espectrometria de massas, de proteínas separadas previamente por gel 1D e 2D. Análises in silico de vias funcionais e redes ligando as proteínas identificadas também foram realizadas. Eosinofilia em sangue periférico, assim como dados clínicos e ecocardiográficos, foram avaliados retrospectivamente. RESULTADOS: Foram selecionados 27 pacientes em estágio avançado da EMF, portadores de disfunção diastólica e/ou valvar. Todas as amostras de plasma analisadas apresentaram níveis detectáveis de pelo menos uma das citocinas avaliadas. As citocinas IL-6, TNF-?, IL-10 e IL-4 foram detectadas em pelo menos 74% das amostras, sendo que os níveis de IL-6, TNF-? e IL-10 se apresentaram significantemente elevados em comparação com os detectados nos indivíduos saudáveis. Foi observada correlação positiva entre os níveis de todas as citocinas avaliadas. Apenas 33% dos pacientes apresentaram algum episódio isolado de eosinofilia periférica ao longo do tempo, sendo que 11% apresentaram hipereosinofilia. Os níveis de IgE total foram semelhantes aos observados na população controle. A análise proteômica permitiu identificar 140 proteínas distintas no tecido endomiocárdico, das quais 18 eram provenientes da matriz extracelular. As análises in silico indicaram IL-6 e TNF-? como dois dos principais indutores da expressão das proteínas identificadas e a principal via canônica envolvida nas proteínas identificadas foi a: \"Resposta de Fase Aguda\". Coincidentemente, os níveis de PCRus encontraram-se significativamente aumentados nos portadores de EMF. CONCLUSÕES: Elevados níveis de citocinas pró e anti-inflamatórias e de PCRus sugerem a presença de perfil inflamatório misto (Th1/Th2) nas fases avançadas da patogênese da EMF. A pouca expressão da eosinofilia descarta sua participação ativa na patogênese da fase avançada da doença, mas não é possível excluir sua participação nas fases iniciais. Nossos dados permitem levantar a hipótese que os níveis elevados de citocinas inflamatórias modulem a expressão de proteínas- incluindo as de fase aguda- no tecido endomiocárdico de pacientes portadores de EMF / INTRODUCTION: Endomyocardial fibrosis (EMF) is a typical disease in tropical countries, characterized by the fibrous deposition in the endomyocardium, with severe clinical manifestations. EMF pathogenesis is still unclear, but one of the major hypothesis suggests that EMF could be a consequence of a chronic inflammatory process with possible involvement of a Th2 immune responses after helminthiasis, mediated by eosinophils, which could contribute to pathogenesis in the early stages of the disease. In this study, we evaluated the inflammatory response profile and the protein expression profile of the affected endomyocardium, to understand the mechanisms involved in this pathogenesis. METHODS: Plasma and serum samples were collected from the 27 patients diagnosed with advanced stage EMFand from healthy controls, to mensured plasma levels of 6 cytokines belonging to the Th1/Th2 cytokine profiles, ultrasensitive C Reactive Protein (CRP), total and allergen-specific serum IgE against prevalent and helminthic allergens. Proteomic analysis of tissue samples obtained from 4 EMF patients submitted to surgical resection of affected endomyocardial tissue allowed the identification of proteins by mass spectrometry, after separation by 1D and 2D electroforesis. In silico analysis of functional pathways and networks connecting the proteins identified in the EMF cardiac tissue was also performed. Blood peripheral eosinophilia, clinical and echocardiography data were evaluated retrospectively. RESULTS: All EMF patients displayed detectable plasma levels of at least one of the cytokines tested. We found that TNF-?, IL-6, IL-4 and IL-10 were each detected in at least 74% of tested sera, and plasma levels of IL10, IL6 and TNF-? were significantly higher than controls. Plasma levels of such cytokines positively correlated with each other. Only 33% of the patients presented any episode of blood eosinophilia along time, and 11% of these patients presented hypereosinophilia. Total IgE levels were similar to those from healthy subjects. Proteomic analysis allowed the identification of 140 distinct proteins from the resected endomiocardium of the EMF patients, 18 of which belonging to the extracellular matrix. In silico analysis indicated IL-6 and TNF-? as two of the major gene expression inducers of the identified proteins in our analysis, and the Acute Phase Response was identified as the major canonical pathway involved with the identified set of proteins. Similarly, CRP levels were significantly increased in the EMF patients. CONCLUSIONS: Increased levels of an inflammatory/anti-inflammatory circulating cytokines (Th1/Th2), along with increased CRP levels, suggested the presence of a mixed inflammatory profile in EMF advanced stage. The number of EMF patients with blood eosinophilia does not support the active participation of eosinophils in pathogenesis of advanced EMF. However, it is not possible to exclude the participation in the pathogenesis early stages. Our data support the hypothesis that the increased levels of inflammatory cytokines modulate protein expression -including proteins of the acute phase response - in the endomyocardial tissue of EMF patients

Os efeitos protetores da imunidade humoral são medidas por uma família de glicoproteínas chamadas anticorpos ou imunoglobulinas. As preparações de imunoglobulina G (IgG) utilizadas em nosso país são importantes. No Brasil, a primeira preparação de IgG foi obtida na Fundação Pró-Sangue Hemocentro de São Paulo em 1993. O presente estudo avaliou preparações de IgG obtidas de misturas de plasma humano de variadas procedências, inclusive a preparação obtida no Brasil. Foram avaliados os seguintes parâmetros: concentração protéica, distribuição das subclasses da IgG, atividade de anticorpos específicos e segurança quanto a agentes patogênicos transmissíveis pelo sangue. Em algumas preparações, a concentração protéica de IgG e a distribuição das suas subclasses estavam fora das especificações. As preparações apresentaram atividade de anticorpos específicos contra os vírus das hepatites A e B, do herpes simples, da rubéola, citomegalovírus; contra a bactéria Streptococcus pyogenes &#946;-hemolítico do grupo A e contra o parasita Toxoplasma gondii. A qualidade de matéria-prima utilizada em algumas das preparações de IgG não foi adequada em função de reações positivas para anticorpos contra alguns agentes infecciosos, tais como HTLV I/II, HAV, HBV, HCV e Treponema pallidum. Esse estudo também mostrou a necessidade de se implantar urgente um programa abrangente para avalição das preparações de IgG a serem consumidas pela população brasileira. / A family of glicoproteins, which are called antibodies or immunoglobulins (IgG), mediates the protective effects of humoral immunity. In Brazil, the IgG for intravenous use are imported from other countries. The first Brazilian immunoglobulin G for therapheutic use was obtained from human plasma at the Fundação Pró-Sangue Hemocentro de São Paulo. The present study was carried out to evaluate different preparations of IgG, human plasmad-derived, include the preparation from Brazil. The protein concentration, IgG subclass distribution, specific antibody activities and safety regarding the main blood transmitted infectious diseases were analyzed. In some preparations, IgG protein concentration and subclass distribution were different from their specifications. Some preparations showed specific antibody activity against the following antigens: A and B hepatitis virus, rubella, herpes simplex virus, citomegalovirus, measles virus, Streptococcus pyogenes &#946;-hemolytic group A and Toxoplasma gondii. The presence of antibodies against antigens such as HTLV I/II, HAV, HBV, HCV and Treponema pallidum has compromissed the quality guaranty of the material-source (plasma) used in some preparations. This study has also showed that a complete and effective program for the quality evaluation of IgG preparations used in Brazil is needed and should be urgently established

A Imunodeficiência Comum Variável (ICV) e a Deficiência Seletiva de Imunoglobulina A (DIgA) são as imunodeficiências primárias humorais de maior freqüência na população mundial. Ambas as doenças são caracterizadas pela ausência ou redução significativa de imunoglobulinas no soro. Embora diversas anormalidades imunológicas tenham sido associadas a estas doenças, nenhuma hipótese unificadora a respeito das bases moleculares das mesmas foi proposta até o presente momento, sendo que o único defeito comum a todos os pacientes é a falha na diferenciação de células B em plasmócitos e conseqüente secreção de anticorpos. Devido à alta incidência de auto-imunidade e alergia em pacientes com ICV e DIgA, no presente trabalho, visamos analisar por citometria de fluxo a população de linfócitos CD4+ com potencial regulador nesses pacientes, para avaliar se possíveis defeitos quantitativos ou funcionais nesta população reguladora poderiam explicar a alta incidência de doenças auto-imunes ou alérgicas associadas a estas imunodeficiências. / Common Variable Immunodeficiency (CVID) and Selective Immunoglobulin A deficiency (IgAD) are the humoral primary immunodeficiencies with the highest incidence in the population. Both diseases are characterized by the absence or significant reduction of serum immunoglobulins. Although several immunological abnormalities have been associated with these diseases, no unifying hypothesis regarding the molecular basis of CVID and IgAD have been proposed to date, and the only defect common to all patients is the failure in differentiation of B cells into plasma cells and consequent secretion of antibodies. Due to the high incidence of autoimmunity and allergy in patients with CVID and IgAD, in the present work we analyzed by flow cytometry the population of CD4+ lymphocytes with regulatory potential in these patients to assess whether possible quantitative or functional defects in this regulatory population could explain the high incidence of autoimmune diseases or allergic reactions associated with these immunodeficiencies.

<p>Увод: Хронична бубрежна инсуфицијенција (ХБИ) се дефинише као структурно или функционално оштећење бубрега у трајању од најмање три месеца и/или смањење јачине гломеруларне филтрације (ЈГФ) испод 60 мл/мин/1.73м2. У доступној литератури постоје различити подаци о присуству оралних манифестација код пацијената са ХБИ у квантитативном и квалитативном погледу. Стање бубрежне дисфункције праћено је променама у протоку и саставу пљувачке што је у последњој деценији допринело испитивању клиничких и лабораторијских показатеља бубрежне болести. Циљ: Циљ студије је био да се испита објективно стање оралне слузокоже, вредности рН, сијалометрије, концентрације урее, креатинина и секреторног имуноглобулина А пљувачке као и орални микробиолошки статус код пацијената са ХБИ. Материјал и методе: Узорак је био сачињен од 50 предијализних (31 мушкарца и 19 жена просечне старости 59,06&plusmn;14,30) и 25 хемодијализних пацијената (18 мушкараца и 7 жена просечне старости 54,92&plusmn;13,60) са постављеном дијагнозом ХБИ, заједно са 25 системски здравих испитаника компарибилних по полу и старости. Поред клиничког прегледа усне дупље спроведен је тест витроадхезије, одређивање интензитета саливације, рН вредности пљувачке и индекса крварења из интерденталне папиле (PBI). На узорцима сакупљене пљувачке, уз помоћ аутоматизованог система Beckman Coulter АУ480 спроведено је лабораторијско одређивање урее и креатинина методом спектрофотометрије и секреторног имуноглобулина А методом имунотурбидиметрије. За микробилошко испитивање коришћен је брис језика и техника оралног испирка. Резултати: Нису утврђене статистички значајне разлике између група према демографско-социјалним подацима. Предијализни испитаници су имали значајно веће присуство промена оралне слузокоже и оралних симптома. Просечне вредности клиренса креатинина су биле значајно мање код оболелих испитаника са бледилом оралне слузокоже, уремичним задахом, ксеростомијом и измењеним осећајем укуса у поређењу са испитаницима без наведених промена. Код предијализних су утврђене значајно смањене вредности сијалометрије према контролним групама и повећане pH вредности према групи здравих испитаника. Просечне концентрације урее и креатинина су се статистички значајно разликовале између испитиваних група. Умерена позитивна корелација је утврђена између серумских и пљувачних концентрација урее и креатинина код предијализних и креатинина код хемодијализних. Према просечним вредностима секреторног имуноглобулина А није било разлика између група. Код пацијената са ХБИ утврђено је значајно веће присуство гљива из рода Candida са предоминацијом non-albicans Candida врста. Закључак: Резултати истраживања указују на важност утврђивања клиничких карактеристика усне дупље код предијализних пацијената. Интензитет саливације, pH вредност и пљувачне концентрације уремијских токсина могу бити поуздани маркери бубрежног оштећења. Једноставан и неинвазиван приступ приликом узорковања пљувачке и поузданост лабораторијске анализе треба да допринесу широј примени пљувачке као компетитивним дијагностичким флуидом серуму. Техника оралног испирка је прецизна квантитативна метода за одређивање степена гљивичне колонизације.</p> / <p>Uvod: Hronična bubrežna insuficijencija (HBI) se definiše kao strukturno ili funkcionalno oštećenje bubrega u trajanju od najmanje tri meseca i/ili smanjenje jačine glomerularne filtracije (JGF) ispod 60 ml/min/1.73m2. U dostupnoj literaturi postoje različiti podaci o prisustvu oralnih manifestacija kod pacijenata sa HBI u kvantitativnom i kvalitativnom pogledu. Stanje bubrežne disfunkcije praćeno je promenama u protoku i sastavu pljuvačke što je u poslednjoj deceniji doprinelo ispitivanju kliničkih i laboratorijskih pokazatelja bubrežne bolesti. Cilj: Cilj studije je bio da se ispita objektivno stanje oralne sluzokože, vrednosti rN, sijalometrije, koncentracije uree, kreatinina i sekretornog imunoglobulina A pljuvačke kao i oralni mikrobiološki status kod pacijenata sa HBI. Materijal i metode: Uzorak je bio sačinjen od 50 predijaliznih (31 muškarca i 19 žena prosečne starosti 59,06&plusmn;14,30) i 25 hemodijaliznih pacijenata (18 muškaraca i 7 žena prosečne starosti 54,92&plusmn;13,60) sa postavljenom dijagnozom HBI, zajedno sa 25 sistemski zdravih ispitanika komparibilnih po polu i starosti. Pored kliničkog pregleda usne duplje sproveden je test vitroadhezije, određivanje intenziteta salivacije, rN vrednosti pljuvačke i indeksa krvarenja iz interdentalne papile (PBI). Na uzorcima sakupljene pljuvačke, uz pomoć automatizovanog sistema Beckman Coulter AU480 sprovedeno je laboratorijsko određivanje uree i kreatinina metodom spektrofotometrije i sekretornog imunoglobulina A metodom imunoturbidimetrije. Za mikrobiloško ispitivanje korišćen je bris jezika i tehnika oralnog ispirka. Rezultati: Nisu utvrđene statistički značajne razlike između grupa prema demografsko-socijalnim podacima. Predijalizni ispitanici su imali značajno veće prisustvo promena oralne sluzokože i oralnih simptoma. Prosečne vrednosti klirensa kreatinina su bile značajno manje kod obolelih ispitanika sa bledilom oralne sluzokože, uremičnim zadahom, kserostomijom i izmenjenim osećajem ukusa u poređenju sa ispitanicima bez navedenih promena. Kod predijaliznih su utvrđene značajno smanjene vrednosti sijalometrije prema kontrolnim grupama i povećane pH vrednosti prema grupi zdravih ispitanika. Prosečne koncentracije uree i kreatinina su se statistički značajno razlikovale između ispitivanih grupa. Umerena pozitivna korelacija je utvrđena između serumskih i pljuvačnih koncentracija uree i kreatinina kod predijaliznih i kreatinina kod hemodijaliznih. Prema prosečnim vrednostima sekretornog imunoglobulina A nije bilo razlika između grupa. Kod pacijenata sa HBI utvrđeno je značajno veće prisustvo gljiva iz roda Candida sa predominacijom non-albicans Candida vrsta. Zaključak: Rezultati istraživanja ukazuju na važnost utvrđivanja kliničkih karakteristika usne duplje kod predijaliznih pacijenata. Intenzitet salivacije, pH vrednost i pljuvačne koncentracije uremijskih toksina mogu biti pouzdani markeri bubrežnog oštećenja. Jednostavan i neinvazivan pristup prilikom uzorkovanja pljuvačke i pouzdanost laboratorijske analize treba da doprinesu široj primeni pljuvačke kao kompetitivnim dijagnostičkim fluidom serumu. Tehnika oralnog ispirka je precizna kvantitativna metoda za određivanje stepena gljivične kolonizacije.</p> / <p>Introduction: Chronic kidney disease (CKD) is defined as structural and functional kidney damage for a period of at least three months and/or reduction of glomerular filtration rate (GFR) under 60 ml/min/1.73m2. There are different data in the available literature in term of quantitative and qualitative presence of the oral manifestation in patients with CKD. Kidney dysfunction is accompanied by changes in the salivary flow and composition, which is in the last decade contributed by examination of clinical and laboratory markers of renal disease. Aim: The aim of the study was to examine condition of oral mucosa, pH value, salivary flow rate, concentration of salivary urea, creatinine, secretory immunoglobulin A and oral microbiological status in patients with CKD. Materials and Methods: The sample was consisted of 50 predialysis (31 males and 19 females, mean age 59,06&plusmn;14,30) and 25 hemodialysis patients (18 males and 7 females, mean age 54,92&plusmn;13,60) with a diagnosis of CKD, along with 25 age and gender matched healthy controls. In addition of clinical examination, tongue blade adhesion test, sialometry, salivary pH test and determination of papilla bleeding index (PBI) were conducted. Saliva samples were collected for laboratory analysis performed by automated system Beckman Coulter AU480. Levels of uremic toxins (urea and creatinine) and secretory immunoglobulin A were determinated by spectrophotometric and immunoturbidimetric method, respectively. Oral swab and oral rinse method were used for microbiological examination. Results: The sociodemographic characteristics of the patients with CKD and healthy controls showed no significant differences. Predialysis subjects had significantly higher presence of oral mucosa changes and oral symptoms. Mean values of creatinine clearence were significantly lower in patients with oral mucosa pallor, uremic fetor, xerostomia and disguesia, compared to patients without listed symptoms. Predialysis patients showed significantly decreased salivary flow rate compared to both control groups and significantly increased pH values compared to healthy controls. Mean concentrations of salivary urea and creatinine were statistically different between the groups. Moderate positive correlation was determined between serum and salivary levels of urea and creatinine in predialysis patients and creatinine in hemodialysis patients. Statistical analysis showed no differences between groups in mean concentration of secretory immunoglobulin A. The rate of oral fungal colonisation was significantly higher in CKD patients with predominance of non-albicans Candida species. Conclusion: The results of the present study indicate the importance of determining the clinical characteristics of oral cavity in predialysis patients. Saliva flow rate, pH value and salivary concentration of uremic toxins could be reliable markers of kidney disease. Simple and non-invasive approach due to saliva sampling and reliability of laboratory test should contribute to a wider application of saliva as a competitive diagnostic fluid. Oral rinse technique is an accurate quantitative method for determining the rate of fungal colonization.</p>

A asma é uma doença heterogênea caracterizada por um processo inflamatório crônico das vias aéreas inferiores que está associado ao desenvolvimento da hiperresponsividade brônquica e remodelamento da via aérea. Atualmente, a asma é considerada uma síndrome, ou ao menos uma doença com diversos fenótipos. Tradicionalmente, dois fenótipos são bem definidos pela clínica e exames subsidiários: asma alérgica e asma não alérgica. Eles são diferentes quanto á idade de início, apresentação clínica, história pessoal e familiar de atopia e resposta ao tratamento. Ao contrário da asma alérgica, cuja fisiopatologia está bem caracterizada, a etiologia e mecanismos envolvidos na asma não alérgica não estão bem elucidados. Algumas possibilidades incluem alergia desencadeada por antígenos desconhecidos (fungos), infecção persistente (Chlamydia trachomatis, Mycoplasma sp) e auto-imunidade. Estudos têm descrito em diferentes populações associações entre a asma e alelos/antígenos HLA classe I e II, mas os resultados têm sido inconclusivos. O objetivo deste estudo foi identificar possíveis associações do antígeno leucocitário humano (HLA) classe I (A, B, C) e II (DR, DQ, DP) em pacientes brasileiros com asma alérgica e não alérgica. Um total de 109 pacientes com o diagnóstico de asma (56 com asma alérgica e 53 com asma não alérgica) que estavam em acompanhamento no Serviço de Imunologia Clínica e Alergia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, e 297 controles (doadores falecidos de órgãos sólidos) tiveram seu sistema HLA classe I (A, B e C) e II (DR, DQ e DP) tipificado. Os pacientes também realizaram espirometria e coletaram sangue para a quantificação da imunoglobulina E (IgE) sérica total e nível sérico de eosinófilos. Além disso, foram avaliados quanto à IgE específica para aeroalergenos através do teste cutâneo de puntura e a pesquisa da IgE sérica específica (ImmunoCAP). O grupo com asma alérgica foi constituído por pacientes que apresentavam resultado positivo para a pesquisa da IgE específica em ambos teste cutâneo de puntura e na investigação in vitro. E o grupo com asma não alérgica apresentava resultados negativos nos dois testes. A comparação do HLA classe I nos grupos estudados identificou frequência significativamente maior do HLA-B*42 e HLA-C*17 no grupo com asma alérgica, enquanto o HLA-B*48 estava estatisticamente associado com o fenótipo não alérgico. Na análise do HLA classe II, o HLA-DPA1*03 e HLA-DPB1*105 apresentou associação com os pacientes com asma alérgica. Concluindo, o estudo observou diferentes associações dos alelos HLA classe I e II com asma alérgica e não alérgica na população brasileira, a qual é caracterizada pela diversidade de origens e miscigenação. Porém, a predisposição genética para asma é poligênica e novos estudos em grandes populações são necessários para confirmar a associação do HLA como fator protetor ou causador da doença / Asthma is a heterogeneous chronic inflammatory disease of lower airways associated with the development of bronchial hyperresponsiveness and airway remodeling. Currently, asthma is regarded as a syndrome or at least a disease with several phenotypes.Traditionally, two phenotypes of asthma have been defined according to clinical and laboratory features: allergic and non-allergic asthma. Each of them has distint age of onset, clinical presentation, personal and family history of allergy and response to therapy. In contrast to allergic asthma, which pathophysiology is well characterized, the etiology and mechanisms involved in non-allergic asthma remain unclear. Some possibilities include allergy triggered by unknow antigens (fungi), persistent infection (Chlamydia trachomatis, Mycoplasma sp) and autoimmunity. Studies have reported associations between asthma and HLA class I and II alleles/antigens in different populations, but the results have been inconclusive. The objective of this study was to identify possible associations of the human leukocyte antigens (HLA) class I (A, B and C) and II (DR, DQ and DP) in Brazilian patients with allergic and non-allergic asthma. A total of 109 patients with asthma (56 with allergic asthma and 53 with non-allergic asthma), who were being followed at the Service of Clinical Immunology and Allergy of the Hospital das Clínicas of the University of São Paulo Medical School, and 297 controls (deceased solid organ donors) had their HLA class I (A,B and C) and II (DR, DQ and DP) typing. Patients performed spirometry and had their blood drawn to measure total serum immunoglobulin E (IgE) levels and eosinophil count. Furthermore, they were assessed for specific IgE to aeroallergens with skin prick test and serum tests (ImmunoCAP). The allergic asthma group was composed of patient presenting positive results for specific IgE in both skin prick test and in vitro assay. And the non-allergic asthma group had negative results in both tests. There were significantly higher frequencies of HLA-B*42 and HLA-C*17 in the allergic asthma group, whereas the HLA-B*48 was associated with the non-allergic group. Regarding HLA class II analysis, HLA-DPA1*03 and HLA DPB1*105 were associated with allergic asthma patients. In conclusion, the study identified different associations of HLA class I and II with allergic and non-allergic asthma in the Brazilian population, which is characterized by diversity of origins and miscegenation. However, the genetic predisposition of asthma is polygenic and new studies on large populations are needed to confirm the role of HLA as a protective or predisposing factor of disease

In this thesis some multivariate spectroscopic methods for the analysis of solutions are proposed. Spectroscopy and multivariate data analysis form a powerful combination for obtaining both quantitative and qualitative information and it is shown how spectroscopic techniques in combination with chemometric data evaluation can be used to obtain rapid, simple and efficient analytical methods. These spectroscopic methods consisting of spectroscopic analysis, a high level of automation and chemometric data evaluation can lead to analytical methods with a high analytical capacity, and for these methods, the term high-capacity analysis (HCA) is suggested. It is further shown how chemometric evaluation of the multivariate data in chromatographic analyses decreases the need for baseline separation. The thesis is based on six papers and the chemometric tools used are experimental design, principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), partial least squares regression (PLS) and parallel factor analysis (PARAFAC). The analytical techniques utilised are scanning ultraviolet-visible (UV-Vis) spectroscopy, diode array detection (DAD) used in non-column chromatographic diode array UV spectroscopy, high-performance liquid chromatography with diode array detection (HPLC-DAD) and fluorescence spectroscopy. The methods proposed are exemplified in the analysis of pharmaceutical solutions and serum proteins. In Paper I a method is proposed for the determination of the content and identity of the active compound in pharmaceutical solutions by means of UV-Vis spectroscopy, orthogonal signal correction and multivariate calibration with PLS and SIMCA classification. Paper II proposes a new method for the rapid determination of pharmaceutical solutions by the use of non-column chromatographic diode array UV spectroscopy, i.e. a conventional HPLC-DAD system without any chromatographic column connected. In Paper III an investigation is made of the ability of a control sample, of known content and identity to diagnose and correct errors in multivariate predictions something that together with use of multivariate residuals can make it possible to use the same calibration model over time. In Paper IV a method is proposed for simultaneous determination of serum proteins with fluorescence spectroscopy and multivariate calibration. Paper V proposes a method for the determination of chromatographic peak purity by means of PCA of HPLC-DAD data. In Paper VI PARAFAC is applied for the decomposition of DAD data of some partially separated peaks into the pure chromatographic, spectral and concentration profiles.

Intense exercise is known to cause alterations in the psychophysiological status of an athlete. Monitoring the health and recovery of an athlete is imperative for the maintenance of performance and reduced fatigue and injury incidence. The physicality associated with select sports results in significant elevations and suppression of psychophysiological biomarkers that are often modulated by game-related impacts, intense training regimes and psychosocial aspects associated with the professional era. The aim of the studies outlined in this thesis were to determine the effectiveness of selected “stress” markers in several sports that result in significant “stress”, and quantify the level of acute and chronic “stress” following individual games and competitions to improve athlete management and recovery. Study one aimed at developing a new strong-cation exchange high performance liquid chromatography (SCX-HPLC) method for the detection and quantification of urinary pterins and creatinine in a body-building cohort completing high intensity resistance training. The method had an intra- and inter-assay variability of 3.04 % and 5.42 % respectively, with visibly clear peaks and no tailing. Urinary neopterin (NP) and 7,8-dihydroneopterin during a week of competitive natural body-building did not significantly change indicating no alteration in immune system function and oxidative stress. It did provide evidence for the use of specific gravity as a similarly reliable method for urine volume correction following exercise. Study two focused on a playoff game of elite amateur rugby. The time course changes of NP, cortisol, salivary immunoglobulin A (sIgA) and myoglobin in 11 elite amateur rugby players were measured up to 86 hours post-game. Cortisol increased 4-fold, myoglobin 2.85-fold, NP 1.75-fold and total NP 2.3-fold, all significant, whilst sIgA did not change. All markers returned to baseline within 17 hours providing valuable information for sample collection schedule optimization. Respiratory elastance was also measured by ventilation for the assessment of exercise induced lung inflammation/injury following the game (Chapter three). There was an increase in elastance in selected individuals that did not correlate with either global positioning system (GPS) or impact data. It was shown however, that a ventilator is capable of measuring respiratory changes in a conscious and healthy individual. Study three focused on the final three games of professional rugby in the 2013 ITM Cup. The acute and cumulative changes in the same four markers were analysed following three home games. There were significant increases in NP, total NP, cortisol and myoglobin along with significant suppression of sIgA (p < 0.05). Large intra- and inter-individual variation existed between players with changes associated with total impacts. Moreover, impact induced muscle damage may account for changes in oxidative status. Specific gravity (SG) was shown to be a more reliable marker for urine volume correction in comparison to creatinine; while some players showed signs of cumulative stress. Study four examined stress in a professional team throughout the 22 week 2014 Super 15 competition. Part one investigated changes in oxidative stress and muscle damage markers to solidify the muscle damage/oxidative status theory postulated in the previous study. Experimental evidence showed iron and myoglobin are separately capable of oxidizing 7,8-dihydroneopterin to NP in vitro. It was then identified that players who suffered the greatest muscle damage as a result of impacts also had the greatest change in oxidative status (NP). This evidence suggests rugby union induces significant alterations in oxidative status that may be exacerbated by the impact induced release of myoglobin. Part two measured urinary NT-proBNP during the last two consecutive home games to identify whether rugby union causes significant cardiovascular stress and if the pre to post-game change can be explained by GPS technology. Significant individualized elevations were observed in games one and two which did not correlate with any GPS measurements or impacts. Concentrations returned to normal ~ 36 hours post-game suggesting no permanent damage to cardiac muscle had occurred. The lack of correlation suggests GPS technology is not an accurate measure of cardiovascular stress in professional rugby union. Part three involved the measurement of cortisol, total NP and sIgA throughout the season to assess the degree of cumulative stress. Samples were taken at regular intervals ~ 36 hours post-game for 22 weeks. Extreme inter-individual variation was present. Select individuals showed continual elevation in immune system activation and psychophysiological stress, whilst others presented with a continual decline in immune system function. Collectively however, minor deviations from baseline in all markers were observed and participation in long distance travel did not significantly affect the psychophysiological status of the group. Together this suggests a season does not cause an accumulation in psychophysiological stress, although careful individual player analysis is warranted. Understanding rugby union positional demands is essential for training program specification and position specific development of players. Part four used GPS, video-analysis and biochemical analysis to identify positional demands in five regular season games. Forwards tended to be involved in more impacts and covered less distance, while backs covered more distance and carried the ball into contact more regularly. There was no difference in the psychophysiological status between positions indicating both aspects of stress (impacts and distance covered) may induce a similar response. Alternatively, individual biological variation may be solely responsible for this change suggesting careful consideration should be given when using traditional work-load measures such as GPS when quantifying “stress”. Part five assessed the effectiveness of varied recovery interventions. Total NP, cortisol, myoglobin and sIgA were measured pre- post- and ~ 36 hours post game to identify which intervention was most effective at returning players to a psychophysiological state that allowed for the resumption of normal training. Findings concluded the immediate post-game strategy employed by the team (cold bath, consumption of protein and carbohydrates, compression garments and eight hours sleep) seemed to provide the greatest psychophysiological improvement regardless of the “next-day” intervention. There was large inter-individual variation and players were still in a state of recovery ~ 36 hours post-game as indicated by the elevated total NP and sIgA concentrations. Study five had four aspects. Develop a new, cost-effective and simple reverse phase HPLC (RP-HPLC) method for the quantification of urinary myoglobin in a clinically relevant range, quantify the level of structural stress following a simulated mixed martial arts (MMA) contest, determine whether cold water immersion attenuates the level of inflammation and muscle damage following a contest, and whether this hypothesized attenuation may be explained by cryotherapy induced mononuclear cell activation suppression in vitro. The RP-HPLC method had an intra- and inter-assay variations from 0.32 - 2.94 %. Linearity was in the range of 5 – 1000 µg/mL which detected significant increases in urinary myoglobin following the MMA contest. Total NP was found to significantly increase following the contest and return to approximately pre-contest levels 24 hours later for the passive group only. Cold water immersion was further found to attenuate the total NP increase in the first two hours post-contest solidifying its use as a recovery technique following intense exercise, while cryotherapy significantly suppressed T-cell activation. This study provides a reliable and repeatable assay for muscle damage quantification in a clinically relevant range, evidence of the physicality associated with MMA, and indicates cold water immersion is a reliable recovery intervention that may impart its positive benefits through T-cell suppression. The data generated by these investigations highlights the necessity for individual physiological analysis. Group data often masks the extreme variation that exists in clinical and exercise trials where treatment and management of athletes is conducted for recovery and performance. Biochemical analysis provides an added sophistication of work-load and psychophysiological assessment that common technological methods cannot emulate. With a lack of correlation between the quantitative changes in specific non-overlapping biomarkers and GPS, video-analysis and questionnaires, it would seem pertinent to develop a non-invasive quantitative approach in elite sport to understand the level of exercise-induced psychophysiological stress for the precise management of athletes.