Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A hansenÃase continua sendo um problema de saÃde mundial, sendo o Brasil o segundo paÃs em maior em nÃmero de casos novos. No Cearà a doenÃa à considerada endÃmica e em 2009 foram diagnosticados 1.952 casos novos, alcanÃando um coeficiente de detecÃÃo geral de 22,8/100.00 habitantes. A doenÃa à causada pelo M. leprae, manifesta-se atravÃs de sinais e sintomas dermatoneurolÃgicos e à transmitida de pessoa a pessoa atravÃs do convÃvio de indivÃduos suscetÃveis com doentes bacilÃferos sem tratamento. A interaÃÃo do M. leprae com os subtipos de cÃlulas T produz citocinas do tipo Th1 e Th2, responsÃveis pelas diferentes formas clÃnicas da doenÃa. PadrÃes de citocinas Th1 (IL-2, IFN-&#947; e TNF-&#945;) foram encontrados em lesÃes de pele das formas tuberculÃides e padrÃes de citocinas Th2 (IL-4, IL-5 e IL-10) foram encontrados em lesÃes das formas virchovianas. A hansenÃase à influenciada por vÃrios fatores, sendo os genÃticos os mais estudados no momento. Os genes das citocinas aparecem como fortes candidatos capazes de influenciar a interaÃÃo patÃgeno- hospedeiro e favorecer ou nÃo o desenvolvimento da doenÃa. A IL-10 à uma citocina anti-inflamatÃria e imunomoduladora que possui regiÃes promotoras bastantes polimÃrficas, contendo regiÃes de microssatÃlites e SNPs que formam vÃrios haplÃtipos que estÃo associados a diferentes nÃveis de produÃÃo de citocina in vitro. VÃrios estudos tentam reportar associaÃÃes entre os polimorfismos de IL-10 e o risco ou proteÃÃo para diversas doenÃas. Contudo, os dados relatados tem sido contraditÃrios e a maioria das associaÃÃes entre os polimorfismos e a produÃÃo dessa citocina sÃo baseados em estudos in vitro. Dessa forma o presente estudo teve o objetivo de definir como os polimorfismos da regiÃo promotora da citocina afetam a produÃÃo in vivo frente à infecÃÃo pelo M. leprae. Foram quantificadas as concentraÃÃes sÃricas de IL-10 de 181 indivÃduos, sendo 77 casos Ãndices de hansenÃase, 74 indivÃduos contactantes e 30 controles saudÃveis. Sendo que destes, 31 possuÃam anÃlise genotÃpica de IL-10 no grupo caso, 33 no grupo contactante e 29 no grupo controle. Os pacientes com anÃlise genotÃpica foram estratificados em baixo, mÃdio e alto produtor da citocina. As diferenÃas nos nÃveis da citocina foram comparadas entre os grupos dentro do espectro da hansenÃase (paucibacilar e multibacilar), dos controles externos, dos controles internos, dos genÃtipos e alelos encontrados nos grupos. Foram realizados testes de Kruskall-Wallis e Mann-Whitney para anÃlise das medianas de IL-10 em pg/mL. NÃo foi encontrada diferenÃa significante entre os grupos caso, contactante e controle (p=0,7450), entre os indivÃduos pauci e multibacilar (p=0,7898), entre os fenÃtipos de nÃvel de produÃÃo de citocina (baixo, mÃdio e alto) (p=0,4355). Foi encontrada diferenÃa significante na produÃÃo de IL-10 entre os alelos -1082A,-819C e -592C do grupo caso em relaÃÃo aos controles (p<0,05) e dos alelos -819C e -592C do grupo caso em relaÃÃo aos contactantes (p<0,05). NÃo foi encontrada diferenÃa significante entre os grupos contactante e controle (p>0,05). Em conclusÃo, os genÃtipos de IL-10, -1082G>A, -819C>T e -592G>C nÃo influenciaram a produÃÃo e/ou o desfecho da infecÃÃo pelo M. leprae. Por outro lado, os alelos -1082A,-819C e -592C determinaram menor produÃÃo de IL-10 em indivÃduos com hansenÃase. / Leprosy is a world problem of health and Brazil has the second higher rates of new cases in the world. It is considered an endemic disease at Cearà and a total of 1,952 new cases were detected, reaching a detection rate of 22.8/100,000 inhabitants. The interaction between the M. leprae and different T cells stimulates the production of a Th1 or a Th2 pattern of cytokines, which are responsible for the different clinical forms of leprosy. Th1 cytokines (IL-2, IFN-&#947; and TNF-&#945;) were found in tuberculoid skin lesions while Th2 cytokines (IL-4, IL-5 and IL-10) were found in lepromatoid lesions. Leprosy is influenced by many distinct factors, among them, genetic factors are the most studied at this moment. Cytokine genes seem to influence the interaction between the pathogen and the host and contribute to the development or not of the disease. The IL-10 is an antiinflammatory and immunomodulatory cytokine which has too much polymorphic promoter regions with microsatellites and SNPs. Different haplotypes are associated to distinct levels of cytokine production in vitro. Many studies reported associations between IL-10 polymorphisms and the risk or the protection against many diseases. However, the data reported have been contradictory and most of the associations between these polymorphisms and the production of IL-10 are showed in in vitro studies. The aim of this study was to evaluate how the promoter region polymorphisms of IL-10 influence the cytokine levels production in vivo, during the M. leprae infection. Serum levels of IL-10 were analyzed by ELISA in 181 individuals, 77 of them were leprosy cases, 74 household contacts and 30 healthy controls. Among them, 31 had IL-10 polymorphism typed in the case group, 33 in household contact group and 29 in healthy controls. Cases with polymorphism typed were stratified in low, medium and high levels of cytokine production. Differences in the IL-10 production were compared among leprosy cases (pauci and multibacillary), household contacts, healthy controls, genotypes and alleles distribution found in the groups. Kruskall-Wallis and Mann-Whitney tests were used to analyze mean values of IL-10 levels. No differences were observed between cases, contacts and controls (p=0.7450), pauci and multibacillary (p=0.7898), phenotypes of IL-10 production (low, medium or high) (p=0.4355). A significant difference in the IL-10 levels between cases and controls was found associated to the alleles -1082A,-819C and -592C (p<0.05) and between cases and contacts associated to the alleles -819C and -592C (p<0.05). No differences were found between contacts and controls (p>0.05). In conclusion, the -1082G>A, -819C>T and -592G>C IL-10 genotypes did not influence the IL-10 production or the M. leprae infection outcome. On the other hand, -1082A,-819C e -592C alleles determined a lower production of IL-10 in cases of leprosy. Keywords: Leprosy; Polymorphisms; IL-10; Serum levels; Contacts

A interface formada por materiais biológicos e materiais sintéticos tem grande importância em aplicações biomédicas, tais como o desenvolvimento de biomateriais para implantes médicos, que tem como processo essencial a deposição de proteínas na superfície dos biomateriais, e ainda não é bem compreendido no nível molecular. Algumas proteínas sofrem mudanças conformacionais após a adsorção na interface sólido-líquido, afetando suas funções ou propriedades, e algumas técnicas podem medir mudanças conformacionais em interfaces sólido. É possível estudar a fluorescência intrínseca de proteínas: a posição do máximo na faixa espectral da fluorescência, a eficiência quântica e o tempo de vida de fluorescência são indicadores de mudanças no ambiente local de grupos de moléculas de proteína fluorescente. Por outro lado, Nanopartículas de ouro têm atraído muita atenção pela sua afinidade com materiais biológicos e suas propriedades ópticas. Nesta tese, estudamos a viabilidade de substratos de vidro, quartzo, mica e ITO (óxido de índio e estanho) modificado com quitosana, phtalocyanines (Ni, Fe e Ni) e poli(alilanina hidroclorada) (PAH) na adsorção de BSA em forma dos filmes produzidos pela técnica camada por camada. O sistema foi estudado por UV-Vis e espectroscopia de fluorescência estática e resolvida no tempo. A caracterização morfológica dos filmes foi realizada por microscopia de força atômica e microscopia óptica. Os resultados mostram que os filmes de BSA / HAP cresceram com eficiência quatro vezes maior do que os filmes feitos de quitosana, que o quartzo tem a melhor janela de trabalho de UV-vis e há uma relação entre o pH da BSA e o tempo vida de fluorescência do filme resultante. As nanopartículas de ouro foram produzidas pela redução química e estabilizada por quatro diferentes métodos. O crescimento das nanopartículas foi monitorado por UV-vis spectroscopy. A carga de superfície das nanopartículas e da BSA foi estimado em vários valores de pH por medidas de potencial zeta. Os resultados indicaram que as nanopartículas têm cargas negativas na faixa de pH estudada. Soluções de BSA foram preparadas em diferentes valores de pH, e levadas para interagir com as nanopartículas de ouro. Os dados de supressão de fluorescência da BSA mostraram uma maior afinidade da BSA com nanopartículas estabilizadas com sacarose, com pH próximo do ponto isoelétrico (IP) estimado para BSA. / The interface formed by biological materials and synthetic materials has great importance in biomedical applications such as the development of biomaterials for medical implants, which has as an essential process of protein adsorption on the surface of biomaterials, and is not yet well understood in the molecular level. Some proteins undergo conformational changes after adsorption at solid-liquid interfaces, affecting their functions or properties, and few techniques can measure conformational changes in solid interfaces. It is possible to study the intrinsic fluorescence of proteins: the position of the maximum in the spectral range of fluorescence, the quantum efficiency and lifetime of fluorescence are indicators of change in the local environment of fluorescent groups of protein molecules. On the other hand, gold nanopartículas have attracted much attention for its affinity with biological materials and their optical properties. In this thesis we study the feasibility of glass substrates, quartz, mica and ITO (Indium tin oxide) modified with chitosan, phtalocyanines (Ni, Fe and Ni) and poly (allylamine hydrochloride) (PAH) on the adsorption of BSA in the form of films produced by the layer by layer technique. The system was studied by UV-Vis and static and time-resolved fluorescence spectroscopy. Morphological characterization of the films was performed by atomic force microscopy and optical microscopy. The results indicate that the films of BSA/PAH grew with efficiency four times greater than the films made of chitosan, that the quartz has the best working window for UV-vis and there is a relationship between the pH of the BSA and lifetime of fluorescence of the resulting film. Gold nanoparticles were produced by chemical reduction and stabilized by four different methods. The growth of nanoparticles was monitored by UV-vis spectroscopy. The surface charge of nanoparticles and the BSA was estimated at various pH values by zeta potential measurements. The results indicated that the nanoparticles have negative charges in the pH range studied. BSA solutions were prepared at various pH values, were taken to interact with gold nanoparticles. Fluorescence quenching data of BSA showed a greater affinity of the BSA with nanoparticles stabilized with sucrose, at pH near the isoelectric point (IEP) estimated for BSA.

A fumonisina B1 (FB1) e uma micotoxina produzida pelo metabolismo secundário de espécies de Fusarium, principalmente F. verticillioides e F. proliferatum, os quais contaminam diversos alimentos antes e apos o processamento, sobretudo o milho e derivados, gerando graves problemas para a Saúde Pública e a qualidade dos alimentos. O objetivo deste trabalho foi avaliar a exposição humana a FB1 presente nos alimentos através da estimativa de ingestão da toxina na dieta e da análise de diferentes biomarcadores presentes em amostras de sangue, urina e cabelo. Além disso, foram investigados os efeitos da toxina através da avaliação de ácido fólico presentes em alimentos e em soro, e os níveis de uréia e creatinina presentes em soro. O estudo foi realizado em dois municípios dos Estados de São Paulo e Santa Catarina, cujos respectivos voluntários foram categorizados como de baixo consumo de derivados de milho (Grupo A, voluntários de Pirassununga/SP) e de alto consumo de derivados de milho (Grupo B, voluntários de Erval Velho/SC). As amostras de alimentos do Grupo A (Pirassununga/SP) foram fornecidas pelos voluntários (n=100) nos meses de Junho/2011, Setembro/2011, Dezembro/2011 e Marco/2012. Os voluntários do Grupo B (Erval Velho/SC) (n=20) forneceram amostras de alimentos no mês de Abril/2012. Em cada grupo, uma lista com 20 alimentos a base de milho foi entregue aos voluntários, para fornecimento de amostras daqueles disponíveis em suas respectivas residências em cada mês de amostragem, totalizando 122 amostras de derivados de milho no Grupo A e 17 amostras no Grupo B coletadas durante o estudo. Adicionalmente, aplicou-se um Questionário de Frequência Alimentar (QFA) e um Inquérito Recordatório de 24 horas (QIR - 24 h) no momento das coletas de amostras. Em cada mês de amostragem de alimentos, foram coletadas amostras de sangue, urina (somente Grupo A) e cabelo dos voluntários, sendo as amostras armazenadas a -20ºC (urina e cabelo) ou -80ºC (sangue) até o momento das análises. As amostras de alimentos foram submetidas a análise de FB1, sendo que as de farinha de milho foram também analisadas quanto ao teor de ácido fólico. Ambas as análises foram feitas através de cromatografia líquida de alta eficiência (CLAE). Em soro, foram avaliadas a relação esfinganina/esfingosina (Sa/So), resíduos de FB1, ácido fólico, uréia e creatinina. Em urina, foram analisados os níveis de FB1, creatinina para correção do volume urinário e a relação Sa/So. Em cabelo, foram analisados os resíduos de FB1 através de CLAE acoplada a espectrometria de massas. Todos os métodos de análise foram submetidos a procedimento de otimização e validação intra--laboratorial. A incidência de FB1 nos alimentos foi, em média, 72% (n=122) nas amostras do Grupo A (Pirassununga/SP) e 35% (n=17) no Grupo B (Erval Velho/SC). Os maiores níveis foram encontrados em amostras de pipoca provenientes do Grupo B, com uma amostra excedendo o limite de tolerância estabelecido no Brasil (2,500 &micro;g kg-1). A ingestão diária provável média (IDPM) de FB1 no Grupo A foi de 63,3 ng kg-1 peso corpóreo (p.c.) dia-1, que corresponde a 3,1% da ingestão provisória máxima tolerável (IPMT) recomendada para fumonisinas (2.000 ng kg-1 p.c. dia-1). A IDPM do Grupo B apresentou uma média de 190,1 ng kg-1 p.c. dia-1 o que corresponde a 9,5% da IDMT. As concentrações de ácido fólico nas amostras de farinha de milho variaram de < 0,3 &micro;g kg-1 (limite de quantificação do método) a 1.705 &micro;g kg-1, com média de 713 &plusmn; 435 &micro;g kg-1. Somente uma amostra apresentou nível de ácido fólico acima do valor mínimo estabelecido pela ANVISA. Em urina, a incidência de FB1 foi de 33,4% (n=251), com níveis médios de 3,19 &plusmn; 3,15 ng mg-1 de creatinina. Não houve correlação (P&gt;0,05) entre as concentrações de FB1 na urina e nos alimentos. Os níveis de esfinganina foram mais elevados em mulheres, com 25,0% (n=116) de amostras positivas, em comparação à urina de homens, 10,4% (n=96). A relação Sa/So apresentou em média 0,91, 0,77 e 0,89 para urina de mulheres, homens e em combinação, respectivamente. Em soro, os níveis de esfingosina foram em média 2,48 ng mL-1 para o Grupo A e 5,01 ng mL-1 para o Grupo B. A relação Sa/So variou de 0,06 a 3,19 com média de 0,79 para o Grupo A e 0,78 para o Grupo B. Embora tenha havido correlação positiva (r=0,574, P&lt;0,05) entre a relação Sa/So no soro e os dados de consumo de milho e derivados obtidos no QIR-24 h, não foram observadas correlações (P&gt;0,05) entre a ingestão de FB1 e a relação Sa/So na urina ou soro. A concentração de ácido fólico no soro variou de 6,7 a 24,0 ng mL-1 (média de 13,4 &plusmn; 5,4 ng mL-1), com ambos os grupos (A e B) apresentando resultados dentro dos valores de referências. Não foram observados níveis detectáveis de FB1 nas amostras de soro. No entanto, FB1 foi detectada em 4 amostras de cabelo humano (7,2%) dos Grupos A e B, cuja concentração média foi de 21,3 &plusmn; 12,1 ng g-1. Em síntese, os resultados obtidos nas análises de biomarcadores de FB1 no presente trabalho estão de acordo com os valores de IDPM encontrados, indicando que a exposição a FB1 nas populações estudadas não representa um risco a saúde. / Fumonisin B1 (FB1) is a mycotoxin produced by the secondary metabolism of Fusarium species, mainly F. verticillioides and F. proliferatum, which contaminates foods before and after processing and causes serious problems to public health and food quality. The aim of this study was to evaluate the human exposure to FB1 in food by means of estimated intake of toxin in the diet, and analysis of different biomarkers in serum, urine and hair. In addition, folic acid in food and blood as well urea and creatinin in serum were investigated to evaluate the toxin effects. The study was conducted in two cities of Sao Paulo and Santa Catarina States, where the respective volunteers were categorized as low-consumers of corn products (Group A, volunteers from Pirassununga/SP) and high-consumers of corn products (Group B, volunteers from Erval Velho/SC). Food samples from Group A (Pirassununga/SP) were provided by volunteers (n=100) in June/2011, September/2011, December/2011 and March/2012. The volunteers from Group B (Erval Velho/SC) (n=20) provided food samples in April/2012. In each group, a list of 20 corn products was given to volunteers, to allow them to check and collect the food items available in their homes at each sampling time. The total number of samples of corn products provided by the volunteers were 122 and 17 in Group A and Group B, respectively. Addicionally, a Food Frequency Questionnaire (FFQ) and a 24-Hours Dietary Recall Questionnaire (24h-DRQ) were applied by the time of sample collections. In each month of food samples collection, samples of blood, urine (only Group A) and hair from the volunteers were collected and storage at -20ºC (urine and hair) or -80ºC (blood) until analysis. Food samples were submitted to determination of FB1, and corn meal samples were also evaluated for folic acid levels. Both analysis were performed by high performance liquid chromatography (HPLC). In serum, analyses included sphinganine/sphingosine ratio (Sa/So), FB1 residue, folic acid, urea and creatinine. In urine, the levels of FB1, creatinine to correct urinary volume and Sa/So ratio were evaluated. In hair, FB1 residues were analysed by HPLC coupled to mass spectrometry. All the analytical methods were submitted to optimization and intra-laboratorial validation procedures. The mean incidences of FB1 in corn products were 72% (n=122) in samples of Group A (Pirassununga/SP), and 35% (n=17) of Group B (Erval Velho/SC). The higher levels were found in popcorn from Group B, with one sample exceeding the tolerance limit established in Brazil (2,500 &micro;g kg-1). The mean probable daily intake (PDIM) of FB1 in Group A was 63.3 ng kg-1 body weigh (b.w.) day-1, which corresponds to 3.1% of provisional maximum tolerable intake (PMTDI) recommended for fumonisins (2,000 ng kg-1 b.w. day-1). PDIM of Group B was 190.1 ng kg-1 b.w. day-1, which represents 9.5% of PMTDI. Folic acid levels in corn meal ranged from &LT; 0,3 &micro;g kg-1 (quantification limit) to 1.705 &micro;g kg-1, with a mean of 713 &plusmn; 435 &micro;g kg-1. Only one sample had levels of folic acid above the minimum established by ANVISA. In urine, the incidence of FB1 was 33,4% (n=251), at mean levels of 3,19 &plusmn; 3,15 ng mg-1 of creatinine. There wasn\'t correlation (P&gt;0.05) between concentrations of FB1 in urine and foods. Sphinganine levels were higher in woman, with 25.0% (n=116) of positive samples in comparison to urine of men, 10.4% (n=96). The mean Sa/So ratios were 0.91, 0.77 and 0.89 for urine of women, men and in combination, respectively. In serum, sphingosine presented a mean of 2.48 ng mL-1 to Group A and 5.01 ng mL-1 to Group B. Sa/So ratio ranged from 0.06 to 3.19 with a mean of 0.79 to Group A and 0.78 to Group B. Although a positive correlation (r=0.574, P&lt;0.05) was found between Sa/So ratio in serum and corn consumption data obtained by 24h-DRQ, no correlation was observed (P&gt;0,05) with FB1 intake and Sa/So ratio in urine or serum. Folic acid concentration in serum ranged from 6.7 to 24.0 ng mL-1 (mean of 13.4 &plusmn; 5.4 ng mL-1), with both groups (A and B) presenting levels within the reference valuies. There were no detectable levels of FB1 in serum samples. However, FB1 was detected in 4 human hair samples (7.2%) of Groups A and B, at a mean concentration was 21.3 &plusmn; 12.1 ng g-1. In summary, the results obtained in the analyses of FB1 biomarkers in the present study are in agreement with the PDIM values found, hence indicating that FB1 exposure in the populations studied do not represent a health concern.

Despite the recognized advances in the treatment of breast cancer, it still accounts for 15% of all cancer-related deaths. 90% of breast cancer deaths are due to unpredicted metastasis. There is neither successful treatment for metastatic patients nor a specific test to predict or detect secondary lesions. Patients with primary tumor will be either over-treated with cytotoxic side effects or under-treated and risk recurrence. This necessitates the need for personalized treatment, which is hard to offer for such heterogeneous disease. Obstacles in treating breast cancer metastasis are mainly due to the gaps exist in the understanding of the molecular mechanism of metastasis. The linear model of metastasis is supported by several observations that reflect an early crosstalk between the primary and secondary tumor, which in turn makes the secondary microenvironment fertile for the growth of disseminated cells. This communication occurs through circulation and utilizes molecules which have not been identified to date. Identifying such molecules may help in detecting initial stages of tumor colonization and predict the target organ of metastasis. Furthermore, these molecules may help to provide a personalized therapy that aims to tailor treatment according to the biology of the individual tumor. Advances in proteomics allows for more reproducible and sensitive biomarker discovery. Proteomic biomarkers are often more translatable to the clinic compared to biomarkers identified using other omics approaches. Further, protein biomarkers can be found in biological fluids making them a non-invasive way to treat or investigate cancer patients. We present in this manuscript our study of the use of a proteomic approach on blood serum samples of metastatic and non-metastatic patients using LC-MS/MS quantitative analysis machine to identify molecules that could be associated with different stages of breast cancer metastasis. We focused on the deferential expression of low molecular weight biomolecules known to reflect disease-specific signatures. We manually analyzed 2500 individual small biomolecules in each serum sample of total of 51 samples. Comparisons between different sample types (from stage I and III Breast Cancer patients in this case) allows for the detection of unique short peptide biomarkers present in one sample type. We built a multi-biomarker model with more sensitivity and specificity to identify the stage of the tumor and applied them on blinded set of samples to validate prediction power. We hope that our study will provide insights for future work on the collection, analysis, and understanding of role of molecules in metastatic breast cancer.

Abstract Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. In some CRC patients, the presence of the tumor elicits a systemic inflammatory response and metabolic derangements that lead to progressive tissue wasting. Systemic inflammation has been associated with decreased survival independent of tumor stage. However, the mechanisms and downstream effects of systemic inflammation in CRC are uncertain. The aim of these studies was to examine the determinants of systemic inflammation in CRC. The study material consisted of tumor and serum samples collected from patients with stage I–IV CRC operated at the Oulu University Hospital (n=336). From preoperative serum samples, the levels of cell death marker keratin 18, matrix metalloproteinase 8 (MMP8), and ten metabolites (apolipoprotein A1 and nine amino acids) were measured. CRC patients with systemic inflammation, assessed using a modified Glasgow Prognostic Score, had elevated serum levels of MMP8 and phenylalanine. On the contrary, the concentrations of apolipoprotein A1, glutamine, and histidine were lower compared to patients without systemic inflammation. Increased serum keratin 18 level associated with systemic inflammation in patients with metastatic disease. Elevated keratin 18 and MMP8 levels and decreased apolipoprotein A1 level were independent predictors of worse survival. These studies describe biomarkers of systemic inflammation that provide insight into the mechanisms of systemic inflammation, have potential prognostic value in CRC, and are possible therapeutic targets. The results suggest that cell death and systemic inflammation are strongly connected in CRC, but the potential mechanistic link between them and tissues involved remain to be elucidated. / Tiivistelmä Paksusuolisyöpä on kolmanneksi yleisin syöpä ja toiseksi yleisin syöpäkuoleman aiheuttaja Suomessa. Osalla potilaista syöpään liittyy systeemisen tulehdusreaktion aktivoituminen ja aineenvaihduntahäiriö, joka johtaa yleiseen näivettymiseen. Systeemisen tulehduksen on havaittu olevan yhteydessä huonoon ennusteeseen riippumatta kasvaimen levinneisyydestä. Systeemisen tulehduksen aktivaatiomekanismit ja vaikutukset paksusuolisyövässä ovat kuitenkin huonosti tunnettuja. Tutkimuksen tarkoituksena oli selvittää systeemistä tulehdusta määrittäviä tekijöitä paksusuolisyövässä. Aineisto koostui Oulun yliopistollisessa sairaalassa leikattujen paksusuolisyöpäpotilaiden (syövän levinneisyysaste I–IV) kasvain- ja seeruminäytteistä (n=336). Seeruminäytteistä mitattiin solukuolemaa osoittavan keratiini 18:n pitoisuudet, matriksin metalloproteinaasi 8 (MMP8):n määrät sekä 10 metaboliitin (apolipoproteiini A1 ja 9 aminohappoa) tasot. Paksusuolisyöpäpotilailla, joilla systeemistä tulehdusta osoittava mGPS-indeksi oli korkea, seerumin MMP8- ja fenyylialaniinitasot olivat koholla. Sen sijaan apolipoproteiini A1:n, glutamiinin ja histidiinin pitoisuudet olivat matalammat verrattuna potilaisiin, joilla ei ollut systeemistä tulehdusta. Kohonnut keratiini 18 -pitoisuus oli yhteydessä systeemiseen tulehdukseen etäpesäkkeistä tautia sairastavilla potilailla. Kohonnut keratiini 18- ja MMP8-taso sekä matala apolipoproteiini A1-pitoisuus liittyivät potilaiden huonoon ennusteeseen. Tutkimuksessa löydettiin systeemisen tulehduksen merkkiaineita, jotka tuovat hyödyllistä tietoa systeemisen tulehduksen mekanismeista ja potilaiden ennusteesta paksusuolisyövässä, ja ovat mahdollisia terapeuttisia kohteita. Tulosten perusteella paksusuolisyövässä solukuolema liittyy vahvasti systeemiseen tulehdukseen, mutta lisätutkimuksia tarvitaan selvittämään näiden tapahtumien mahdolliset syy-seuraussuhteet sekä tapahtumaan liittyvät kudokset.

Rising temperatures all over the world has correlated with more frequent heat stroke related injuries and death. This statistic not only applies to humans, but to canines as well, who have similar body temperature thresholds. Recent studies have demonstrated that serum histones, released after cell death from heat stroke, play a role in heat stroke related injuries and death. This proposal aims to determine the severity of the effects caused by serum histone release after heat stroke by exposing selected canine cell lines to cell lysate and purified histones H2A, H2B, H3, and H4, which have been found to be associated with heat stroke injuries. Effectiveness of the histones will be determined by measuring the levels of apoptosis, NETosis, and necrosis in the cells, as well as the expression levels of heat shock proteins. Further research will also be done to determine whether toll-like receptors present on the cell surface are responsible for the mechanism utilized by serum histones to damage tissue in the body.

Diagnosing disease in equine neonates poses a challenge for the equine industry because of the nonspecific manifestations of many diseases and the rapid deterioration that occurs. The differential diagnostic procedure requires many laboratory tests, whose results take days to receive. Serum amyloid A (SAA) is the only major acute phase protein identified in the horse; it exists in low levels in the healthy horse and increases over 100 fold in response to inflammatory stimulus 6-8 hours post stimulus. A point of care test allows veterinarians to obtain a SAA concentration within minutes that indicates the existence of infection. Being able to test and quantify this protein at the onset of illness may reduce the time before treatment is initiated and therefore increase the chance of survival for the equine neonate, which would greatly help a large problem in the industry.

Thesis (M.Sc. (Chemical Pathology)) --University of Limpopo, 2013 / Objectives The present study investigated how the serum lipids responded to a high-fat meal in black South African subjects with different APOE genotypes, a population that until recently was reported to be consuming a traditional diet of low fat and high carbohydrates. Methods Sixty students (males and females) of the University of Limpopo, Turfloop Campus were successfully genotyped using Restriction Fragment Length Polymorphism (RFLP) and grouped into four APOE genotype groups; ε2, ε2/ε4, ε3 and ε4. Only thirty-three subjects volunteered to participate in the oral fat-tolerance test (OFTT), but two were excluded for having abnormal total cholesterol (6.05 mmol/l) and LDL cholesterol (3.12 mmol/l) so only 31 subjects were left. The numbers per group were ε2=5, ε2/ε4=8, ε3=9 and ε4=9. After an overnight fast blood was drawn for measurements of baseline serum parameters. Subjects were administered a high fat meal 30 minutes after the baseline blood sample was drawn. Blood was drawn at intervals of 20, 40, 60, 120, 180, 240, 300 and 360 minutes for measurements of postprandial serum parameter levels. Serum parameters measured were triglyceride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, glucose and insulin. Results Mean levels of serum lipids at baseline in mmol/l were as follows; group 1[TG=0.69(0.55-0.81), TCHOL=3.10±0.29, HDL-C=1.12±0.32, LDLC= 1.67±0.28]; group 2 [TG=0.61(0.53-1.00), TCHOL=2.98±0.53, HDLC= 1.20±0.37, LDL-C=1.43±0.37]; group 3 [TG=0.67(0.28-0.86), TCHOL=2.96±0.54, HDL-C=1.22±0.30, LDL-C=1.46±0.47]; group 4 [TG=0.76(0.51-1.16), TCHOL=3.27±0.51, HDL-C=1.12±0.17, LDLC= 1.79±0.47]. There was no significant difference in the mean levels of baseline triglyceride, total cholesterol, low density lipoprotein cholesterol, and ix high density lipoprotein cholesterol between the APOE groups hence no significant difference in the response to a fatty meal. Conclusions There was no significant change in serum lipid concentrations after a fatty meal in individuals with different APOE genotypes in a population that consume a traditional diet of low fat and high carbohydrates. Due to the small sample size, the results should be interpreted with caution. A larger study is recommended to ascertain the role of APOE genotypes on serum lipid response to a fatty meal in Black South African population.

Thesis (M.Sc. (Chemical Pathology)) --University of Limpopo, 2013 / Objectives: The present study investigated how the serum lipids responded to a high-fat meal in black South African subjects with different APOE genotypes, a population that until recently was reported to be consuming a traditional diet of low fat and high carbohydrates. Methods: Sixty students (males and females) of the University of Limpopo, Turfloop Campus were successfully genotyped using Restriction Fragment Length Polymorphism (RFLP) and grouped into four APOE genotype groups; ε2, ε2/ε4, ε3 and ε4. Only thirty-three subjects volunteered to participate in the oral fat-tolerance test (OFTT), but two were excluded for having abnormal total cholesterol (6.05 mmol/l) and LDL cholesterol (3.12 mmol/l) so only 31 subjects were left. The numbers per group were ε2=5, ε2/ε4=8, ε3=9 and ε4=9. After an overnight fast blood was drawn for measurements of baseline serum parameters. Subjects were administered a high fat meal 30 minutes after the baseline blood sample was drawn. Blood was drawn at intervals of 20, 40, 60, 120, 180, 240, 300 and 360 minutes for measurements of postprandial serum parameter levels. Serum parameters measured were triglyceride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, glucose and insulin. Results Mean levels of serum lipids at baseline in mmol/l were as follows; group 1[TG=0.69(0.55-0.81), TCHOL=3.10±0.29, HDL-C=1.12±0.32, LDLC= 1.67±0.28]; group 2 [TG=0.61(0.53-1.00), TCHOL=2.98±0.53, HDLC= 1.20±0.37, LDL-C=1.43±0.37]; group 3 [TG=0.67(0.28-0.86), TCHOL=2.96±0.54, HDL-C=1.22±0.30, LDL-C=1.46±0.47]; group 4 [TG=0.76(0.51-1.16), TCHOL=3.27±0.51, HDL-C=1.12±0.17, LDLC= 1.79±0.47]. There was no significant difference in the mean levels of baseline triglyceride, total cholesterol, low density lipoprotein cholesterol, and high density lipoprotein cholesterol between the APOE groups hence no significant difference in the response to a fatty meal. Conclusions There was no significant change in serum lipid concentrations after a fatty meal in individuals with different APOE genotypes in a population that consume a traditional diet of low fat and high carbohydrates. Due to the small sample size, the results should be interpreted with caution. A larger study is recommended to ascertain the role of APOE genotypes on serum lipid response to a fatty meal in Black South African population.

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors.