Rozprawy doktorskie na temat „Potential biomaker”

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Mestrado em Biomedicina Molecular
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques (senile plaques) and intracellular neurofibrillary tangles formed by hyperphosphorylated TAU protein. TAU protein when hyperphosphorylated loses the ability to bind to microtubules and can be released into peripheral fluids. This process leads to neuronal degradation and neuronal death. Phosphorylation at threonine 231 has been shown to be specific for AD and to precede assembly of paired helical filaments in the human brain. In order to understand more about this residue we analysed SH-SY5Y cells undifferentiated and in differentiated cells induced by retinoic acid (RA). Treatment with RA increased expression of TAU phosphorylated at Thr231 (TAUpThr231) as determined by Western blot analysis. We further explored TAU phosphorylation by immunocytochemistry and noticed that in undifferentiated SH-SY5Y cells, TAUpThr231 was located mainly in the nucleus. In contrast, TAU and TAUpThr231 was redistributed to the neurites and in the soma of SH-SY5Y cells, which were induced to differentiate by retinoic acid (RA). In order to evaluate the potential of TAUpThr231 as a biomarker, we measured TAUpThr231 in CSF by a sandwich enzyme immunoassay and observed that the ratio of TAUpThr231/TAU levels discriminated significantly the AD group for the non-AD group. These findings indicate that TAUpThr231/t-TAU ratio levels may be a valuable marker for the clinical diagnosis of AD, irrespective of age and gender.
A doença de Alzheimer (AD) é uma doença neurodegenerativa progressiva caracterizada pela presença de placas de amilóide extracelulares (placas senis) e tranças neurofibrilhares intracelulares formadas pela proteína TAU hiperfosforilada. A proteína TAU quando hiperfosforilada perde a capacidade de se ligar a microtúbulos e pode ser libertada para fluidos periféricos. Este processo leva à degradação neuronal e à morte neuronal. A fosforilação na treonina 231 tem-se demonstrado ser específica para a AD e preceder a formação de filamentos helicoidais emparelhados no cérebro humano. Para melhor perceber a função deste resíduo e a contribuição para a localização da TAU, analisámos células SH-SY5Y indiferenciadas e células diferenciadas, pela adição de ácido retinoico (RA). O tratamento com RA aumentou a expressão de TAU fosforilada na Thr231 (TAUpThr231) conforme determinado por Western blot. Explorámos ainda a fosforilação da TAU por imunocitoquímica e percebemos que em células SH-SY5Y indiferenciadas, a phosphoTAU231 estava localizada principalmente no núcleo. Em contraste, TAU e phosphoTAU231 foram redistribuídas para as dendrites e citosol das células SH-SY5Y diferenciadas pelo ácido retinoico (RA). Para avaliar o potencial deste resíduo como biomarcador; medimos TAUpThr231 em CSF por meio de um imunoensaio enzimático em sanduíche e observámos que a proporção de níveis de TAUpThr231 / TAU discriminou de forma significativa o grupo AD do grupo não-AD. Essas descobertas podem indicar que os níveis da relação TAUpThr231 / t-TAU podem ser um marcador valioso para o diagnóstico clínico de AD, independentemente da idade e do género.

Mestrado em Biomedicina Molecular
DNA methylation is the major studied epigenetic mechanism and consists in the addiction of a methyl group to 5’ carbon position of the cytosine ring. Methylation of promoter gene regions are directly correlated with gene silencing, which can allow the development of certain diseases since relevant genes may be inhibited due to its promoter methylation; being that the opposite can also occur. This epigenetic mechanism has been linked to cancer and recently it is also being pointed to have an important role in neurological disorders, such as Alzheimer’s Disease (AD). Furthermore, aging is the major risk factor for AD, and also the process underlying many of the epigenetic alterations. As AD etiology and pathological development is not complete understood, alterations in epigenetic mechanisms like DNA methylation may underlie the basis of gene expression alterations. Hence, in this study, possible AD patients and age- and sex-matched controls were used. Genomic DNA was extracted from blood samples and the global methylation levels were determined using an ELISA-type assay, The possible AD patients exhibit lower methylation levels (0,75%±0,29) than age- and sex-matched controls (0,86%±0,29). Gene specific methylation profiles of AD related genes, namely APP and ApoE, was carried out using Combine Bisulfite Restriction Analysis (COBRA), Methylation Sensitive-High Resolution Melting (MS-HRM) and Bisulfite Direct Sequencing. The APP promoter region did not reveal any methylation by COBRA and MS-HRM. For the ApoE promoter region evaluated no differences in the methylation levels could be detected between patients and controls using these two techniques. However, by sequencing analysis of this ApoE promoter region, differences arise in specific CpG sites, indicating a tendency for increased methylation of CpG1, CpG3 and CpG 5 and a decreased methylation of CpG6 in AD patients when compared with age- and sex-matched controls. In this study AD patients exhibited a tendency for lower global methylation levels when compared to control individuals and the ApoE promoter region exhibited a tendency for a pattern of methylation that maybe related to AD pathology.
A metilação de DNA é, de todos os mecanismos epigenéticos, o mais estudado e consiste na adição de um grupo metil ao carbono da posição 5 do anel de uma citosina. A metilação da região promotora de genes está diretamente relacionada com silenciamento genético, podendo conduzir ao desenvolvimento de determinadas patologias, uma vez que genes importantes não são expressos devido a este mecanismo. Este mecanismo epigenético tem vindo a ser estudado a nível do cancro e, recentemente, tem adquirido alguma importância ao nível de doenças neurológicas, como a doença de Alzheimer (DA). O envelhecimento é o maior factor de risco para a DA e ao mesmo tempo está na base de muitas das alterações nos mecanismos epigenéticos. Como a patologia e etiologia da DA ainda não é completamente conhecida, alterações ao nível de mecanismos epigenéticos, como a metilação de DNA, podem ajudar a entender as alterações de expressão de genes nesta doença. Neste estudo, utilizaram-se amostras de pacientes com possível DA e controlos com idades semelhantes. O DNA genómico foi extraído de amostras de sangue e os níveis de metilação global foram avaliados por um ensaio de ELISA. Os pacientes apresentaram uma tendência para uma diminuição da metilação global (0,75%±0,29) relativamente aos controlos (0,86%±0,29). Os perfis de metilação dos genes envolvidos na DA, APP e ApoE, foram obtidos por Combine Bisulfite Restriction Analysis (COBRA), Methylation Sensitive - High Resolution Melting (MS-HRM) e Bisulfite Direct Sequencing (BDS). Para a região promotora do gene ApoE, não foram encontradas diferenças entre pacientes e controlos assim como a região promotora do gene APP não apresentou metilação através do método COBRA e MS-HRM. De igual modo, a região promotora do gene ApoE também não apresentou diferenças usando estas 2 técnicas. No entanto, a BDS evidenciou diferenças em locais CpGs específicos, mostrando que existe uma tendência para o aumento da metilação nos CpGs 1, 3 e 5; e para diminuição da metilação no CpG 6 dos pacientes relativamente aos indivíduos control. Este estudo permitiu verificar uma tendência para de diminuição da metilação global dos pacientes com possível DA relativamente aos controlos e também a existência de um possível padrão de metilação da região promotora do gene ApoE, que pode estar relacionado com a patologia da DA .

Prostate cancer is a leading cause of cancer-related death in men, and while many men diagnosed with the disease will have an indolent clinical course, 20-25% of men will experience disease recurrence which is invariably lethal. There is an urgent need for prognostic biomarkers that will predict disease recurrence and risk-stratify patients upon diagnosis, allowing for personalised therapies. This thesis attempts to identify new prognostic biomarkers for prostate cancer and investigates their patterns of protein expression in human primary prostate tumour tissue. Cancer stem cells are cancer cells thought to be uniquely capable of self-renewal and tumorigenicity, and may have a role in tumour recurrence. Using a literature searching approach, potential biomarkers related to stem cells, cancer stem cells or recurrence in prostate cancer were identified, and ALDH7A1, BMI1, SDC1, MUC1-C, Nestin and ZSCAN4 were chosen for investigation. An in silico approach was also used for biomarker identification, with RS1 and SLC31A1 selected as their mRNA was found to be upregulated in recurrent tumours. The expression patterns of all 7 potential biomarkers were examined by immunohistochemistry on prostate tumour tissue and benign tissue from prostate biopsies and prostatectomies. BMI1, ALDH7A1, MUC1-C and Nestin showed no relationship to recurrence or other clinical features. RS1 protein levels increased in patients with recurrence within 5 years, negatively correlated with AR expression, and a meta-analysis showed that the RS1 gene was amplified in up to 32% of castration-resistant prostate tumours. ZSCAN4 was heterogeneously expressed in a subset of 26% of prostate tumours with unclear characteristics and was not expressed in benign tissue, but was not associated with recurrence. Finally, SDC1 expression was lost in tumour epithelium, but a population of unidentified SDC1-expressing cells were found in the stroma of a third of tumours, and an increased burden of these cells was associated with primary Gleason pattern 5 tumours. These cells do not overlap with common epithelial, mesenchymal or stromal lineages, but may be migratory. In summary, the data presented in this thesis identifies 3 potential new biomarkers for prostate cancer, and provides the basis for future characterisation of their wider roles in the disease.

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed July 9, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Abstract   The prostate gland, a male organ, situated right under the urine bladder, is involved in male reproduction. It can also be the place for more or less serious diseases such as inflammation, abnormal growth and cancer. Especially prostate cancer is very common in the Western world. Today PSA is the most widely used marker for detection of prostate cancer. Unfortunately, this method is not specific enough. Therefore, there is a need for a better marker for screening of malignant prostate cancer. The marker should be specific both for the organ prostate and for the cancer disease. One promising marker is the prostasome, a small vesicle emanating from epithelial cells in the ejaculatory ducts in the prostate. The aim of this project was to set up an ELISA and test a number of antibodies for their ability to work as suitable capture or detection antibodies. As blocking agent different concentrations of BSA were tested. Biotin-Streptavidin conjugate was used in the detection step. Two surface proteins, PSCA and PSMA were used as capture antigens; they are specific for prostasomes. Clusterin, a prostasomal surface-bound protein, was used as antigen for the secondary antibody in the assay. With this experimental setup the detection limit was 2500ng/mL, which is probably not enough to detect prostasomes in cancer. The development of the ELISA did not reach its final stage, a ready-to-use assay, during this project. We have not yet the knowledge of optimal antibody concentrations and the other test parameters are also at experimental state.

Mestrado em Bioquímica - Métodos Biomoleculares
A superfamília das desidrogenases dos aldeídos (ALDH) é constituída por 19 enzimas cuja principal função é a proteção do organismo contra aldeídos tóxicos. As ALDHs têm sido associadas ao desenvolvimento de múltiplas doenças. As síndromes mielodisplásicas (SMD) são caracterizadas por hematopoiese ineficaz associada a citopenias no sangue periférico e elevada predisposição para transformação leucémica. A leucemia mieloide aguda (LMA) é caracterizada por crescimento anómalo de células mieloides imaturas no sangue e na medula óssea. A fisiopatologia das SMDs e LMAs é um processo complexo de múltiplas etapas que envolve alterações genéticas e epigenéticas numa ampla variedade de genes associados à diferenciação, proliferação, auto-renovação e apoptose celulares. Uma vez que as ALDHs estão envolvidas em alguns destes processos biológicos, a desregulação destas enzimas pode influenciar o desenvolvimento de SMD e LMA. O objetivo deste estudo é avaliar a expressão génica das ALDHs em doentes com SMD e LMA de modo a verificar seu potencial como biomarcadores de diagnóstico e/ou prognóstico destas doenças. Neste contexto, analisou-se a expressão da ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH7A1, ALDH16A1 e ALDH18A1 por PCR de transcriptase reversa. Os genes diferencialmente expressos (ALDH3A2, ALDH3B1, ALDH4A1 e ALDH18A1) foram quantificados, por PCR em tempo real, em 54 doentes, 34 com SMD e 20 com LMA, e em 34 controlos saudáveis. A análise estatística foi realizada com recurso aos testes de Kolmogorov-Smirnov, Mann-Whitney, Kruskal-Wallis e análise ROC. As diferenças foram consideradas significativas quando p<0.05. Os resultados mostraram que para as ALDH1A3, ALDH1L1, ALDH1L2, ALDH3A1, ALDH3B2 e ALDH5A1 não existem diferenças de expressão entre doentes com SMD, doentes com LMA e controlos. Os doentes com SMD e LMA apresentam níveis de expressão de ALDH3A2 (SMD: mediana 1.9251; distância interquartil 1.28; p=0.000618; LMA: mediana 1.5096; distância interquartil 0.99; p=0.008) e ALDH4A1 (SMD: mediana 0.1841; distância interquartil 0.47; p=0.01134; LMA: mediana 0.1635; distância interquartil 0.78; p=0.124) superiores aos observados nos controlos (ALDH3A2: mediana 0.4624; distância interquartil 1.53; ALDH4A1: mediana 0.0388; distância interquartil 0.12). Por outro lado, os doentes com SMD apresentam níveis de expressão mais elevados de ALDH3B1 (mediana 1.6445; distância interquartil 1.39) do que os doentes com LMA (mediana 0.4541; distância interquartil 0.47; p=0.000314) e controlos (mediana 0.3521; distância interquartil 0.51; p=5.9942E-07). Os diferentes subtipos de SMD apresentam expressão diferencial de ALDH3A2 e ALDH3B1. Além disso, os doentes com SMD e com LMA com alterações mielodisplásicas não expressam ALDH18A1. Seguidamente avaliou-se a expressão das ALDHs nos doentes de SMD estratificados de acordo com o WPSS (WHO classification-based Prognostic Scoring System) e verificou-se que os doentes de muito baixo risco apresentaram maior expressão de ALDH3B1 (mediana 17.6934; distância interquartil 16.32) comparativamente aos de risco intermédio (mediana 1.2352; distância interquartil 2.55; p=0.010) . Por fim, a expressão das isoformas de ALDH não parece influenciar a sobrevivência global de doentes com SMD e LMA ou a evolução de SMD para LMA. Em conclusão, este trabalho sugere que as isoformas de ALDH apresentam expressão diferencial em doentes com SMD e LMA relativamente a indivíduos saudáveis e entre si. A expressão de ALDH3B1 poderá ser um potencial biomarcador de diagnóstico de SMD. Além disso, uma vez que nenhum doente com SMD e LMA com alterações mielodisplásicas apresenta expressão da ALDH18A1, a expressão desta isoforma poderá ser um bom biomarcador de diagnóstico de mielodisplasia. Por fim, a expressão de ALDH3A2 demostrou ser um possível biomarcador de diagnóstico de LMA. No entanto, estudos adicionais com um maior número de amostras são necessários para provar o potencial dessas enzimas como biomarcadores de diagnóstico.
Aldehyde Dehydrogenase (ALDH) superfamily is a group of 19 enzymes critical to the protection against toxic aldehydes, and have been associated with multiple diseases. Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis associated with peripheral blood cytopenias, and a predisposition toward leukemic transformation. Acute myeloid leukemia (AML) are characterized by disordered growth of immature myeloid blood cells in the blood and bone marrow. The pathophysiology of both, MDS and AML, is a complex multistep process that involves genetic and epigenetic abnormalities in a wide variety of genes associated with differentiation, cellular proliferation, self-renewal, and apoptosis. Since ALDHs are involved in some of these biological processes, the deregulation of these enzymes may influence MDS and AML development. The aim of the study is to evaluate the gene expression of ALDHs in patients with MDS and AML in order to verify their potential as a biomarker for the diagnosis and/or prognosis of these diseases. To this end, we did a preliminary analysis of the expression levels of ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH7A1, ALDH16A1, and ALDH18A1. Then, we analyzed gene expression of ALDH3A2, ALDH3B1, ALDH4A1, and ALDH18A1 in 54 patients, 34 MDS and 20 AML, and 34 healthy controls. ALDH expression levels were analyzed using Reverse Transcriptase-PCR and differentially expressed genes were quantified by qPCR. The statistical analysis was carried out by the Kolmogorov-Smirnov Test, Mann-Whitney test, Kruskal-Wallis test, and ROC analysis. A value of p < 0.05 was considered significant. The results indicate that ALDH1A3, ALDH1L1, ALDH1L2, ALDH3A1, ALDH3B2, and ALDH5A1 did not show differences in their expression between MDS, AML, and controls. ALDH3A2, ALDH3B1, ALDH4A1, and ALDH18A1 had differential expression among study groups and were quantified by real time PCR. MDS and AML patients showed higher expression of ALDH3A2 (MDS: median 1.9251; interquartile range 1.28; p=0.000618; AML: median 1.5096; interquartile range 0.99; p=0.008) and ALDH4A1 (MDS: median 0.1841; interquartile range 0.47; p=0.01134; AML: median 0.1635; interquartile range 0.78; p=0.124) in comparison with controls (ALDH3A2: median 0.4624; interquartile range 1.53; ALDH4A1: median 0.0388; interquartile range 0.12). On the other hand, the expression of ALDH3B1 was higher in MDS patients (median 1.6445; interquartile range 1.39) than in AML patients (median 0.4541; interquartile range 0.47; p=0.000314) and controls (median 0.3521; interquartile range 0.51; p=5.9942E-07). ALDH3A2 and ALDH3B1 also showed statistically significant differences between the different subtypes of MDS. Additionally, patients with MDS and AML with myelodysplasia related changes (AML-MRC) did not expressed ALDH18A1. When we compared MDS patients according to WHO classification-based prognostic scoring system (WPSS) risk groups it was found that patients with very low risk had higher expression of ALDH3B1 (median 17.6934; interquartile range 16.32) in comparison with patients with intermediate risk (median 1.2352; interquartile range 2.55; p=0.010). Furthermore, the expression of ALDH isoforms does not appear to influence MDS and AML patient’s overall survival or MDS evolution to AML. In summary, ALDH isoforms have differential expression patterns in MDS and AML patients when compared with controls and each other. The ALDH3B1 is a potential diagnostic biomarker of MDS. Since none MDS and AML-MRC patients expressed ALDH18A1, the expression of this isoform may be a good diagnostic biomarker. Finally, ALDH3A2 could be a diagnostic biomarker of AML. However, further studies with a higher number of participants are needed to prove the potential of these enzymes as diagnostic biomarkers.

Laboratory test results are important in making decisions regarding a patient's diagnosis and response to treatment. These tests often measure the biomarkers found in biological fluids such blood, urine, and saliva. Immunoassay is one type of laboratory test used to measure the level of biomarkers using specific antibodies. Microfluidics offer several advantages such as speed, small sample volume requirement, portability, integration, and automation. These advantages are motivating to develop microfluidic platforms of conventional laboratory tests. I have fabricated polymer microfluidic devices and developed immunoassays on-chip for potential cancer markers. Silicon template devices were fabricated using standard photolithographic techniques. The template design was transferred to a poly(methyl methacrylate) (PMMA) piece by hot embossing and subsequently bonded to another PMMA piece with holes for reservoirs. I used these devices to perform microchip immunoaffinity electrophoresis to detect purified recombinant thymidine kinase 1 (TK1). Buffer with 1% methylcellulose acted as a dynamic coating that minimized nonspecific adsorption of protein and as sieving matrix that enabled separation of free antibody from antibody-TK1 complexes. Using this technique, I was able to detect TK1 concentration >80 nM and obtained separation results within 1 minute using a 5 mm effective separation length. Detection of endogenous TK1 in serum is difficult because TK1 is present at the pM range. I compared three different depletion methods to eliminate high abundance immunoglobulin and human serum albumin. Cibacron blue columns depleted abundant protein but also nonspecifically bound TK1. I found that ammonium sulfate precipitation and IgG/albumin immunoaffinity columns effectively depleted high abundance proteins. TK1 was salted out of the serum with saturated ammonium sulfate and still maintained activity. To integrate affinity columns in microfluidic devices, I have developed a fast and easy strategy for initial optimization of monolith affinity columns using bulk polymerization of multiple monolith solutions. The morphology, surface area, and porosity, were qualitatively assessed using scanning electron microscopy. This method decreased the time, effort, and resources compared to in situ optimization of monoliths in microfluidic devices. This strategy could be used when designing novel formulations of monolith columns. I have also integrated poly(ethylene glycol dimethacrylate-glycidyl methacrylate) monolith affinity columns in polymer microfluidic devices to demonstrate the feasibility of extracting human interleukin 8 (IL8), a cancer biomarker, from saliva. Initial results have shown that the affinity column (~3 mm) was successfully integrated into the devices without prior surface modification. Furthermore, anti-IL8 was immobilized on the surface of the monolith. Electrochromatograms showed that 1 ng/mL of IL8 can be detected when in buffer while 10 ng/mL was detected when IL8 was spiked in saliva. Overall, these findings can be used to further develop immunoassays in microfluidic platforms, especially for analyzing biological fluids.

Mastocytosis is a myeloproliferative neoplasm characterized by infiltration of clonally derived mast cells in different tissues. According to mast cells localization, it is possible to discriminate cutaneous mastocytosis (CM) from systemic mastocytosis (SM), the latter involving at least an extracutaneous organ, like bone marrow, liver, spleen and gastrointestinal tract. Some of the SM patient can develop also cutaneous lesions (SM+C). Oral cavity is commonly involved in the symptomatology. Disease classification is often tricky. In the first part of this thesis, in order to highlight possible qualitative/quantitative modifications of the salivary proteome associated to the different forms of the disease, we investigated salivary samples collected from 6 CM, 35 SM patients, among which 8 with only systemic symptoms (SM-C) and 27 with both systemic and cutaneous symptoms (SM+C), and 48 healthy controls by a top-down proteomic approach. Low-resolution HPLC-ESI-MS analysis of the acid soluble fractions of saliva highlighted different proteomic profiles in the three patients’ groups, showing that the salivary samples of the patients were characterized by a down-regulation of peptides and proteins involved in the homeostasis and defense of the oral cavity, and in the innate immunity and in inflammation not only in the oral cavity but at systemic level, such as aPRPs, statherin, histatins and cystatins. Only two proteins with regulatory roles in the innate immunity and inflammation, S100A8 and antileukoproteinase, resulted up-regulated in patients differently to all the other salivary proteins analyzed, suggesting the establishment of a response by the organism to the injuries caused by the disease. Interestingly, some differences have been found among the patients in the concentration of α-defensins 1, thymosin β-4, and the truncated forms of cystatin D-R26 variant, and some truncated forms of P-B and statherin. Correlation between the protein/peptide levels and tryptase concentration evidenced that acidic PRPs, statherin and P-B fragments, and cystatin D-R26 des1-5 correlated positively just in SM-C group, while thymosin β-4 correlated negatively. Since the interesting data on cystatin D, in the second part of the thesis I focused on the characterization of the salivary protein complex aggregating to the cystatin D-C26 variant (named by us SIC-D). Indeed, the C-26 variant is usually undetectable in acid soluble fraction of saliva but measurable in whole saliva. Pools of whole saliva from 4 CM, 3 SM-C, 14 SM+C, and 20 sex/age matched healthy controls, were submitted to immunoprecipitation with cystatin D-C26 antibody followed by SDS-PAGE/western-blot under reducing and non-reducing conditions. Since the low volume of CM samples, the tryptic digestion, and the nano-HPLC-high-resolution-MS/MS analysis were performed only in SM-C, SM+C and control samples. The quantitative comparison was performed with Proteome Discoverer 2.2 software. SIC-D included 44 proteins, among which IgA, IgG, PIgR, annexins, α-defensin 1/2, S100A8, carbonic anhydrase 6, prolactin-inducible protein, lysozyme C and dermicidin. Several qualitative/quantitative differences were highlighted with respect to controls and between the two patient groups. The most relevant were: all the patients exhibited lower levels of IgA, PIgR, DMBT-1 and S100A8 than controls, but higher levels of IgG, α-defensins 1/2 and carbonic anhydrase 6. The highest level of cystatin D-C26 was found in SM+C patients, which were different from SM-C for annexin A2. Both SM-C and SM+C showed the presence of antileukoproteinase and S100A14. The results on the acid-soluble fraction of saliva and the preliminary results on the SIC-D complex are promising in order to find candidate markers able to discriminate the different forms of mastocytosis.

Introduction: The diagnosis of Arrhythmogenic Cardiomyopathy (AC) is challenging and often late after disease onset. It relies on a scoring system of major and minor criteria, requiring several clinical, instrumental and genetic tests. Diagnosis confirmation is often obtained by invasive procedures like endomyocardial biopsy and electroanatomical mapping. At present time, no circulating biomarkers are available for the diagnosis of AC. We hypothesized that circulating microRNAs (miRNAs), which have been already demonstrated as circulating biomarkers of many cardiac diseases (e.g. heart failure, myocardial infarction, atrial fibrillation) may be used as potential diagnostic tools in AC. Aims: 1. To screen the level of expression of miRNAs in plasma samples of AC and non-AC male subjects. 2. To assess the specificity of potential miRNAs in diagnosing AC. In particular to identify the differential expression of plasma miRNAs in AC patients vs. healthy controls (HC) and/or patients with ventricular arrhythmias of different aetiologies: idiopathic ventricular arrhythmias (IVT) and patients with ischemic ventricular arrhythmias (IC). 3. To evaluate a possible correlation between miRNAs expression and the severity of the disease in terms of ventricular function and fibro-adipose replacement of the myocardium by means of ElectoAnatomic voltage Mapping [EAM] and Late Gadolinium Enhancement (LGE) detected by Contrast Enhancement Cardiac Magnetic Resonance (CE-CMR) 4. To assess in cardiac stromal mesenchimal cells culture how different levels of expression of the identified miRNAs may regulate the onset and progression fibro-adipogenesis. Methods: All patients with a history of ventricular arrhythmias referred to Arrhythmia Center of the Cardiology Center Monzino (Milan), for transcatheter ablation were enrolled in the study as well as a control cohort of patients matched in terms of age and sex. All patients underwent: - CE-CMR evaluating the volumes and function of the right and left ventricle (RV and LV), the presence and extension of LGE; - Electroanatomic voltage mapping (CARTO) of RV and LV. - Endomyocardial biopsy, when indicated, to evaluate the presence and extension of fibro-fatty infiltration of the RV. - Blood sampling for molecular analysis of 320 miRNA expression. Blood samples (5ml) were collected in EDTA coated tubes and the total RNA was extracted from plasma. The expression of each single miRNA was evaluated using TaqMan microRNA assays (Life Technologies) following manufacturer’s instructions. Validated miRNAs expression data were analyzed using GraphPad Prism version 5.03 for Windows and reported as mean ± standard deviation of the mean (SD). Cardiac Mesenchymal Stromal Cells (C-MSCs) from AC ventricular samples have been obtained in our laboratory and have been used as in vitro model of AC and we decided to evaluate miR-320a in this model. In order to assess C-MSCs involvement during adipogenesis we planned an in vitro experiment to simulate AC development. We maintained the same plated number of C-MSCs in adipogenic medium for 3 days. Results: In the present study a total of 114 male subjects were enrolled: 35 patients were affected by AC, 35 were HC, 20 were affected by IVT and 24 were affected by IC. The level of expression of 368 miRNAs was screened in plasma of 3 symptomatic AC patients and 3 age- and sex-matched healthy donors: 150 miRNAs were found expressed in all screened plasma samples and 14 miRNAs resulted putatively regulated. Among the top 4 regulated miRNAs, considering both relative fold change and statistical significance, miR-320a was confirmed to be regulated in an initial validation step, performed by qRT-PCR in the plasma of 16 HC and 16 AC patients, as defined by current guidelines. Therefore its expression was analyzed in all HC and AC patients. MiR-320a showed a statistically significant lower expression in AC patients compared to HC (0.42±0.04, p=0.008) and with a cut-off value of ΔCt <-5.55 presented a sensitivity of 65% specificity of 80% to discriminate AC patients. We did not find any statistical significance in the level of expression of miR-320a between HC and IVT (fold 1.09±0.5 p=ns) as well as between HC and IC (fold 0.74±0.22 p=ns). In evaluating the expression of miR-320a in terms of the severity of the disease in patients with AC we did not find any statistically significant correlation with major arrhythmic events as well as no correlation with RV function. A significant correlation was found between impairment of the LV function and ΔCt expression (r2=0.20 p<0.04). Evaluating miR-320a expression in 12 patients with AC we found a trend of statistically significance with the extension of scar areas detected by unipolar electroanatomic mapping and LGE (p=0.07). The results from the in vitro study on C-MSC, showed a lower expression of miR-320a in AC C-MSCs compared to non-AC C-MSCs (0.44±0.08, p=ns). Conclusions: This is the first study that evaluates the diagnostic potential of circulating miRNAs in AC. Plasma levels of mir-320a are consistently lower in AC patients compared to HC, IVT and IC subjects and has a fairly good accuracy in discriminating AC vs. IVT patients. Low miR-320a plasma concentrations may represent a new potential biomarker for AC. Plasma concentration of miR-320a seems to demonstrate an inverse correlation with AC severity. MiR-320a regulation in a cardiac cellular model of AC during induced adipogenesis may pave the way to future mechanistic studies on the epigenetic control of AC adipogenesis.
Introduzione: La diagnosi di Cardiomiopatia Aritmogena (CA) risulta essere spesso una sfida per il cardiologo clinico e viene talvolta effettuata in ritardo rispetto all'insorgenza dei sintomi. Si basa sull’identificazione di criteri diagnostici ottenuti attraverso l’utilizzo di diverse indagini clinico-strumentali, talvolta invasive e tramite test genetici. Ad oggi nessun marcatore bioumorale circolante è stato validato e utilizzato quale criterio diagnostico. Abbiamo ipotizzato che i microRNA (miRNA) circolanti, già validati nella diagnosi di molte altre malattie cardiache (insufficienza cardiaca, infarto miocardico, fibrillazione atriale) possano essere utilizzati come potenziale strumento diagnostico nella CAVD. Obiettivi: 1. Eseguire uno screening di miRNA in campioni di plasma di soggetti maschi sani o affetti da CA. 2. Valutare la specificità e la sensibilità dei miRNA nel riconoscimento della CA. In particolare identificare i diversi livelli d’espressione dei miRNA plasmatici nei pazienti con CA rispetto ai controlli sani (CS) e/o ai pazienti con aritmie ventricolari a diversa eziologia (tachicardia ventricolare idiopatica (TVI) o conseguente a cardiopatia ischemica (CI). 3. Valutare la possibile correlazione tra l'espressione dei miRNA e la gravità della malattia espressa in termini di numerosità degli eventi aritmici maggiori, funzionalità ventricolare destra e sinistra e in termini di estensione della sostituzione fibro-adiposa ottenuta mediante mappaggio elettroanatomico e risonanza magnetica cardiaca (RMC). 4. Valutare come la differente espressione dei miRNA circolanti identificati possa regolare lo sviluppo e la progressione della sostituzione fibro-adiposa in colture di cellulle cardiache mesenchimali stromali (C-MSC). Metodi: Sono stati arruolati nello studio tutti i pazienti con aritmie ventricolari, riferiti all’Unità di Elettrofisiologia del Centro Cardiologico Monzino (Milano) per essere sottoposti ad ablazione transcatetere e controlli sani. Tutti i pazienti sono stati sottoposti a: - RMC per valutazione dei volumi e funzionalità del ventricolo destro e sinistro e per determinazione dell’estensione delle “scars”. - Mappaggio elettroanatomico intracavitario bipolare e unipolare (CARTO). - Biopsia endomiocardica, quando indicata per valutazione della presenza e l’estensione della sostituzione fibro-adiposa del ventricolo destro. - Prelievo di sangue venoso l'analisi molecolare di 320 miRNA. I campioni di sangue (5 ml) sono stati raccolti in provette EDTA e l'RNA totale è stato estratto dal plasma. L'espressione di ogni singolo miRNA è stata valutata utilizzando saggi TaqMan microRNA (Life Technologies) seguendo le istruzioni del produttore. L’espressione dei MiRNA validati è stata analizzata utilizzando GraphPad Prism versione 5.03 per Windows e riportati come media ± deviazione standard della media (SD). Le cellule stromali mesenchimali cardiache (C-MSC) sono state ottenute da un campione bioptico di pazienti con sospetta CA. Al fine di valutare il coinvolgimento C-MSC durante adipogenesi è stato eseguito un esperimento in vitro per simulare lo sviluppo della patologia. Risultati: 114 soggetti maschi sono stati arruolati nello studio: 35 soggetti affetti da CA, 35 soggetti sani, 20 affetti da TVI e 24 da CI. Il livello d’espressione di 368 miRNA circolanti è stato valutato su campioni di plasma di 6 soggetti (3 affetti da CA e soggetti sani). Di 150 miRNA espressi in tutti i campioni di plasma, 14 miRNA sono risultati regolati. Tra i 4 miRNA maggiormente regolati, il miR-320a è stato validato inizialmente mediante qRT-PCR nel plasma di 32 soggetti (16 controlli sani e 16 soggetti affetti da CA) In seguito abbiamo valutato l’espressione di miR-320a in tutti i soggetti dello studio. I livelli d’espressione del miR-320A sono risultati statisticamente inferiori nei soggetti affetti da CA rispetto ai controlli sani (0,42 ± 0,04, p = 0,008) con un valore di cut-off (ΔCt <-5.55) il miR-320a ha presentato una sensibilità del 65% e una specificità dell’ 80% nel discriminare I pazienti con CA. Non abbiamo trovato nessuna differenza statisticamente significativa nell’espressione di miR-320a tra soggetti sani e affetti da TVI (1,09 ± 0,5 p = ns) come tra soggetti sani e affetti da CI (0.74 ± 0.22 p = ns). Nei soggetti affetti da CA, non abbiamo trovato alcuna correlazione tra i livelli d’espressione di miR-320 a e il verificarsi di eventi aritmici maggiori così come con gli indici di funzionalità ventricolare. Una correlazione statisticamente significativa è stata riscontrata tra i livelli di espressione del miR-320a e la funzione ventricolare sinistra (FE)(r2=0.20 p<0.04). In 12 dei pazienti con CA abbiamo trovato un trend di significatività statistica tra i livelli di miR-320 a e le aree di “scar” al mappaggio elettroanatomico unipolari e la presenza di LGE (p=0,07). Dallo studio in vitro sulle C-MSC abbiamo evidenziato una ridotta espressione di miR-320 nelle cellule ottenute dai pazienti con CA rispetto alle cellule ottenute dagli altri soggetti (0,44 ± 0,08, p=ns) Conclusioni: Questo è il primo studio in cui sia stata valutata l’utilità diagnostica dei miRNA circolanti nella CA. I livelli plasmatici d’espressione del miR-320a sono costantemente più bassi nei soggetti affetti da CA rispetto a soggetti sani o con cardiopatia ischemica o affetti da tachicardia ventricolare idiopatica e si è dimostrato in grado di discriminare con una buona accuratezza i soggetti affetti da CA rispetto ai soggetti affetti da TVI. I nostri dati preliminari suggeriscono miR-320a quale potenziale marcatore bioumorale di CA e sembrerebbero evidenziare una correlazione inversa tra i livelli d’espressione di miR-320a e la gravità della patologia. La creazione di un modello cellulare di adipogenesi indotta in cellule mesenchimali stromali cardiache mediante regolazione di miR-320a potrebbe aprire la strada a futuri studi meccanicistici sul controllo epigenetico dell’adipogenesi nella CA.

Yes
Background: Carbon (δ13C) and nitrogen (δ15N) isotope ratios of collagen from teeth and bone are used to study human nutrition and health. As bones are constantly remodelling throughout life, isotopic values of bone collagen represent an average of several years. In contrast, human teeth do not remodel and their primary dentine contains only the isotopic data from the time of formation. In contrast to all other bones, human auditory ossicles also appear not to remodel. As they develop in utero and finish formation in the first 2 years of life, their collagen should also represent isotopic values of these two relatively short periods. Aim: By comparing δ13C and δ15N data from ossicles and incremental dentine, this study aims to investigate how two developmental periods of the ossicles, in utero and the first 2 years of life, reflect in collagen obtained from the ossicles. Subject and methods: Ossicle and tooth samples of 12 individuals aged 0.5 ± 0.4 years to 13 ± 1 years from the nineteenth century St. Peter’s burial ground in Blackburn were collected and processed to obtain bulk bone and incremental dentine collagen which was measured for δ13C and δ15N. Results: Averaged δ13C and δ15N of ossicles are lower when compared to every age group except after 3 years of age. Average offset between ossicles and dentine of different groups ranges from 0.4–0.9‰ for δ13C and from 0.3–0.9‰ for δ15N, with highest counterbalance at birth and after the first 5 months after birth. Conclusions: There appears to be a systematic offset between the dentine and ossicle data. It seems that the second phase of development does not influence the isotopic values of collagen significantly and the data we are obtaining from ossicles represents the in utero period.
Research grant from The Society for the Study of Human Biology.

Hyperfosforylering av biomarkörproteinet Tau förekommer i flera neurodegenerativa sjukdomar som kallas Taupathies. Proteinets huvudfunktion i människokroppen är att modulera flexibilitet och stabilitet för axonal-mikrotubulin. I Taupathies utlöser hyperfosforyleringen av Tau instabilitet och neurodegenerationen. I dagens läge kan hyperfosforylering av treonin 217 (P217) endast mätas i hjärnan. I den här studien undersöks hyperfosforyleringen av treonin 217 (P217). I syfte att se om nivåerna av P217 är mätbara i cerebrospinalvätska (CSV) och i blodet. Samt för att evaluera hur nivåer av P217 förändras i olika Taupathies, genom att testa hjärnprover från friska kontroller och olika Taupathies. Studien görs för att öka kunskapen om effekten av hyperfosforylering av treonin 217 i Taupathies och för att bidra med en ny provtagningsmetod för P217. Simoa HD-1 Analyzer var instrumentet som användes för analyserna av P217. Det är ett instrument som kan upptäcka onormala nivåer av biomarkörer genom kvantifiering, med hjälp av antikroppar och ett enzym. Enzymet kallas Streptavidin β-galaktosidas och omvandlar en befintlig P217-molekyl i proven till en fluorescerande produkt. Genom Simoa HD-1 Analyzer utvecklades en ultrasensitiv analys med antikropparna P217 och Tau 12, som kunde upptäcka mycket låga nivåer av P217 i hjärnan, CSF och i blod. Förändring av P217-nivåer hittades även i olika Taupathies. De Taupathies med de högsta nivåerna av P217 var Progressiv supranukleär pares, Corticobasal degeneration och Globular glial Taupathies.
Hyperphosphorylation of the biomarker protein Tau occurs in many neurodegenerative diseases called Taupathies. The proteins main function in the human body is to modulate flexibility and stability for axonal microtubules. In Taupathies the hyperphosphorylation of the Tau triggers instability and neurodegeneration. Nowdays hyperphoshorylation on threonine 217 (P217) can only be measured in the brain. In this study the hyperphoshorylation on the phosphorylation site of threonine 217 (P217) is examined. In aim to see if levels of P217 is measurable in cerebrospinal fluid (CSF) and in blood. As well to evaluate how P217 variate in different Taupathies, through the use of brain samples from healthy controls and different Taupathies. The study is made for the purpose of enhancing the pure knowledge about the effect of hyperphosphorylation on threonine 217 in Taupathies and to contribute with a new sampling method for P217. Simoa HD-1 Analyzer was the key instrument of the analyses of P217. It’s an instrument which can detect abnormal levels of biomarkers through quantification, with help of antibodies and an enzyme. The enzyme is called Streptavidin β-galactosidase and converts an existing P217 molecule in the samples to a fluoresce product. Through the use of Simoa HD-1 Analyzer an ultrasensitive assay with antibodies P217 and Tau 12 was developed which could detect very low levels of P217 in brain, CSF and in blood. Variation of P217 levels was also found in different Taupathies. The Taupathies with the highest levels of P217 was Progressive supranuclear palsy, Corticobasal Degeneration and Globular glial Taupathies.

Bladder cancer is an important global disease. The gold standard for diagnosis is cystoscopy and biopsy; both are invasive and require highly trained personnel. In a majority of cases, treated patients are followed up by frequent cystoscopies lasting several years. The discovery of biomarkers indicating which individuals should proceed to cystoscopy would be an important addition to bladder cancer management. The odour of urine is produced by volatile organic compounds, (VOCs), detectable by gas chromatography and mass spectrometry (GC-MS). An analysis of the VOCs in urine from various groups of individuals including patients with bladder cancer is undertaken in search of possible discriminating compounds, which could be harnessed in future as a potential screening tool or adjunct in bladder cancer management. Methods First void urine was obtained from 64 patients with new non-muscle invasive bladder cancer, 71 cancer free patients with haematuria and 51 asymptomatic volunteers. After equilibration, the headspace above these pH adjusted urine samples was extracted for 20minutes, using a carboxen / polydimethylsiloxane solid phase micro-extraction fibre (SPME). This was followed by desorption and VOC identification by GC-MS. Results Urine headspace VOCs under acidic conditions, (pH of modified urine 2), were found to be discriminating. Identified compounds were analyzed using forward stepwise discriminant analysis: 9 VOCs when used together, gave 84.7% correct classification of samples (Haematuria control v Bladder cancer) with no change on cross validation of results. The calculated sensitivity and specificity of this model is 76.6% and 92.9% respectively, with Positive predictive value of 90.7% and Negative predictive value of 92.9%. These results are comparable, and in some cases better than those obtained using commercially available urinary bladder cancer biomarkers. Conclusion Volatile organic compounds in urinary head space change with the development of bladder cancer. Urinary VOCs are exciting novel potential biomarkers in the detection of bladder cancer.

High-throughput technologies in biomedical sciences, including gene microarrays, supposed to revolutionise the post-genomic era, have barely met the great expectations they inspired to the biomedical community at first. Current efforts are still focused toward improving the technology, its reproducibility and accuracy. In the meantime, computational techniques for the analysis of the data from these technologies have achieved great progresses and show encouraging results. New approaches have been developed to extract relevant information out from these results. However, important work needs to be further conducted in order to extract even more meaningful and relevant information. These techniques offer great possibilities to explore the overall dynamic held within a living organism. The potential information contained in their output can reveal important leads at deciphering the interconnection, interaction or regulation influences that can exist between several molecules. In front of an increasing interest of the scientific community toward the exploration of these dynamics, some groups have started to develop solutions based on different technologies to extract these information related to interactions. Here we present an Artificial Neural Network-based methodology for the study of interactions in gene transcriptomic data. This will be applied and validated in a breast cancer context. This manuscript will discuss the methodological optimisation to identify biomarkers of interest from high-throughput transcriptomic technologies; and it will show how the algorithms were brought forward to identify the potential relationship that may exist between the markers identified. It will illustrate and highlight the robustness of the methods by discussing some examples of application in different breast cancer studies. The present thesis will show that despite the great difficulty to obtain gold validation to prove the robustness of the approach; it has been possible to identify some relevant features able to highlight the promises held by this preliminary development of the method. The results obtained by trying to identify the correlated component within an artificial dataset suggest some interesting ability of the approach. Additionally, when applied to the van’t Veer dataset (van’t Veer et al., 2002), the list of selected transcripts held two different isoforms for two different genes, and the method identified the strong correlation between the 2 forms. Finally, the results involving the transcripts for DTL, TK1 and CDC45L have been shown to overlap with the result of a similar work from Gevaert et al. (2006) on the van’t Veer dataset using a different method involving a Bayesian network with Markov blanket. Ultimately, this thesis will try to discuss the advantages or limitations as well as the potential application and future hopes around the methods introduced.

Mestrado em Biomedicina Molecular
Current clinical AD diagnosis criteria used to identify the disorder in patients who have already overt the advanced stage of dementia. This is too late for some kind of successful disease adjustment and consequently, early recognition of AD needs to be improved. Therefore, it is really needed different approaches for discovering new AD biomarkers, such as the application of metabolomics techniques (e.g. FTIR). The potential of FTIR in the clinical field has recently received particular attention, since it uses vibration frequencies of molecules present in the analysed sample to produce a metabolic fingerprint, which is then specific for each sample. This dissertation aims to identify biochemical alterations that might be related to dementia, being possible to distinguish between control and disease groups of plasma samples, through application of FTIR methodology at the 4000-600 cm-1 spectral region. Using plasma samples makes the process being minimally invasive, with other relevant clinical advantages. Besides the collection of blood samples used in this project, the volunteers were also submitted to cognitive evaluation trough Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR), with other relevant clinical information being also collected. All the analysed samples were matched by age and sex. For a better discrimination between control and disease samples, Principal Component Analysis (PCA) was applied to spectra data at specific regions, namely 3500-2700 cm-1, 1700-1400 cm-1, and 1200-900 cm-1. This allowed to identify the main pathological changes that occurred at the biochemical level during neurodegenerative disorder development. In the former spectral region, disease samples presented a higher content of saturated lipids in relation to the unsaturated ones, which translates in a high potential brain damage. Besides, it was also noted the presence of carboxylic acids that are usually related to lipid hyperoxidation, production of reactive carbonyls and proteins structural and functional alterations. In turn, the spectral region 1700-1400 cm-1 allowed to identify differences in protein conformation between control and disease samples, and these last ones were still related with occurrence of protein aggregates. In other hand, the 1200-900 cm-1 region could be associated to cellular damage provoked by oxidative stress in disease samples. As an important note to take, FTIR analysis could have the potential to be applied in future not only for cognitive impairment diagnosis but also for identification of disease stage and prognostic evaluation, besides assessment of disease developing risk for control subjects.
Os atuais critérios clínicos de diagnóstico da doença de Alzheimer geralmente identificam a patologia apenas quando os pacientes já atingiram a fase de demência, ou seja, o estágio mais avançado da doença. Isto revela-se demasiado tarde para que qualquer tipo de terapêutica seja bem-sucedida e, consequentemente é necessário desenvolver uma metodologia de diagnóstico que permita um reconhecimento mais precoce da doença. Desta forma, é preciso adotar diferentes abordagens para a descoberta de novos biomarcadores para a doença de Alzheimer, tais como a utilização de técnicas de metabolómica (ex. FTIR). O potencial do FTIR no campo clínico recebeu recentemente particular atenção, sendo que esta técnica utiliza as frequências de vibração das moléculas presentes na amostra analisada para produzir uma “impressão digital” metabólica, a qual é específica para cada amostra. Esta dissertação tem como objetivo a identificação de potenciais alterações bioquímicas que possam estar relacionadas com demência, tornando possível a distinção entre grupos controlo e de doentes no seio do conjunto de amostras de plasma analisadas. A utilização de amostras de plasma torna o processo minimamente invasivo, de entre outras vantagens clínicas. Para além da colheita das amostras de sangue utilizadas neste projeto, os voluntários foram submetidos a uma avaliação cognitiva através da realização do MMSE e do CDR, com outra informação clínica relevante a ser também recolhida. Todas as amostras analisadas foram emparelhadas de acordo com a idade e o sexo. Para uma melhor distinção entre amostras controlo e de doentes foi aplicada a metodologia de PCA aos dados dos espectros obtidos em regiões específicas, nomeadamente em 3500-2700 cm-1, 1700-1400 cm-1, e 1200-900 cm-1. Isto permitiu identificar as principais alterações patológicas que ocorrem a nível bioquímico durante o desenvolvimento da doença neurodegenerativa. Na primeira região espectral referida, as amostras dos doentes apresentaram um maior conteúdo de lípidos saturados comparativamente aos não saturados, o que se traduz num potencial risco cerebral maior. Para além disso, foi observada a presença de ácidos carboxílicos, usualmente relacionados com hiperoxidação de lípidos, produção de carbonilos reativos e alterações estruturais e funcionais de proteínas. Por sua vez, a região espectral 1700-1400 cm-1 permitiu identificar diferenças na conformação de proteínas entre amostras controlo e de doentes, tendo estas últimas sido ainda relacionadas com a ocorrência de agregados proteicos. Por outro lado, a região 1200-900 cm-1 pôde ser relacionada com presença de danos celulares provocados por stress oxidativo nas amostras de doentes. Como importante nota a reter, a análise por FTIR pode ter o potencial para ser aplicada no futuro não apenas para diagnóstico de disfunção cognitiva, mas também para identificação do estádio da doença e realização de prognósticos, para além da avaliação do risco de desenvolvimento da doença em sujeitos controlo.

A population based case-control study was designed to test the hypothesis that cases of squamous cell carcinoma (SCC) had lower concentration of cis linoleic (LA) in their red blood cell membranes and it would remain a predictor of SCC after adjusting for other risk factors. The probability of LA as a potential biomarker for SCC was investigated because it is the precursor of arachidonic acid (AA), a secondary messenger for proteins involved in cell growth and proliferation. Ras oncogene perpetually activates the two phospholipases A-2 and C, causing release of AA from the membrane phospholipids. Red blood cell membranes were used as the medium for assessing the concentration of cis linoleic acid because donation of blood was well accepted by the participants. Tissue availability, and the low cost of operation were the other factors to utilize red blood cell membranes. Cases who had confirmed pathological diagnosis of SCC between December 1991-October 1994 were identified and consecutively sampled from the Arizona Skin Cancer Registry. Controls were recruited from the same geographic area using a random digit dialing technique. Subjects, limited to White-Anglo European descendent, were frequency matched by their age (± 5 years). Participants were screened for use of steroids, prescribed non-steroid anti-inflammatory drugs and history of chronic illness. Data on sun exposure habits, medication usage, lifestyle habits, and family history of SCC were collected during a personal interview. A 15 cc blood sample was collected. The composition of fatty acids in the red blood cell membranes was analyzed using gas-liquid chromatography techniques. Statistical analysis revealed that concentration of LA was significantly lower in the red blood cell membranes of the cases than the controls. However, cases had higher concentration of linolenic and trans linoleic acids. The concentration of 17 other fatty acids were equal, indicating that the general dietary pattern of cases and controls were similar. Use of logistic regression statistics showed that LA remained a significant independent risk factor for SCC (OR = 2.83, 95% Cl = 1.38-5.97) after adjusting for other risk factors. Based on this preliminary research, it is cautiously suggested that LA could be used as a potential biomarker of SCC.

EVALUATION OF REDOX POTENTIAL AS A NOVEL BIOMARKER OF OXIDATIVE STRESS, INFLAMMATORY RESPONSE, AND SHOCK USING NANOPOROUS GOLD ELECTRODES Background: Redox potential is a chemical species’ affinity for electrons. Increased oxidant concentration is associated with disease1,2, yet there is not a way to measure systemic redox status.3 Redox potentiometry uses metal electrodes that do not work in blood because protein molecules adhere on the metal surface, blocking electron exchange. Methods: Nanoporous gold electrodes have large surface areas that allowed electron exchange to continue in blood.4 Redox potential was measured in blood with ascorbic acid, in cardiac bypass patients and pigs undergoing hemorrhagic shock and resuscitation. Results: Blood redox decreased with ascorbic acid addition, both in vitro and in vivo. It was more positive in patients undergoing cardiac surgery compared to healthy volunteers. Conclusions: Preliminary studies were limited, but appear to show correlation to disease processes and medical therapies. More work needs to be done to further examine the relation of redox to disease and treatment.

The thesis presents the development of immunochemical methods for detection of trichlorophenols (TCP) in environmental and biological samples. An indirect enzyme linked immunosorbent assay (ELISA) for 2,4,5-TCP has been developed after a rational design of the immunizing hapten chemical structure, and the screening of 12 competitor haptens. The effect of the conjugation degree of the competitors and their homology with the target analyte, the physicochemical parameters (pH, ionic strength), the concentration of detergent, the time of incubation and the specificity were studied.

Two immunoassays (2,4,5- and 2,4,6-TCP ELISAs) were evaluated for the analysis of water, milk, serum and urine. Drinking water was analyzed directly after buffering the sample. The strong matrix effects in milk samples requires the sample clean up. Human serum can be analyzed after protein precipitation with absolute ethanol. The strong matrix effect of urine and its variability for samples from different individuals suggested the introduction of a purification step prior to ELISA. The C18- solid phase extraction (SPE) is an effective clean up method to remove an important part of the nonspecific interferences present in urine. The C18-SPE-ELISA method allows accurate quantification of TCPs in urine of occupationally exposed persons. SPE based on immunosorbents (immunoaffinity extraction, IAE) have been developed in single and 96-column formats. IAE is an effective clean up method to remove all nonspecific urinary interferences. The IAE step was optimized regarding sample volume, loading level, type of urine hydrolysis washing and elution conditions. The selectivity of the immunosorbents can be modulated by the washing conditions. The immunosorbents have sufficient capacity to effectively extract 2,4,6-TCP from urine samples of occupationally exposed persons and the general population. The HTS-IAE-ELISA method allows the processing of 100 samples/day with very good precision and accuracy. The method was validated with GC-MS and applied to the biomonitoring of three groups of population from Catalonia.

A quenching fluorescence immunoassay based on the laser-induced fluorescence detection in microdroplets (LIF-microdroplet-QFIA) for 2,4,6-TCP has been developed as a novel biodetection system. This approach offers significant improvement in method detectability compared to the microplate immunoassays and is the first application urine samples that can be directly analyzed after sample dilution.

Canine degenerative myelopathy(DM) is a late onset neurodegenerative disease that primarily affects German Shepherd dog (GSD), though a number of other specific breeds are also affected. The underlying cause of the disorder remains elusive, though recent advances have implicated a mutation of superoxide dismutase 1(Sod1) in the aetiology, also implying DM is a potential orthologue of human amyotrophic lateral sclerosis. The identification of the Sod1 mutation raises the index of suspicion for an individual animal, however it is not specifically diagnostic as a proportion of dogs homozygous for the Sod1 mutation do not develop DM. Therefore, there is a clinical need for the development of specific biomarker(s) for DM to support genetic test. The aim of this study was to establish potential biomarkers for DM by exploring canine cerebrospinal fluid (CSF). A dual strategy was adopted;1) Evaluation of potential ALS biomarkers in DM CSF, 2) Identification of novel biomarker(s) in DM CSF. The cases selected in this project had a presumptive diagnosis of DM and were homozygous for Sod1 mutation. Preliminary characterisation by Western blot and mass spectrometry identified four protein candidates in DM CSF, comprised of cystatin C, transthyretin (dimeric and monomeric TTR), haptoglobin and clusterin. Since the validity of these putative biomarkers may be influenced by pre-analytical variables that may arise from the clinical environment, we therefore assessed the impact of three potential sample handling practices on these four proteins. The results from these experiments demonstrate that dimeric TTR and clusterin were affected by sample handling conditions. Therefore, an appropriate protocol for CSF sample handling was established. Western blot analyses indicated that clusterin is the most viable biomarker candidate for DM. Clusterin was significantly elevated in DM CSF when compared to a range of neurological conditions. The second potential candidate for DM biomarker is TTR, which is potentially reduced, an observation similar to those found in ALS CSF. The relationship of these proteins in the pathogenic mechanisms that underpin DM is unclear. However, based on observations on ALS, it is reasonable to speculate that their alterations are associated with a toxic gain of function of the mutant SOD1 protein. The successful characterisation of clusterin and TTR in DM CSF may therefore represent components of a panel of emerging biomarkers that may combine to distinguish DM in the clinic and provide further insights into the disease mechanisms.

The global population with dementia is rapidly increasing around the world.The major risk factor for dementia is aging. There is currently no treatmentavailable and the cost of symptomatic treatment is high. There is a growinginterest in possible clinical applications of non-invasive methods that are safeand easy-to-perform in diagnosis of dementia. The purpose of this paper is toinvestigate the usage of transcranial magnetic stimulation (TMS) withelectroencephalography (EEG) to diagnose dementia in early stages of thedisease. Early diagnosis is needed to reduce the costs of symptomatic care.When investigating the usage of TMS-EEG technology, we will look at how wecan distinguish dementia in different neurodegenerative diseases between eachother. More research is needed to suggest an accurate parameters fordiagnosis of dementia with this type of technology.

As the aging population is increasing worldwide, so is the prevalence of neurodegenerativediseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia(FTD) and amyotrophic lateral sclerosis (ALS). Reliable biomarkers able to aid the diagnosis anddifferentiation of these diseases are needed in order to start the right treatment as early as possible.Due to its representative state of the central nervous system, cerebrospinal fluid (CSF) is afavorable sample material for biomarker discovery within neurodegenerative diseases. Alteredprotein levels of this body fluid might serve as a biomarker, but further validation of earlierfindings is needed. The aim of this project was to validate earlier studies suggesting potentialprotein biomarkers in CSF. From a list of 80 potential biomarkers in the CSF of patient samples,eight were chosen to be included in this validation effort. By utilizing a suspension bead array ina sandwich assay setup, 21 antibodies were tested in an initial screening. Antibody pairs that couldmeasure the protein levels in a dilution dependent manner was further optimized before individualpatient samples were analyzed. Sandwich assays targeting the three proteins Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) and Beta-synuclein (SNCB) were successfully developed andcorrelated to earlier generated data using a suspension bead array with a single binder setup.Therefore, the earlier findings of elevated levels of AMPH and SNCB in AD patients and CHIT1in ALS patients were successfully validated.
Prevalensen av neurodegenerativa sjukdomar såsom Alzheimers sjukdom (AD), Parkinsonssjukdom (PD), frontallobsdemens (FTD) och amyotrofisk lateralskleros (ALS) ökar i takt med denåldrande populationen. Pålitliga biomarkörer som kan hjälpa till vid diagnostiseringen av dessasjukdomar behövs för att starta rätt behandling så tidigt som möjligt. Ryggmärgsvätska, enkroppsvätska tillhörande det centrala nervsystemet, kan ge en inblick i det centrala nervsystemetstillstånd. Förändrade proteinnivåer i denna kroppsvätska skulle därför kunna fungera sombiomarkörer. Målet i detta projekt var att validera tidigare föreslagna proteinbiomarkörer iryggmärgsvätska. Utifrån en lista av 80 tidigare analyserade proteiner i ryggmärgsvätska hospatienter, inkluderades åtta proteiner i detta valideringsförsök. En antikroppsbaserad så kalladsandwich assay användes i en suspension bead array för att testa 21 stycken antikroppar i ett initialtscreeningsförsök. Antikroppspar som kunde mäta proteinnivåer på ett spädningsberoende vis i detinitiala screeningsförsöket optimerades vidare innan den utvecklade sandwich assayn användes föratt analysera proteinnivåer i individuella prover. Sandwich assays gentemot Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) och Beta-synuclein (SNCB) kunde bli framtagna ochkorrelerade gentemot tidigare genererat data från en single binder assay på ett framgångsrikt sätt.Projektet kunde därmed validera tidigare fynd som indikerat förhöjda nivåer av AMPH och SNCBi AD patienter, samt förhöjda nivåer av CHIT1 i ALS patienter.