Small intestinal neuroendocrine tumours (SI-NETs) are rare, hormone producing and proliferate slowly. Patients usually display metastases at time of diagnosis, the tumours are difficult to cure, and the disease course is unpredictable. The gene expression pattern was investigated in paper I, with emphasis on aggressive disease and tumour progression. Expression microarrays were performed on 42 tumours. Unsupervised hierarchal clustering revealed three clusters that were correlated to clinical features, and expression changes from primary tumour to metastasis. Eight novel genes, ACTG2, GREM2, REG3A, TUSC2, RUNX1, TGFBR2, TPH1 and CDH6 may be of importance for tumour progression. In paper II, expression of ACTG2 was detected in a fraction of SI-NETs, but not in normal enterochromaffin cells. Inhibition of histone methyltransferase and transfection of miR-145 induced expression and no effect was seen after DNA methylation or selective EZH2 inhibition in vitro. miR-145 expression was reduced in metastases compared to primary tumours. Overexpression of ACTG2 inhibited cell growth, and inducing ACTG2 may have therapeutic effects. TCEB3C (Elongin A3) is located on chromosome 18 and is imprinted in some tissues. In paper III a reduced protein expression was detected. The gene was epigenetically repressed by both DNA and histone methylation in a tumour tissue specific context. The expression was also induced in primary cell cultures after DNA demethylation and pyrosequencing revealed promoter region hypermethylation. Overexpression of TCEB3C inhibited cell growth by 50%, suggesting TCEB3C to be a tumour suppressor gene. In paper IV, 69 biomarkers were analysed in blood serum using multiplex proximity ligation assay. Nineteen markers displayed different levels between patients and controls. In an extended cohort, ELISA analysis showed elevated serum levels of Mindin, DcR3 and TFF3 in patients and protein expression in tumour cells. High levels of DcR3 and TFF3 were associated with poor survival, and DcR3 may be a marker for liver metastases. Mindin, DcR3, and TFF3 are potential novel diagnostic biomarkers for SI-NETs.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

The serum fraction of blood is an ideal material in which to search for novel biomarkers for disease. It is easily obtained through relatively non-invasive means, routinely collected, and a rich treasure-trove of information about the health of an individual. Cells react to signal molecules, take up nutrients, and release waste products, fragments that are the result of proteolysis, and other molecules out into the bloodstream. If these components are unique to the cells in question, that part of the complex mixture that is the blood stream can potentially characterize the health of the tissue or organ those cells are a part of. Serum is dense with proteins that span over ten orders of magnitude in size and abundance. The top 22 most abundant proteins in serum account for 99% of the total protein. These abundant proteins are well-characterized and not useful in a search for novel biomarkers for disease. Removal of these large proteins is accomplished using an organic-solvent precipitation step. Analyzing the resultant mixture of low-molecular-weight serum peptides using cLC-MS produces large, data-rich, and very complex data files. We have developed a manual analysis method we have developed that is capable of performing all of the processing steps necessary to identify novel biomarkers for disease as well as a method for the sequencing of low-abundance, highly charged peptide species without additional sample preparation. These methods are applied to two serum sample sets collected to investigate two pregnancy-related diseases: preterm birth, and preeclampsia. Three novel biomarkers of preterm birth have been identified and a combination of these with 5 previously studied markers can predict women who will have preterm birth with a sensitivity of 89.9% and a specificity of 81.0%. Nineteen different molecular species have been identified that predict women at risk for preeclampsia with a p-value of <0.05. Weighted combinations of various groups of the 19 biomarkers can increase the sensitivity up to 96% and the specificity up to 100%. The use of cLC-MS in the search for novel serum biomarkers of pregnancy-related disease allows for seamless integration from potential biomarker selection to polypeptide sequence identification.

Acute occlusion of the superior mesenteric artery (SMA) is a condition associated with high mortality and morbidity. The aim of this thesis is to evaluate diagnostic and therapeutic approaches for acute SMA occlusion. In a prospective study of patients with suspected intestinal ischemia, no biomarker was sufficiently accurate to detect this condition. In a second retrospective study, pancreatic amylase and troponin-I were elevated in a substantial proportion of patients with verified SMA occlusion. In an experimental animal model of acute SMA occlusion, microarray studies of ischemic small bowel wall were used to characterize the mRNA response to ischemia. Thrombospondin, Monocyte Chemoattractant Protein 1 and Gap Junction Alpha 1 were consistently up-regulated in all pigs with intestinal ischemia. Genes encoding previously proposed biomarkers for intestinal ischemia were either up-regulated, such as lactate dehydrogenase and creatine kinase, or down-regulated, such as intestinal fatty acid binding protein and glutathione S-transferase. In a study of the role of computed tomography in the diagnosis of SMA occlusion, it was shown that computed tomography with intravenous contrast was associated with improved survival. A retrospective analysis of all acute SMA revascularizations in Sweden 1999-2006 revealed that D-dimer was elevated in all 35 measured cases.  Endovascular surgery was associated with better outcome than open surgery, both in short and in long term. The presence of postoperative short bowel syndrome was a strong independent risk-factor for decreased long-term survival. Conclusions: Data affirm that D-dimer may serve as an exclusion test for acute SMA occlusion, whereas elevated troponin-I and pancreatic amylase are potential diagnostic pitfalls. Contrast-enhanced computed tomography of the visceral arteries seems to be the best diagnostic method. Endovascular surgery is an option to open surgery in selected cases, and was associated with favourable outcome.

Objectif: Évaluer l'efficacité du dépistage de l’hypertension gestationnelle par les caractéristiques démographiques maternelles, les biomarqueurs sériques et le Doppler de l'artère utérine au premier et au deuxième trimestre de grossesse. Élaborer des modèles prédictifs de l’hypertension gestationnelle fondées sur ces paramètres. Methods: Il s'agit d'une étude prospective de cohorte incluant 598 femmes nullipares. Le Doppler utérin a été étudié par échographie transabdominale entre 11 +0 à 13 +6 semaines (1er trimestre) et entre 17 +0 à 21 +6 semaines (2e trimestre). Tous les échantillons de sérum pour la mesure de plusieurs biomarqueurs placentaires ont été recueillis au 1er trimestre. Les caractéristiques démographiques maternelles ont été enregistrées en même temps. Des courbes ROC et les valeurs prédictives ont été utilisés pour analyser la puissance prédictive des paramètres ci-dessus. Différentes combinaisons et leurs modèles de régression logistique ont été également analysés. Résultats: Parmi 598 femmes, on a observé 20 pré-éclampsies (3,3%), 7 pré-éclampsies précoces (1,2%), 52 cas d’hypertension gestationnelle (8,7%) , 10 cas d’hypertension gestationnelle avant 37 semaines (1,7%). L’index de pulsatilité des artères utérines au 2e trimestre est le meilleur prédicteur. En analyse de régression logistique multivariée, la meilleure valeur prédictive au 1er et au 2e trimestre a été obtenue pour la prévision de la pré-éclampsie précoce. Le dépistage combiné a montré des résultats nettement meilleurs comparés avec les paramètres maternels ou Doppler seuls. Conclusion: Comme seul marqueur, le Doppler utérin du deuxième trimestre a la meilleure prédictive pour l'hypertension, la naissance prématurée et la restriction de croissance. La combinaison des caractéristiques démographiques maternelles, des biomarqueurs sériques maternels et du Doppler utérin améliore l'efficacité du dépistage, en particulier pour la pré-éclampsie nécessitant un accouchement prématuré. / Objective: To evaluate the screening efficacy of maternal demographic characteristics, serum biomarkers and uterine artery Doppler (uaD) during the first and the second trimester for the hypertensive disorders of pregnancy. To elaborate prediction models of these diseases based on the combination of selected maternal demographic characteristics, maternal serum biomarkers and uaD indexes. Methods: This is a prospective pregnant cohort study of 598 singleton nulliparous consecutive women. UaD investigation was performed by transabdominal sonography between 11+0 and 13+6 weeks, and between 17+0 and 21+6 weeks. All the serum samples for measurement of several placental biomarkers were collected at the first trimester. Maternal demographic characteristics were recorded at the same time. Receiver operating characteristic curves and predictive values were used to analyze the predictive powers of the above parameters. Different combinations and their logistic regression predictive models were analyzed. Results: Among 598 women, 20 developed preeclampsia (3.3%), 7 developed early-onset preeclampsia (1.2%), 52 developed gestational hypertension (8.7%), 10 developed gestational hypertension with delivery before 37 weeks (1.7%). Second trimester uterine artery pulsatility index was the best predictor with statistical significance for all the outcomes. In the multivariable logistic regression analysis, the best predictive value in the first and second trimester was obtained for the prediction of early onset preeclampsia. The combined screening showed significantly better results compared to either maternal parameters or Doppler alone. Conclusion: As a single marker, second trimester Doppler has the highest predictive value for hypertensive disorders, preterm birth and SGA. Combination of the maternal demographic characteristics, maternal serum biomarker and uaD improves the screening efficacy, especially when this necessitates early delivery.

RÉSUMÉ Objectif: Évaluer l'efficacité du dépistage de l’hypertension gestationnelle par les caractéristiques démographiques maternelles, les biomarqueurs sériques et le Doppler de l'artère utérine au premier et au deuxième trimestre de grossesse. Élaborer des modèles prédictifs de l’hypertension gestationnelle fondées sur ces paramètres. Methods: Il s'agit d'une étude prospective de cohorte incluant 598 femmes nullipares. Le Doppler utérin a été étudié par échographie transabdominale entre 11 +0 à 13 +6 semaines (1er trimestre) et entre 17 +0 à 21 +6 semaines (2e trimestre). Tous les échantillons de sérum pour la mesure de plusieurs biomarqueurs placentaires ont été recueillis au 1er trimestre. Les caractéristiques démographiques maternelles ont été enregistrées en même temps. Des courbes ROC et les valeurs prédictives ont été utilisés pour analyser la puissance prédictive des paramètres ci-dessus. Différentes combinaisons et leurs modèles de régression logistique ont été également analysés. Résultats: Parmi 598 femmes, on a observé 20 pré-éclampsies (3,3%), 7 pré-éclampsies précoces (1,2%), 52 cas d’hypertension gestationnelle (8,7%) , 10 cas d’hypertension gestationnelle avant 37 semaines (1,7%). L’index de pulsatilité des artères utérines au 2e trimestre est le meilleur prédicteur. En analyse de régression logistique multivariée, la meilleure valeur prédictive au 1er et au 2e trimestre a été obtenue pour la prévision de la pré-éclampsie précoce. Le dépistage combiné a montré des résultats nettement meilleurs comparés avec les paramètres maternels ou Doppler seuls. Conclusion: Comme seul marqueur, le Doppler utérin du deuxième trimestre a la meilleure prédictive pour l'hypertension, la naissance prématurée et la restriction de croissance. La combinaison des caractéristiques démographiques maternelles, des biomarqueurs sériques maternels et du Doppler utérin améliore l'efficacité du dépistage, en particulier pour la pré-éclampsie nécessitant un accouchement prématuré. Mot clés: Hypertension gestationnelle, Doppler utérins, Biomarqueurs sériques maternels, Caractéristiques démographiques maternelles, Dépistage, Modèle prédictif Multivarié. / ABSTRACT Objective: To evaluate the screening efficacy of maternal demographic characteristics, serum biomarkers and uterine artery Doppler (uaD) during the first and the second trimester for the hypertensive disorders of pregnancy. To elaborate prediction models of these diseases based on the combination of selected maternal demographic characteristics, maternal serum biomarkers and uaD indexes. Methods: This is a prospective pregnant cohort study of 598 singleton nulliparous consecutive women. UaD investigation was performed by transabdominal sonography between 11+0 and 13+6 weeks, and between 17+0 and 21+6 weeks. All the serum samples for measurement of several placental biomarkers were collected at the first trimester. Maternal demographic characteristics were recorded at the same time. Receiver operating characteristic curves and predictive values were used to analyze the predictive powers of the above parameters. Different combinations and their logistic regression predictive models were analyzed. Results: Among 598 women, 20 developed preeclampsia (3.3%), 7 developed early-onset preeclampsia (1.2%), 52 developed gestational hypertension (8.7%), 10 developed gestational hypertension with delivery before 37 weeks (1.7%). Second trimester uterine artery pulsatility index was the best predictor with statistical significance for all the outcomes. In the multivariable logistic regression analysis, the best predictive value in the first and second trimester was obtained for the prediction of early onset preeclampsia. The combined screening showed significantly better results compared to either maternal parameters or Doppler alone. Conclusion: As a single marker, second trimester Doppler has the highest predictive value for hypertensive disorders, preterm birth and SGA. Combination of the maternal demographic characteristics, maternal serum biomarker and uaD improves the screening efficacy, especially when this necessitates early delivery. Key words: Hypertensive disorders of pregnancy, Doppler, Maternal serum biomarkers, Maternal demographic characteristics, Screening, Multivariable predictive model.

Made available in DSpace on 2016-06-02T20:19:23Z (GMT). No. of bitstreams: 1 5772.pdf: 2838907 bytes, checksum: dd99c753250191bec4e2415172771de9 (MD5) Previous issue date: 2014-02-24 / Financiadora de Estudos e Projetos / Background: Muscle changes of paretic limb resulting from stroke lead to changes in the mechanical properties of muscles, such as muscle weakness and increased resistance to stretching. Studies that characterize the components of the muscle tissue, as the noncontractile, and biomarkers related to proliferation of connective tissue (such as TGF-&#946;1 and myostatin) are clinically relevant and necessary for understanding of the resistance to stretching of paretic muscles. Therefore, the aim of this study was to evaluate the serum concentration of TGF-&#946;1 and myostatin, the percentage volume of non-contractile tissue and passive peak torque and resistance to stretching (stiffness) of the extensor and flexor muscles of the knee in chronic hemiparesis. Methods: Cross-sectional study. Fourteen subjects with chronic hemiparesis post stroke and fourteen healthy paired-subjects participated in this study. Paretic, non-paretic and control limbs were evaluated. MRI images were obtained in all subjects and the percentage volume of non-contractile tissue of the quadriceps and hamstrings was measured. Serum TGF-&#946;1 and myostatin concentrations were quantified by ELISA method. Passive torque peak and resistance during stretching (stiffness) of extensors and flexors muscles of knee were assessed at 60°/s using isokinetic dynamometer. Results: An increase in the percentage volume of non-contractile tissue in VM and VL of paretic limb compared to non-paretic limb was observed (p<0,05). Also, an increase was observed in the percentage volume of non-contractile tissue in SS in paretic limb compared to control limb (p<0,05). No differences were observed in serum TGF-&#946;1 and myostatin concentrations in hemiparetic group compared to the control group (p> 0.05). Regarding passive torque, there was an increase in peak torque and resistance during passive stretching of extensor muscles with increasing ROM of paretic, non-paretic and control limbs (p<0,05), but no differences were found among limbs (p>0,05). In relation to flexor muscles, there was also an increase in peak torque along the ROM of the three limbs (p<0,05). However, non-paretic limb has lower values of peak torque than control and paretic limbs in lower ROM (p<0,05). Paretic limb increases resistance in a more accentuated pattern at intermediate ROM (50-40º) compared to control (p=0,02). A moderate correlation was observed between TGF-&#946;1 serum concentration and flexor peak torque of paretic limb, considering complete ROM (p=0,01; r=0,736). Conclusion: Paretic muscles, extensors and knee flexors, although they have increased noncontractile tissue, exhibit similar resistance to stretching the muscles of healthy subjects. Knee flexor muscles of the non-paretic limbs have less passive stretch resistance compared to healthy subjects without changes in non-contractile content. Furthermore, no changes in serum concentrations of TGF-&#946;1 and myostatin in chronic hemiparetic compared to healthy subjects were observed. / Contextualização: Alterações na musculatura do membro parético em decorrência do Acidente Vascular Cerebral (AVC) podem resultar em mudanças nas propriedades mecânicas da musculatura, como fraqueza muscular e aumento da resistência ao alongamento. Estudos que caracterizem os componentes do músculo, como o tecido não contrátil, e biomarcadores relacionados à proliferação de tecido conjuntivo (como TGF-&#946;1 e miostatina) são necessários e clinicamente relevantes para o entendimento da resistência ao alongamento de músculos paréticos. Portanto, o objetivo do presente estudo foi avaliar a concentração sérica de TGF-&#946;1 e miostatina, o volume percentual de tecido não contrátil e o pico de torque passivo e resistência ao alongamento ( stiffness ) dos músculos extensores e flexores do joelho de hemiparéticos crônicos. Materiais e Métodos: Estudo transversal. Quatorze sujeitos com hemiparesia crônica pós-AVC e quatorze sujeitos saudáveis pareados participaram deste estudo. Os membros parético, não parético e controle, foram avaliados. Imagens por ressonância magnética foram obtidas em todos os sujeitos e o volume percentual de tecido não contrátil dos músculos do quadríceps e dos isquiotibiais foi mensurado. As concentrações séricas de TGF-&#946;1 e miostatina foram quantificadas pelo método ELISA. O pico de torque passivo e a resistência ao alongamento passivo ( stiffness ) dos músculos extensores e flexores do joelho foram obtidos a 60°/s em dinamômetro isocinético e avaliados ao longo de intervalos de ADM. Resultados: Foi observado um aumento no volume percentual de tecido não contrátil nos músculos vasto medial (VM) e vasto lateral (VL) do membro parético comparado ao não parético (p<0,05). Ainda, houve um aumento no volume percentual de tecido não contrátil nos músculos semitendinoso e semimembranoso (SS) do membro parético comparado ao membro controle (p<0,05). Não foram observadas diferenças nas concentrações séricas de TGF-&#946;1 e miostatina entre os grupos (p>0,05). Houve um aumento no pico de torque e na resistência ao alongamento passivo extensor com o aumento da amplitude de movimento (ADM) nos membros parético, não parético e controle, sem diferenças entre os membros (p>0,05). Em relação aos flexores, também houve um aumento no pico de torque ao longo da ADM nos membros parético, não parético e controle. No entanto, o membro não parético apresenta valores de pico de torque menores que o parético e controle em amplitudes menores (p<0,05). O membro parético aumenta a resistência de forma mais acentuada em ADM intermediária (50-40º) comparado ao controle (p=0,02). Houve correlação moderada entre a concentração sérica de TGF-&#946;1 e o pico de torque flexor do membro parético, considerando-se a ADM completa (p=0,01; r=0,736). Conclusão: Músculos paréticos, extensores e flexores do joelho, apesar de possuírem aumento de tecido não contrátil, apresentam resistência ao alongamento semelhante a músculos de indivíduos saudáveis. Músculos flexores do joelho dos membros não parético apresentam menor resistência passiva ao alongamento comparado a indivíduos saudáveis, sem alterações no conteúdo não contrátil. Além disso, indivíduos hemiparéticos crônicos não apresentam alterações nas concentrações séricas de TGF-&#946;1 e miostatina comparados a indivíduos saudáveis.