Treament with oral anticoagulation is highly effective in reducing the burden of thromboembolic complications in several clinical conditions. The number of patients receiving oral anticoagulation is growing steadily. InSwedenabout 1.5 percent of the population receives treatment. Although the treatment is highly effective in preventing thromboembolic complications, it is also associated with a substantial increase in the risk of bleeding. In clinical practice every physician has to balance the potential benefit of treatment against the risk of bleeding complications in the individual patient. To aid in this decision making, risk scores addressing the likelihood of thromboembolic events, as well as the risk of bleeding complications, have been developed. These scores are imperfect and, to some degree limited by the fact that the risk factors predictive of thromboembolic events are also often associated with bleeding complications. The addition of biomarkers has the potential to increase the predictive ability of risk scores and further enhance the net benefit of oral anticoagulant treatment in the individual patient. In this thesis several potential biomarkers for thromoboembolic and haemorrhagic complications of anticoagulant therapy have been investigated in a longitudinal cohort study of 719 patients with a median follow-up time of 4.2 years. Thrombomodulin is a key component in the generation of activated protein C and hence, a coagulation inhibitor. Conversely, it is also a key component in the inhibition of fibrinolysis by activation of trombin-activated fibrinolysis inhibitor. In warfarin-treated patients we demonstrate that thrombomodulin predicts an increased risk of bleeding complications, but not cardiovascular events. Thus, thrombomodulin has potential as a biomarker specifically for bleeding complications. Von Willebrand factor plays a central and intricate role in the aggregation of platelets and low levels of VWF have been associated with bleeding as a manifestation of von Willebrand’s disease. In our study we noted that high levels of von Willebrand factor predict an increased risk of cardiovascular as well as all-cause mortality, possibly as an expression of endothelial dysfunction. We also noted that high levels of WVF seem to be associated with serious bleeding complications. Decreased renal function is usually measured by an increase in the levels of creatinine and cystatin C, or a decrease in the calculated glomerular filtration rate. A decrease in kidney function is regarded as a marker of an increased risk of bleeding complications. We investigated all the mentioned markers of kidney function and no association with bleeding complications became apparent. However, a clear association between a decrease in kidney function and mortality was noted. Our findings indicate that the emphasis on impaired kidney function as a risk marker needs to be shifted from bleeding complications toward thromboembolic events. Fibrinolysis is important in containing coagulation and several constituents of the fibrinolytic pathway have been shown to predict cardiovascular events and mortality. We found that fibrinolytic factors seem to predict cardiovascular events in patients with oral anticoagulation and that D-dimer also predicts bleeding complications. In conclusion, we have found several biomarkers which exhibit different predictive abilities in patients with oral anticoagulation. It is likely that biomarkers, either alone, in combination, or as ancillary components of risk scores, can contribute to improved risk stratification in patients with oral anticoagulation.

Alzheimer’s is a widespread neurodegenerative disease, growing larger and larger in the world. Once developed, the disease has no cure. To this day, there is only mitigating drugs. To be able to start this treatment rapidly, a method to distinguish healthy individuals from prospective Alzheimer’s diseased needs to be developed. Cerebrospinal fluid is thought to contribute to the development of such method, through its close substitution of fluids, molecules and proteins from the brain. It may provide a progressive marker of the disease, a substance differently expressed in the healthy and diseased; a biomarker. The aim of the present study was to develop and evaluate a stable method for degradation and analysis of peptides and proteins in the cerebrospinal fluid using mass spectrometry techniques, such as selective reaction monitoring. Mass spectrometry is often used after a first dimension separation with liquid chromatography. Three degradation methods were evaluated, resulting in a protocol with the detergent DOC being the most beneficial. Tryptic peptides occurred in a concentration of 10 % w/v due to the SDS-PAGE gels and database searches concomitantly. The elution pattern from the liquid chromatography enables a narrow selection in the sensitivity for each peptide. Chromatographic preferences such as column, hydrophobicity and time span was determined, and unwanted peptides filtered away. A specific protein, the amyloid precursor protein APP, is thought to play a significant part in the development of Alzheimer’s disease. The protein is located in the neurons, cleaved and processed to produce the neurodegenerating plaques found in the brain at the diseased. Three isoforms are found in the neurons, APP695, APP751 and APP770. When cleaved, a shorter soluble tryptic peptide is generated from all APP isoforms. This was the target for the current study, as a potential progressive biomarker. The method developed was able to separate and distinguish the soluble APP751 isoform, but not the APP695 or APP 770 isoforms, most probably due to glycosylations of the two resembling isoforms.

El càncer de pròstata (CP), és la segona causa de mort per malaltia oncològica en els homes del món occidental. S’estima que un de cada sis homes desenvoluparà un càncer d’aquest tipus al llarg de la seva vida. El CP afecta com el seu nom indica, a la pròstata. La pròstata, és un òrgan glandular de l’aparell genital-urinari masculí. Té la mida d’una nou i es localitza sota de la bufeta, envoltant la uretra i davant del recte. La seva funció és secretar productes que s’afegiran al líquid seminal amb la finalitat de nodrir i protegir els espermatozous. El actual diagnòstic del CP es basa en una triada diagnòstica que consta de; l’anàlisi dels nivells de PSA (Prostate Specific Antigen) en sèrum, el tacte rectal (TR) i finalment la biòpsia prostàtica. Quan els nivells de PSA en sèrum es situen per sobre de 4 ng/mL i/o el TR és sospitós, l’uròleg pot estimar quina és la probabilitat que el pacient estigui afectat per un CP i per tant decidir la necessitat de practicar o no una biòpsia prostàtica, que permetrà establir el diagnòstic definitiu. La introducció del test del PSA, a finals dels anys 80, s'ha traduït en una millora del diagnòstic precoç del CP, moment en el qual les opcions de tractament per el són eficaces. No obstant això, malgrat aquesta detecció primerenca, la mortalitat per CP no ha disminuït significativament en els últims anys. El principal problema del PSA com a marcador d’screening és que aquest presenta un nivell d’especificitat baix (30% aprox) i alhora, un valor predictiu negatiu baix. Això es tradueix en que al voltant d'un terç de tots els homes sotmesos a una biòpsia seran positius per CP. Com a resultat dels seus persistents nivells sèrics de PSA, però els resultats negatius de la biòpsia, aquests homes es sotmeten a repetides biòpsies. Tot i el importants avenços en la investigació de biomarcadors de CP, alguns homes encara estan sobre-diagnosticats de CP “indolent”, mentre que d’altres moren de malaltia agressiva que s'ha diagnosticat massa tard. Aquesta situació es coneix com "dilema diagnòstic". És per tot això, que el càncer de pròstata, es beneficiaria de l’existència de nous marcadors d’screening més específics i alhora d’un diagnòstic menys invasiu. Per altra banda, una millora en el diagnòstic evitaria un gran nombre de biòpsies innecessàries i conseqüentment un important estalvi econòmic en el cost sanitari actual. La recerca de nous marcadors en el càncer de pròstata suposa un camp de treball important en la detecció precoç d’aquest tipus de càncer. Donada la situació de la pròstata a l’organisme, sota la bufeta i envoltant la uretra, les secrecions i inclús les mateixes cèl·lules prostàtiques, ja siguin normals o malignes, poden trobar-se presents en l’orina. És per això que considerem l’orina com una font important d’informació, a través de la qual es podria arribar a determinar quina situació s’està donant a l’òrgan en qüestió. Altres estudis evidencien l’existència de potencials biomarcadors en l’orina que podrien ajudar en la millora del diagnòstic del CP. A nosaltres ens ocupa l’estudi d’aquelles molècules de RNA així com de les molècules protèiques que es troben a l’orina. Suposem doncs, que un massatge prostàtic enriqueix la mostra d’orina de tot tipus de molècules proteiques. Així doncs, la nostra hipòtesi de treball recolza que, l’orina després d’un massatge prostàtic pot ser el fluid ideal per a la recerca de nous biomarcadors capaços de discriminar entre pacients amb o sense càncer de pròstata. L’objectiu principal de l’estudi és diagnosticar el CP assimptomàtic per us sistema minimament invasiu, que consisteix en l’anàlisi de l'ARN o de la proteïna en l'orina obtinguda després d’un massatge prostàtic i, superar la baixa especificitat de l’actual sistema d’screening (PSA en sèrum) mitjançant l'ús de biomarcadors addicionals (RNA o proteina) per tal de reduir el nombre de biòpsies innecessàries (reduir els costos socio-econòmics, així com la reducció dels efectes secundaris no desitjats). 1. ANÀLISI TRANSCRIPTÒMICA: 1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al., Prostate. 2010 Dec 1;70(16):1760-7. En el primer estudi, mitjançant l’anàlisi de transcripts per RTqPCR en 215 (34% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), es va determinar la utilitat de PSGR (Prostate Specific G-coupled receptor), previament descrit com a sobre-expressat en mostres de teixit de CP, com un nou biomarcador, comparable a PCA3 (ja conegut), per a la detecció del CP en orines (taula 1). Per l'anàlisi de la corba ROC vàrem determinar els valors de l’Area sota la corba (AUC) de PSGR (AUC 0,68) i PCA3 (AUC 0,66). Fixant la sensibilitat a 95%, aconseguim un valor d’especificitat de 15% per PSGR i el 17% de PCA3. Alhora vàrem veure que al combinar els dos biomarcadors (PSGRvPCA3) mitjançant un anàlisi “multiROC”, els resultats van millorar significativament en termes de rendiment del biomarcador (AUC 0,73) com de la seva especificitat (34%). En conclusió, podem dir que PSGR presenta un comportament similar al biomarcador estàndard per CP en orina (PCA3), però, és necessàri la realització d’estudis futurs per millorar encara més el rendiment d'aquesta prova. 1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. En un segon estudi, vàrem estudiar la possibilitat de multiplexar diferents biomarcadors, per tal de ser capaços de guanyat especifcitat sense perdre sensibilitat en la detecció del CP. Donada l’heterogeneïtat del CP la idea d’utilitzar diferents marcadors per a la seva detecció es presenta atractiva. Mitjançant l’anàlisi de transcripts, per RTqPCR, en 215 (37% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), vàrem determinar la utilitat de PSMA (Prostate Specific Membrane Antigen), un marcador descrit anteriorment com a sobre-expressat en el CP, com a biomarcadors per a la detecció de CP en orina. L’anàlisi de corbes ROC revela un rendiment similar PSMA (AUC 0,62), PSGR (AUC 0,65) i PCA3 (AUC 0,60) (Taula 1). Per tal de millorar l'eficàcia diagnòstica d'aquests biomarcadors es van combinar PSMAvPSGRvPCA3 (3M), utilitzant un anàlisi de multiROC. L’AUC del 3M (AUCm) (0,74) millora notablement, en comparació amb els valors individuals per a cada un dels marcadors. D'altra banda, si combinem 3M amb el valor de PSA en sang l’AUCm (0,77) va ser encara millor (taula 11). Si fixem la sensibilitat a 96%, l'especificitat del model 3M manté una especificitat del 34%, mentre que no es va observar augment en d’aquest valor quan ho combinem amb PSA en sang (34%) (Taula 11). El comportament d'aquests tres marcadors en una subpoblació de l’estudi que presentava nivells de PSA en sang entre 4-10 ng/mL i sense informació d’una biòpsia prèvia "zona gris del PSA" és de PSMA (AUC 0,74), PSGR (AUC 0,66) i PCA3 (AUC 0,61). Per al model combinat (3M), l’AUCm és de 0,82 (Taula 11). Fixant la sensibilitat al 96%, l'especificitat del model combinat (3M) manté una especificitat del 50%. En conclusió, aquests resultats demostren que la combiació de diferents marcadors proporciona una major especificitat a la prova sense comprometre el valor de sensibilitat. Expresat en el nombre de biopsies que s’haurien pogut estalviar, els valors se situen al voltant del 34% de biòpsies estalviades. 1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia” Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. El tercer estudi, està centrat en la utilitat, del ja conegut biomarcador per CP en orina (PCA3), per a l’identificació una lessió pre-maligna a la pròstata, així com la seva utilitat en la selecció dels homes amb biòpsies negatives, que requereixen la repetició del procediment. La troballa de les lesions de HGPIN (High grade preneoplastic intraepithelial lesion) és una indicació freqüent de la repetició de la biòpsia. Tot i la controvèrsia al voltant de la definició de HGPIN com a lessió pre-maligna i la necessitat de repetició de la biòpsia, encara avui en el nostre hospital s’indica el control exhaustiu del pacient, així com la repetició de la biòpsia en un periode de relativament curt de temps. Per a aquest estudi es van seleccionar un subgrup de pacients amb HGPIN (n=64) i com a grups control, es van seleccionar els homes amb CP (n=83) i homes amb patologia benigne de la pròstata (BP) (n=97). Després de l’anàlisi dels transcripts de PCA3 per RTqPCR en mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), i de l'anàlisi de la corba ROC, es van obtenir els següents resultats; HGPIN vs CP (AUC 0,63) i BP vs PC (AUC 0,71). Aquests resultats demostren la capacitat dels transcripts de PCA3, en orina obtinguda deprés d’un massatge prostàtic, per a l’identificació d’homes amb CP així com la identificació de pacients amb HGPIN. 2. ANÀLISI PROTEÒMICA COMPARATIVA L’objectiu principal d’aquesta segona part és determinar un perfil proteòmic a l’orina capaç de diferenciar entre pacients amb CP i controls sans. Per dur a terme aquest objectiu ens vàrem plantejar fer una part inicial de discovery, que vàrem complementar amb “data mining” i finalment vàrem verificar mitjançant una tècnica proteòmica independent. 2a) Per la part de discovery es va dur a terme l’anàlisi comparativa de “Differential in Gel Electrophoresis (DIGE). Es van fer dos experiments independents, amb mostres de sobrenedants d’orines (on trobem la fracció soluble de les proteïnes). En el primer experiment es van comparar 6 mostres de proteïna total de pacients amb CP i 6 mostres controls. En el segon experiment es van comparar 9 mostres, de proteïna pre-tractada amb una tècnica de depleció per les proteïnes majoritàries, de pacients amb CP i 9 mostres de pacients control. Un total de 24 proteïnes (9 sobre- i 15 infra-expresades) vàren ser identificades per MALDI-TOF. Una anàlisi informàtic de les proteïnes va mostrar que la majoria d'aquestes proteïnes identificades es secreten els components de diverses xarxes ben conegudes, el càncer funcionals i la inflamació, com ara NFКB, PDGFBβ i β-catenina. D'altra banda, a causa del fet que hem utilitzat sobrenadants d'orina, on podem trobar proteïnes solubles secretades, la majoria de les proteïnes identificades (62%) es van localitzar en l'espai extracelular. Mitjançant una tècnica de proteòmica dirigida (Selected Reaction Monitoring: SRM) es van quantificar de forma relativa, sense l’adició d’estandards interns marcats, un total de 15 (dels 24 biomarcadors) en una població independent de 50 mostres (38% amb CP). Després d'una anàlisi de regressió logística de les dades obtingudes a través de l'assaig de SRM, es va obtenir un panell de set pèptids que corresponen a 5 proteïnes, capaços de distingir entre les mostres de CP i mostres controls amb una sensibilitat del 95% i una especificitat del 78 %. 2b) La segona part de l’estudi de proteómicas es basa en la verificació de les proteïnes prèviament qualificades i alhora l’estandarització dels mètodes de preparació de mostres per al seu posterior anàlisis mitjançant la tècnica de SRM. Les 5 proteïnes que formen part del panell identificat, juntament amb altres proteïnes biologicament rellevants, definides a la nostre fase de discovery, i juntament amb altres proteïnes descrites a la literatura, vàren ser escollides per una segona fase de qualificació i verificació en una població independent de 107 pacients, mitjançant un assaig de SRM en el qual es pretén quantificar de forma absoluta (adició de pèptids estàndards marcats), un total de 42 proteïnes. Prèviament a l’evaluació d’aquestes proteïnes, es va dur a terme una estandarització del protocol d’extracció de proteïna en mostres d’orina, amb l’objectiu de maximitzar la senyal obtinguda en els experiments de SRM. La precipitació amb Acetonitril (a temperatura ambient) seguida de la digestió de les proteïnes en solució amb condicions de ratio 1:10 (trypsine:protein), “over-night” i l’adició de “NAcetyl cysteine”, proporcionen els millors valors de quantificació absoluta. L’anàlisi estadístic de les dades generades d’aquesta segona fase de qualificació i verificació de les proteïnes està encara inacabat, de manera que ara per ara no podem conclou-re res. En conclusió, les dos linies experimentals que aquí es presneten (ARN i proteïnes) represeten resultats prometedors. No obstant això, estudis de validació en poblacions independents (del tipus estudi multicèntric) són necessaris per acabar amb un biomarcador o un panell de biomarcadors vàlid per al diagnòstic del CP. Per altra banda, el seguiment dels pacients ja analitzats poden relevalar dades interessant, com la utilitat pronòstica d’aquests biomarcadors. Els resultats obtinguts haurien de tenir una aplicació clínica ràpida que, juntament amb els paràmetres d’screening actual (nivells sèrics de PSA i DRE), podrien ajudar en la pressa de decisions per CP. / The prostate gland is a walnut-sized organ of the male urinary-genital tract that is located below the bladder surrounding the urethra and in front of the rectum. The most important disease affecting prostate is prostate cancer (PCa). PCa is the most common cause of cancer death, and it is the second most common cause of cancer death among men in the Western world. The risk of developing this type of cancer during a lifetime is estimated at 1 in 6 men in the US, and the risk of death due to this disease is 1 in 36. The current diagnosis of PCa is based on a triad consisting of diagnosis, analysis of the levels of PSA (Prostate Specific Antigen) in serum, the digital rectal examination (DRE) and finally the prostate biopsy (PB). When serum PSA levels are above 4 ng/mL and / or when DRE is suspected, the urologist can estimate how likely the patient is affected by PCa and therefore decided the need to practice or not a PB, which is the gold standard for PCa diagnosis. The introduction of PSA testing in the late 80s, has resulted in an improvement of early diagnosis of PCa, at which time options for treatment are effective. However, despite this early detection, mortality PCa has not decreased significantly in the last 50 years. The main limitation of serum PSA as a tumor marker is its lack of specificity (around 30%) at the cut-off value of 4 ng/mL, and also a low negative predictive value (NPV), which results in a high rate of negative biopsies. Elevated PSA levels can also be attributed to other factors such as benign prostatic hyperplasia (BPH), prostatitis, etc. As a consequence of the current screening parameters, around 2/3 of the approximately 1,300,000 biopsies made yearly in the United States and 390,000 in Europe are unnecessary. In contrast, the false positive rate of a biopsy is about zero, although the false negative rate in the first biopsy may oscillates around 20%. As a result of their persistently elevated PSA levels, but negative biopsy results, these men undergo repeated biopsies to rule out PCa. This situation is called the “PSA dilemma”. For these reasons, PCa would benefit from the existence of new markers for screening and also a more specific diagnosis less invasive. Furthermore, an improvement in diagnosis would avoid many unnecessary biopsies and consequently a significant savings in the cost of health today. The search for new markers in PCa is an important field of work in the early detection of this cancer. Urine has been defined as a liquid biopsy of the urogenital tract, and it can provide much more information about these organs (including the prostate) than a tissue biopsy. Urine obtained after DRE can easily serve as a mirror of what is happening within the prostate. Furthermore, urine collection can be accomplished without disruption of clinical standard practice. It can also be repeated several times throughout the course of the prostatic disease. For all of these reasons, urine can serve as a potential source of prostate disease biomarkers. Nevertheless, using urine for biomarker discovery represents an important technical challenge, both in transcriptomic and proteomic approaches. Although there are some studies that focus on those approaches and biomarker discovery and identification, there still exists some controversy regarding the standardization of collection procedures, sample processing, storage and normalization. We hypothesized that the utilization of targeted genomic and proteomic techniques on urine samples from patients suspected of having PCa can provide a pattern of biomarkers able to efficiently distinguish between the presence or absence of a prostate carcinoma and, further, can help to identify clinically significant prostate cancer patients. The main objective of this study is to diagnose asymptomatic PCa by non/minimally-invasive means using RNA or Protein in urine after prostate massage, and to overcome the low specificity of PSA by the use of additional biomarkers to reduce the number of unnecessary biopsies (reduce financial costs for society, reduction in unwanted secondary effects). 1. TRANSCRIPTOMIC APPROACH: In recent years, the explosion of genomic and transcriptomic approaches have resulted in increased biomarker discovery. The recent discovery of Prostate Cancer Gene 3 (PCA3) in urine as a biomarker for the detection of PCa and studies to determine its applicability in routine diagnosis represent a significant success for the scientific community in this field. First, we wanted to characterize a new urine candidate biomarker (PSGR) to be compared with PCA3, and second, we planned to use a panel of biomarkers, in order to improve diagnostic accuracy. Finally, we proposed to better characterize the well-known biomarker PCA3 as a tool for the early detection of pre-neoplastic PCa lesions, such as High grade prostatic intraepithelial neoplasia (HGPIN). 1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al., Prostate. 2010 Dec 1;70(16):1760-7. PSGR is a member of the G-protein coupled OR family. PSGR has previously been described to be highly prostate tissue-specific and over-expressed in PCa tissue. Our aim was to test whether PSGR could also be detected by RTqPCR in urine sediment obtained after prostate massage (PM). A total of 215 urine samples were collected from consecutive patients (34% with PCa), who presented for PB due to elevated serum PSA levels (> 4 ng/mL) and/or an abnormal DRE. These samples were analyzed by RTqPCR. By univariate analysis we found that PSGR and PCA3 were significant predictors of PCa. A Reciever Operator Characteristics (ROC) curve was used to assess the outcome predictive values of the individual biomarkers. We obtained the following Area Under the Curve (AUC) values: PSGR (0.68) and PCA3 (0.66). Both markers individually overcame the AUC value for serum PSA (0.60). Finally, we combined those markers to test if a combination of both biomarkers could improve the sensitivity of PCA3 alone. By using a multivariate extension analysis, multivariate ROC (MultiROC), the outcome predictive values of the paired biomarkers were assessed. We obtained an AUC value of 0.73 for the combination of PSGR and PCA3 (PSGRvPCA3). Then, we tested whether a combination of PSGR and PCA3 could improve specificity by fixing the sensitivity at 95%. We obtained specificities of 15% (PSGR) and 17% (PCA3) for each individual marker and 34% for PSGRvPCA3. In summary, a multiplexed model that included PSGR and PCA3 improved the specificity for the detection of PCa, especially in the area of high sensitivity. This could be clinically useful for determining which patients should undergo biopsy. 1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. Much evidence points to the fact that a single marker may not necessarily reflect the multifactorial and heterogeneous nature of PCa. The principle that underlies the combined biomarker approach is consistent with tests offered for the detection of PCa in tissue specimens and takes into consideration the heterogeneity of cancer development based on a diagnostic profile. The combined model that results from these combinations provides overall increased sensitivity without decreasing the specificity. Following the same approach than in our previous work, we combined three biomarkers to maximize individual specificities. Prostate Specific Membrane Antigen (PSMA), another well-known PCa biomarker, was used to test whether a combination of PSGR, PCA3 and PSMA was able to improve the specificity of the current diagnostic technique. We analyzed post-PM urine samples from 154 consecutive patients (37% with PCa), who presented for PB due to elevated serum PSA levels (>4 ng/mL) and/or an abnormal DRE. We tested whether the putative PCa biomarkers PSMA, PSGR, and PCA3 could be detected by RTqPCR in the post-PM urine sediment. By univariate analysis, we found that the PSMA, PSGR, and PCA3 scores were significant predictors of PCa. We then combined these findings to test if a combination of these biomarkers could improve the specificity of an actual diagnosis. Using a multiplex model (PSGRvPCA3vPSMA), the area under the MultiROC curve (AUCm) was 0.74, 0.77 with PSA and 0.80 with PSA density (PSAD). Fixing the sensitivity at 96%, we obtained a specificity of 34%, 34% with PSA and 40% with PSAD. Afterwards, we specifically tested our model for clinical usefulness in the PSA diagnostic ‘‘gray zone’’ (4–10 ng/mL) on a target subset of 82 men with no prior biopsy (34% with PCa) and a target subset of 77 men with the PSAD information (35% with PCa). Using a multiplex model, the AUCm was 0.82, 0.89 with PSAD. Fixing the sensitivity at 96%, we obtained a specificity of 50% and 62% with PSAD in the gray zone. This model would allow 34% of the patients to avoid unnecessary biopsies in the gray zone (42% when using PSAD). 1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia” Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. An ideal biomarker for the early detection of PCa should also differentiate between men with isolated HGPIN and those with PCa. PCA3 is a highly specific PCa gene, and its score in post-PM urine seems to be useful in ruling out PCa, especially after a negative PB. The biopsy finding of an HGPIN is a frequent indication that the PB should be repeated. The aim of this study was to determine the efficacy of post-PM urine PCA3 scores for ruling out PCa in men with previous HGPIN. The PCA3 score was assessed by RTqPCR in 244 post- PM urine samples collected from men subjected to PB (64-isolated HGPIN, 83-PCa, and 97-benign pathology findings (BP)). The median PCA3 score was 1.56 in men with BP, 2.01 in men with isolated HGPIN and 9.06 in men with PCa. A significant difference was observed among the three scores (p < 0.001) and also between HGPIN and PCa (p = 0.008); however, no differences were observed between HGPIN and BP (p = 0.128). The AUC in the ROC analysis was 0.71 in the subset of men with BP and PCa, while it decreased to 0.63 when only men with isolated HGPIN and PCa were included in the analysis. Finally, the median of the PCA3 scores was assessed in men with previously diagnosed unifocal HGPIN (2.63) and in men with previously diagnosed multifocal HGPIN (1.59). No differences were observed between unifocal and multifocal HGPIN (p = 0.56). In conclusion, the efficacy of post-PM urine PCA3 scores in ruling out PCa in men with HGPIN is less than in men with BP. For this reason, when HGPIN is found at PB, these results should be taken into consideration, in order to establish the clinical usefulness of the PCA3 score as a tool for avoiding unnecessary repeated biopsies. 2. PROTEOMIC APPROACH: The high-throughput proteomic analysis of urine samples has recently become a popular approach for the identification of novel biomarkers. Proteins secreted by cancer cells, also referred to as "cancer cell secretomes," are a promising source for biomarker discovery. A great advantage to these cancer-secreted proteins and/or their fragments is that in most cases, they enter body fluids, such as blood or urine, and therefore, can be measured via non-invasive assays. Since the protein products of PCa cells can be detected in urine, their use as a proximal body fluid in the detection of PCa is very attractive. 2a.“The Discovery and Qualification of a Panel of Urine Biomarkers for Prostate Cancer Diagnosis”. In this study we used DIGE proteomic analysis on 30 age-matched, post-PM urine supernatant specimens, in order to identify the differentially expressed proteins in patients with PCa. 24 potential biomarkers were identified, the majority of which were secreted proteins associated with several wellknown, functional cancer pathways. Qualification of 15 of the 24 identified biomarker candidates was then undertaken by relative quantification using an SRM-based assay (target) on 50 post-PM urine supernatant samples (38% with PCa). After statistical analysis, 7 peptides, corresponding to 5 different proteins, were selected. A multiplex ROC curve using those 7 peptides showed an AUC value of 0.93. Fixing the sensitivity at 95%, we achieved a specificity of 78%. 2b. “Qualification and Verification of Urine PCa Candidate Biomarkers with Selected Reaction Monitoring”. The qualification and verification of candidate biomarkers is a critical stage in the great biomarker discovery pipeline. Credentialed biomarkers that have successfully passed through this stage are considered verified biomarkers, which are of high value for translation into large-scale, clinical validation studies. The evaluation of biomarkers in body fluids necessitates the development of robust methods to quantify proteins in body fluids, using large sets of samples. In the present study, we performed the qualification of a set of 42 candidate biomarkers for PCa diagnosis on a set of 107 post- PM urine supernatant samples (36% with PCa) using SRM-based absolute quantification. Before that, urine sample preparation and analytical procedures were optimized for SRM methodology. We standardized preparation of the urine protein samples for SRM analysis by using 9 different protocols. Our final goal was to obtain a panel of biomarkers that alone, or in combination with the existing PCa biomarker, would help us to better define patients with PCa. In addition, due to the large number of samples and their pathological conditions, we would also be able to define candidate prognostic markers. However, this study has yet to be completed. In conclusion, the data presented in this dissertation represent a significant advance in the standard care for PCa diagnosis. Our two approaches (RNA- and Protein-based) have begun to yield promising results, as both have levels of specificity that exceed those of PSA. However, validation studies on larger, multi-centric cohorts of urine samples are needed to end up with a valid PCa biomarker. The obtained results should have a rapid application in the clinics and potentially influence, together with actual screening parameters (serum PSA and DRE), decisions that could improve the health system, as well as clinical, managerial and/or public practices for health outcomes in PCa.

Η ανάπτυξη των ανθρώπινων δραστηριοτήτων έχει οδηγήσει τα τελευταία χρόνια στην παραγωγή και απελευθέρωση πολλών τοξικών ουσιών στο θαλάσσιο περιβάλλον. Τα δίθυρα μαλάκια, όπως το Mytilus galloprovincialis, μπορούν να συσσωρεύουν ένα μεγάλο εύρος ρυπαντών και να αποκρίνονται με ποικίλες αλλαγές στη φυσιολογία τους, οι οποίες μπορούν να μετρηθούν ως βιοδείκτες έκθεσης ή αποτελέσματος. Το περιβαλλοντικό stress ωθεί πολλούς οργανισμούς στην αρνητική ρύθμιση της πρωτεϊνοσύνθεσης, αποθηκεύοντας ανενεργά ριβοσώματα, τα οποία επανενεργοποιούνται ταχέως όταν οι συνθήκες βελτιωθούν. Έτσι, ένας αποτελεματικός τρόπος εκτίμησης της απόκρισης των οργανισμών στο μολυσματικό stress, είναι η ανάλυση της κατάστασης ριβοσωμικής συσσώρευσης, όπως επίσης και η ικανότητα των ριβοσωμάτων για έναρξη της πρωτεϊνικής σύνθεσης, που αντανακλούν την κατάσταση της μεταφραστικής ενεργότητας. Στην παρούσα μελέτη έγινε εκτίμηση των επιπέδων ρύπανσης του Πατραϊκού Κόλπου, καταποντίζοντας μύδια (M. galloprovincialis) σε δύο σταθμούς στον Πατραϊκό Κόλπο και μία περιοχή αναφοράς. Ο Σταθμός 1 βρίσκεται στις εκβολές του ποταμού Γλαύκου και είναι υπό την επίδραση βιομηχανικών, αγροτικών και αστικών πηγών ρύπανσης. Ο Σταθμός 2 βρίσκεται στον Άγιο Βασίλειο, παρ’ ότι δε φέρει οργανική μόλυνση, έχει αυξημένα επίπεδα βαρέων μετάλλων και κυρίως Cr. Η περιοχή αναφοράς βρίσκεται κοντά στο Γαλαξείδι και δεν επηρεάζεται από πηγές ρύπανσης. Για την εκτίμηση των επιπέδων ρύπανσης στις περιοχές αυτές, εφαρμόστηκε μία σειρά βιοδεικτών που περιλαμβάνει το περιεχόμενο μεταλλοθειονινών, τη σταθερότητα της λυσοσωμικής μεμβράνης και τη συχνότητα μικροπυρήνων. Επίσης, προσδιορίστηκε η συγκέντρωση μετάλλων στο νερό και στους ιστούς των μυδιών, όπως επίσης και το ποσοστό πολυσωμάτων και η ικανοτητα των ριβοσωμάτων για έναρξη της μετάφρασης σε κύτταρα πεπτικού αδένα. Επιπλέον, έγινε στατιστική εκτίμηση της συσχέτισης μεταξύ των επιπέδων ρύπανσης και των εξεταζόμενων βιοδεικτών. Η ανάλυση των βιοδεικτών έδειξε οτι υπάρχει μία διαφοροποίηση στα επίπεδα ρύπανσης ανάμεσα στις διαφορετικές περιοχές του Πατραϊκού Κόλπου, με το μεγαλύτερο βαθμό ρύπανση να εντοπίζεται στο Σταθμό 1. Οι εποχιακές διακυμάνσεις που παρατηρούνται στις τιμές των βιοδεικτών μπορούν να αποδοθούν σε εξωγενείς παράγοντες, όπως τα επίπεδα της ρύπανσης και/ή σε ενδογενείς παράγοντες, όπως ο αναπαραγωγικός κύκλος των μυδιών. Η στατιστική επεξεργασία των αποτελεσμάτων έδειξε οτι υπάρχει σημαντική συσχέτιση (θετική ή αρνητική ανάλογα με το βιοδείκτη) μεταξύ των επιπέδων ρύπανσης και των βιοδεικτών. Το χαμηλότερο πολυσωμικό περιεχόμενο σε συνδυασμό με τη μειωμένη ικανότητα των ριβοσωμάτων για έναρξη της μετάφρασης που παρατηρήθηκε στο Σταθμό 1, καταδεικνύει την επίδραση του περιβαλλοντικού stress στην πρωτεϊνική σύνθεση στην περιοχή αυτή και εισηγείται τη χρήση των διαταραχών της μετάφρασης ως εργαλείο σε μελέτες βιοπαρακολούθησης. Τα παρόντα δεδομένα σε σύγκριση με εκείνα των τελευταίων πέντε ετών αποκαλύπτουν μία προοδευτική μείωση των επιπέδων ρύπανσης του Πατραϊκού Κόλπου, οφειλόμενη πιθανώς στην απομάκρυνση πολλών εργοστασίων από την περιοχή, και στη λειτουργία σταθμού βιολογικού καθαρισμού. / The rapid increase of anthropogenic activities has led to the production and release of several harmful compounds into the marine environment. Bivalve mollusks such as Mytilus galloprovincialis, are able to accumulate in their tissues a wide range of pollutants and respond in a broad range of alterations that can be measured as exposure or effect biomarkers. Such changes are usefull warning tools in marine environment monitoring. Environmental stress forces many organisms to downregulate translation by storing inactive ribosomes, which are rapidly reactivated when conditions improve. Therefore, an effective way to reveal responses to pollution stress is to analyse the aggregation state of ribosomes which reflects the translational efficiency status. Parallel determination of the capability of ribosomes to initiate protein synthesis may help to understand the mechanism of translation downregulation. In the present study, the pollution status of Patraikos Gulf was assessed by caging mussels (M. galloprovincialis) in two stations of the gulf and in a reference area. Station 1 was near the estuaries of Glafkos River, influenced by industrial, agricultural and urban sources. Station 2 located in Agios Vassileios, had no apparent organic pollution, but was enriched in heavy metals, particularly Cr. Reference area was near Galaxidi, far from pollution sources. To verify the degree of pollution in these areas, a battery of biomarkers was assessed, including metallothionein content, lysosomal membrane stability and micronucleus frequency. In addition, metal ion concentrations in the surrounding waters and in mussel tissues as well as polysome content and the efficiency of ribosomes to initiate translation in digestive gland cells were estimated. Furthermore, an effort was made to reveal the correlation between pollution and the employed biomarkers. Biomarker analysis revealed a differentiation in the degree of pollution among different sites of Gulf of Patras. Seasonal variations in the mean values of the biomarkers can be attributed to exogenous factors, like pollution levels, and/or endogenous factors like reproductive cycle of the mussels. Statistical analysis of the data showed a significant correlation (positive or negative, depending on the biomarker) between the pollution levels and the biomarkers.The lower polysome content in combination with the reduced efficiency of the ribosomes to initiate translation observed in Station 1, indicates the effects of pollution stress on protein synthesis in this area and encourages the evaluation of the translational perturbations as a tool in biomonitoring studies. The present data, compared to those collected the past five years, reveal a progressive pollution depression in Patraikos Gulf, propably caused by the removal of many factories previously contaminating this area, and the recent operation of a local sewage cleaning station.

d-Limonene is a lipid-soluble bioactive food component found in citrus peel. It has demonstrated strong chemopreventive effects in rodent mammary, gastric, skin, liver, and lung cancers and is correlated with a significantly reduced risk for human squamous cell skin carcinoma. Because d-limonene is fat-soluble, it may have enhanced chemopreventive activity in fatty tissues such as breast. No previous methodology to quantify d-limonene in the adipose had been developed which significantly limited d-limonene tissue distribution research. For this research, an assay to extract d-limonene from adipose tissue and quantify with GC/MS was developed and validated. Linear calibration curves were established over the range of 0.0-2,526 ng d-limonene. Extraction recovery was 80%. Satisfactory within day precision (RSD 6.75 to 9.56%) and accuracy (% difference of 2-4%) were achieved. This sensitive, accurate, and precise assay to quantify d-limonene in adipose can be used for future human or animal fatty tissue deposition studies.

No description available.

Background: Hypertension is a pathological increase in blood pressure that affects nearly 30% of the U.S. population and is a primary modifiable risk factor for cardiovascular disease. Despite advancements in prevention and treatment, hypertension is still one of the most common conditions around the world, and for a majority of cases the causal mechanisms remain to be fully elucidated. A growing body of literature suggests that oxidative stress status may play an etiological role in many chronic conditions, including hypertension. Specifically, a systemic overabundance of reactive oxygen species may give rise to endothelial dysfunction, increased sodium and H2O retention, and alterations in sympathetic outflow, leading to an increase in blood pressure. Purpose: The main objective of this study is to investigate the prospective association between F2-isoprostanes, a validated biomarker of oxidative status, and development of hypertension in a large, multi-centered, multi-ethnic cohort of adults aged 40-69 at baseline. Methods: This is a secondary data analysis that utilized previously collected data from the Insulin Resistance Atherosclerosis Study. 844 participants were included in the analysis. Briefly, four urinary F2-isoprostane isomers (F2-IsoP1, F2-IsoP2, F2-IsoP3, and F2-IsoP4) were quantified using liquid chromatography/ tandem mass spectrometry and adjusted for urinary creatinine levels. Hypertension was assessed at baseline and follow-up visits and defined as systolic blood pressure > 140 mm Hg and/or diastolic blood pressure > 90 mm Hg and/or currently taking antihypertensive medications. Crude associations between study population characteristics and hypertensive status were analyzed with the chi-square and Wilcoxon-rank sum tests. Crude associations between study population characteristics and F2-isoprostane levels were analyzed with Wilcoxon-rank sum, Kruskal-Wallis, and Spearman’s rank correlation measures. Finally, the adjusted prospective associations between hypertensive status and F2-isoprostane concentrations were modeled using logistic regression. Results: Of the 844 participants who were included in the study, 258 (31%) were classified as hypertensive at baseline. Among the 586 participants who were normotensive at baseline, 123 (21%) developed hypertension over the five-year study period. Importantly, none of four F2-isoprostane isomers predicted a significant increase in the odds of developing hypertension, as indicated by their odds ratio 95% confidence intervals; F2-IsoP1: (0.85, 1.31), F2-IsoP2: (0.62, 1.13), F2-IsoP3: (0.80, 1.27), and F2-IsoP4: (0.84, 1.29). Conclusion: Previous studies have investigated the association between oxidative status and hypertension prevalence, however the cross sectional nature of the study designs have made it difficult to establish temporality between exposure and outcome. To our knowledge, this is the first study to model the odds of developing hypertension as a function of F2-isoprostane levels. The results of this study suggest that oxidative status is not involved in the development of hypertension.

Chronic Kidney Disease (CKD) is common and is associated with increased risk of progression to end stage renal disease, cardiovascular disease and death. CKD is a heterogeneous condition and accurately predicting an individual’s risk for adverse outcomes remains a challenge. Over the past decade there has been a focus on the identification of novel biomarkers that may help improve risk stratification and the prediction of clinical endpoints in this population. The overall aim of this research project was to investigate a series of novel biomarkers in patients from the Chronic Renal Insufficiency Standards Implementation Study (CRISIS), a prospective observational study of outcome in all cause non-dialysis dependent CKD 3-5. The biomarkers selected for this project were Anti-Apolipoprotein A-1 (Anti-apoA-1 IgG), fetuin-A, fibroblast growth factor-23 (FGF23), high sensitivity cardiac troponin T (HS-cTnT), kidney injury molecule-1 (KIM-1), N-terminal pro-brain natriuretic peptide (NT-proBNP), neutrophil gelatinase associated lipocalin (NGAL) and osteoprotegerin (OPG). These biomarkers were chosen to address the three clinical endpoints of progression, cardiovascular disease and death with biomarkers considered both individually and as groups of related markers. The first aim of this project was to examine associations between the novel biomarkers and the clinical characteristics of the CRISIS population. The second aim was to investigate the associations between novel biomarkers and the study endpoints. In the case of FGF23 longitudinal measurements were analysed and in all other cases associations between baseline levels of the markers and clinical outcomes were considered. The third aim was to consider whether the biomarkers investigated in this project actually improve parameters of risk stratification and model discrimination, thereby demonstrating a potential to improve the prediction of outcome events in the CKD population. Many of the biomarkers were independently associated with one or all of the clinical outcomes considered. Despite these associations, it was more difficult to demonstrate clear improvement in risk classification or the prediction of clinical endpoints. Baseline models of standard biochemical and clinical parameters performed very well so even biomarkers that were strongly associated with clinical outcomes resulted in only small incremental improvements in the prediction of outcome events. It is now important to focus on defining how biomarkers may fit into clinical decision pathways.

1) Metallothioneins (MTs) are frequently proposed as biomarkers for metal exposure and toxicity in molluscs. However, various biotic and abiotic factors influencing the rate of MT synthesis, are not well understood. The objectives of this study are to investigate the effects of biotic factors (size, sex, growth rate, nutritional state, prey type) and abiotic factors (temperature, Cd or oxidative exposure) on MT induction in Nucella lapillus and Littorina littorea, and to evaluate the usefulness of applying MT as a monitoring tool. In this study, total MTs in tissue samples were quantified using the silver saturation method. 2) Induction of MT was monitored in N. lapillus during and after exposure to Cd. N. lapillus were exposed to 500 μg Cd 1-1 (2.2% of 96h LC50) for 60 d and then placed into clean seawater for 110 d. The concentration of MT in the whole animal increased during the exposure period, peaked at Day 70, and then declined gradually. The half-life of MT was ca. 40 d. Cd concentration increased throughout the period of exposure and while in clean seawater, levelling off only after Day 120, indicating that Cd concentration could not be regulated by N. lapillus. Highest MT induction and Cd accumulation were found in the Leiblein gland of N. lapillus, suggesting that measurement of MT induction in this tissue may prove useful as a sublethal biological response to Cd contamination. 3) The combined effects of Cd and water temperature on the oxygen consumption rate (MO2) and biochemistry of fasted N. lapillus were investigated. Inhibition of MO2 by Cd increased with increasing temperature and decreasing animal size. Cd exposure caused significant reductions in glycogen concentrations in N. lapillus at both temperatures (5 & 10°C). Cd-exposed N. lapillus showed significantly higher MT concentrations in the Leiblein gland at 10°C but not at 5°C, indicating that MT synthesis is temperature dependent. Reduction in MO2 may be directly linked to Cd-induced mucus production, structural damage to gills and reduction in oxygen carrying capacity of haemocyanin. However, metabolic depression, including low MO2, glycogen stores and activity in Cd-exposed N. lapillus, may be a strategy to minimise the uptake and toxicity of Cd, and energy expenditure to spare energy reserves for detoxification and maintenance. 4) The influences of nutritional state and prey type on the survival, growth, Cd accumulation, MT induction and glycogen stores in N. lapillus were studied. Prolonged starvation and Cd exposure synergistically reduced the survivorship of N. lapillus, but feeding could help N. lapillus to combat Cd toxicity and minimise mortality. Extended fasting also caused tissue wastage, leading to higher concentrations of Cd and MT in tissues, whereas fed animals increased in weight and had lower Cd and MT concentrations because of the tissue dilution effect. Prey type significantly affected growth rate of N. lapillus and indirectly influenced Cd accumulation, MT induction and glycogen stores. Eating mussels promoted better growth and higher glycogen reserves them eating barnacles. Individual growth rate decreased with increasing Cd accumulation. Cd-exposed survivors grew faster and consumed more than control animals, implying that these survivors may have better fitness and greater tolerance to Cd toxicity. 5) Investigation of the effect of hydrogen peroxide (H2O2), and the combined effect of H2O2 and Cd on MT induction and condition index (CI) in N. lapillus was conducted. Exposure to either Cd or H2O2 alone induced synthesis of MT or MT-like proteins in N. lapillus. Exposure to high H2O2 (1000 ppm) alone or combined with Cd, and exposure to Cd (0.50 ppm) or H2O2 (2.0 ppm), resulted in significant weight loss, indicated by a reduction of CL However, CIs of N. lapillus exposed to 0.5 ppm Cd + 2.0 ppm H2O2 or 0.25 ppm Cd + 2.0 ppm H2O2, were similar to that of the control suggesting that Cd antagonistically reduces toxicity caused by H2O2 since Cd-induced MT may have a protective function against hydroxyl radicals.

The human brain is a complex and powerful organ, directing every aspect of life from somatosensory and motor function to visceral responses to higher order cognition. Neurological and psychiatric disorders often disrupt normal functioning. While the clinical symptoms of such disorders are known, their biological underpinnings are not as clearly characterized. Structural neuroimaging is a powerful, non-invasive tool that can play a critical role in finding biomarkers of these illnesses. Currently, variations in pre-processing techniques yield inconsistent and conflicting results. As neuroimaging is a nascent branch of medical research, gold standards in imaging methodologies have not yet been established. Quantitatively validating and optimizing the way these images are preprocessed is the first step towards standardization. Voxel-based morphometry (VBM) is one technique that is commonly used to compare whole-brain structural differences between groups. Statistical tests are used to compare intensities of voxels throughout all brain scans in each group. In order to ensure that comparable voxels are being tested, the images must be fitted into a common space, which is done through image preprocessing. Spatial normalization to templates is an early pre-processing step that is executed unreliably as many options for both templates and normalization algorithms exist. To determine the effect variations in template usage may cause, we utilized a VBM approach to detect simulated lesions. Template performance was analyzed by comparing the accuracy with which the lesion was detected.