Development of Novel Assays for Measuring Different Molecular Forms of Prostate Specific Antigen

Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer and second leading cause of cancer-related death of men in developed countries. Measurement of prostate specific antigen (PSA) is a very sensitive method for diagnosing and monitoring of prostate cancer (PCa), but the specificity needs improvement. Measurements of different molecular forms of PSA have been shown to improve differentiation between PCa and benign prostatic diseases. However, accurate measurement of some isoforms has not been achieved in previous assays. The aim of the present study was to develop new assays that reliably measure enzymatically active PSA, PSA-α1-chymotryposin (PSA-ACT) and PSA-α1-protease inhibitor (PSA-API), and to evaluate their diagnostic value. Double-label immunofluorometric assays using a novel monoclonal antibody (MAb) and another antibody to either free PSA (fPSA) or total PSA (tPSA) were developed and used to measure PSA-ACT and fPSA or tPSA at the same time. These assays provide enough sensitivity for measurement of PSA-ACT in sera with low PSA levels. The results obtained confirmed that proportion of PSA-ACT to tPSA (%PSA-ACT) was as useful as proportion of fPSA to tPSA (%fPSA) for discrimination between PCa and benign prostatic hyperplasia (BPH). We developed an immunoassay for detection of PSA-API based on proximity ligation, which improved assay sensitivity 10-fold compared with conventional assays. Our results confirmed previous findings that the PSA-API level is somewhat lower in men with than without PCa, and the combination of %fPSA and proportion of PSA-API to tPSA (%PSA-API) provides diagnostic improvement compared with either method alone. Assays based on this principle should be applicable to other immunoassays in which the nonspecific background is a problem. An immunopeptidometric sandwich assay (IPMA) was developed to measure the enzymatically active PSA. This assay showed high specificity, but sensitivity was not good enough for measurement of PSA concentrations in the gray zone, 2-10 µg/L, in which tPSA does not efficiently differentiate between PCa and BPH. We further developed a solid-phase proximity ligation immunoassay, which provided a 10-fold improvement in sensitivity. This proof of concept study shows that peptides reacting with proteins are potentially useful for sensitive and specific measurement of protein variants for which specific MAbs cannot be obtained.

Herpes Simplex Virus Infection, Pathological Pain and Recurrent Lymphocytic Meningitis

Background: Aims of the study were: (i) to characterise the clinical picture, immunological features and changes in brain morphology and function in patients with widespread unilateral pain and HSV-infections, and (ii) to analyse the prevalence, clinical symptoms and immunological predisposing factors of HSV-2 induced recurrent lymphocytic meningitis (RLM) in Southern Finland. Patients and methods: Patients for the studies were recruited from the Pain Clinic, and from the Department of Neurology, at Helsinki University Central Hospital. Plasma concentrations of IgM, IgA, IgG, and IgG1-4, and serum concentrations of C3, C4 were measured. Serological anti-HSV-1 and -2 antibody status was tested. C4 genotyping, HLA-A, HLA-B and HLA-DRB1 typing, MBL2 genotyping, and IgG1 and IgG3 allotyping (Gm) were performed. Clinical neurological examination, quantitative sensory testing, skin biopsy, and functional magnetic resonance imaging were also performed. Results: HSV probably has a role in the generation of a pathological pain state. Low serum IgG1 and IgG3 levels, made the patients vulnerable for recurring HSV infections. Both functional and structural changes were observed in the brain pain-processing areas in the patients: they had less pain-related activity in the insular cortices bilaterally, in the anterior cingular cortex (ACC), and in the thalamus, and the gray matter density was lower in the ACC, in the frontal and prefrontal cortices. In the meningitis studies it was shown that RLM is more common and less benign than previously reported, and that neuropathic pain is frequently present both during and after meningitis episodes. HLA-DRB1*01, HLA-B*27, and low IgG1 levels are predisposing factors for RLM. Conclusions: Patients are vulnerable to recurrent HSV infections because of subtle immunological abnormalities. HSV causes diverse clinical manifestations. First, the herpes simplex virus, or the inflammatory process triggered by it, may cause pathological widespread pain probably by activating glial cells in the CNS. In these patients, signs of alterations in the brain pain-processing areas can be demonstrated by functional brain imaging methods. Secondly, HSV-2 induced RLM is a rare complication of HSV-2 virus. The predisposing factors include low IgG1 subclass levels, HLA-DRB1*01 and HLA –B*27 genotypes. Neuropathic pain is frequently associated with RLM.

Opioid dependence: Brain structure and function : a magnetic resonance imaging, neuropsychological, and electromagnetic study

Background: Opiod dependence is a chronic severe brain disorder associated with enormous health and social problems. The relapse back to opioid abuse is very high especially in early abstinence, but neuropsychological and neurophysiological deficits during opioid abuse or soon after cessation of opioids are scarcely investigated. Also the structural brain changes and their correlations with the length of opioid abuse or abuse onset age are not known. In this study the cognitive functions, neural basis of cognitive dysfunction, and brain structural changes was studied in opioid-dependent patients and in age and sex matched healthy controls. Materials and methods: All subjects participating in the study, 23 opioid dependents of whom, 15 were also benzodiazepine and five cannabis co-dependent and 18 healthy age and sex matched controls went through Structured Clinical Interviews (SCID) to obtain DSM-IV axis I and II diagnosis and to exclude psychiatric illness not related to opioid dependence or personality disorders. Simultaneous magnetoencephalography (MEG) and electroencephalography (EEG) measurements were done on 21 opioid-dependent individuals on the day of hospitalization for withdrawal therapy. The neural basis of auditory processing was studied and pre-attentive attention and sensory memory were investigated. During the withdrawal 15 opioid-dependent patients participated in neuropsychological tests, measuring fluid intelligence, attention and working memory, verbal and visual memory, and executive functions. Fifteen healthy subjects served as controls for the MEG-EEG measurements and neuropsychological assessment. The brain magnetic resonance imaging (MRI) was obtained from 17 patients after approximately two weeks abstinence, and from 17 controls. The areas of different brain structures and the absolute and relative volumes of cerebrum, cerebral white and gray matter, and cerebrospinal fluid (CSF) spaces were measured and the Sylvian fissure ratio (SFR) and bifrontal ratio were calculated. Also correlation between the cerebral measures and neuropsychological performance was done. Results: MEG-EEG measurements showed that compared to controls the opioid-dependent patients had delayed mismatch negativity (MMN) response to novel sounds in the EEG and P3am on the contralateral hemisphere to the stimulated ear in MEG. The equivalent current dipole (ECD) of N1m response was stronger in patients with benzodiazepine co-dependence than those without benzodiazepine co-dependence or controls. In early abstinence the opioid dependents performed poorer than the controls in tests measuring attention and working memory, executive function and fluid intelligence. Test results of the Culture Fair Intelligence Test (CFIT), testing fluid intelligence, and Paced Auditory Serial Addition Test (PASAT), measuring attention and working memory correlated positively with the days of abstinence. MRI measurements showed that the relative volume of CSF was significantly larger in opioid dependents, which could also be seen in visual analysis. Also Sylvian fissures, expressed by SFR were wider in patients, which correlated negatively with the age of opioid abuse onset. In controls the relative gray matter volume had a positive correlation with composite cognitive performance, but this correlation was not found in opioid dependents in early abstinence. Conclusions: Opioid dependents had wide Sylvian fissures and CSF spaces indicating frontotemporal atrophy. Dilatation of Sylvian fissures correlated with the abuse onset age. During early withdrawal cognitive performance of opioid dependents was impaired. While intoxicated the pre-attentive attention to novel stimulus was delayed and benzodiazepine co-dependence impaired sound detection. All these changes point to disturbances on frontotemporal areas.

Effects of Metabolic Risk Factors and Type 1 Diabetes on Brain Glucose and Brain Metabolites

The metabolic syndrome and type 1 diabetes are associated with brain alterations such as cognitive decline brain infarctions, atrophy, and white matter lesions. Despite the importance of these alterations, their pathomechanism is still poorly understood. This study was conducted to investigate brain glucose and metabolites in healthy individuals with an increased cardiovascular risk and in patients with type 1 diabetes in order to discover more information on the nature of the known brain alterations. We studied 43 20- to 45-year-old men. Study I compared two groups of non-diabetic men, one with an accumulation of cardiovascular risk factors and another without. Studies II to IV compared men with type 1 diabetes (duration of diabetes 6.7 ± 5.2 years, no microvascular complications) with non-diabetic men. Brain glucose, N-acetylaspartate (NAA), total creatine (tCr), choline, and myo-inositol (mI) were quantified with proton magnetic resonance spectroscopy in three cerebral regions: frontal cortex, frontal white matter, thalamus, and in cerebellar white matter. Data collection was performed for all participants during fasting glycemia and in a subgroup (Studies III and IV), also during a hyperglycemic clamp that increased plasma glucose concentration by 12 mmol/l. In non-diabetic men, the brain glucose concentration correlated linearly with plasma glucose concentration. The cardiovascular risk group (Study I) had a 13% higher plasma glucose concentration than the control group, but no difference in thalamic glucose content. The risk group thus had lower thalamic glucose content than expected. They also had 17% increased tCr (marker of oxidative metabolism). In the control group, tCr correlated with thalamic glucose content, but in the risk group, tCr correlated instead with fasting plasma glucose and 2-h plasma glucose concentration in the oral glucose tolerance test. Risk factors of the metabolic syndrome, most importantly insulin resistance, may thus influence brain metabolism. During fasting glycemia (Study II), regional variation in the cerebral glucose levels appeared in the non-diabetic subjects but not in those with diabetes. In diabetic patients, excess glucose had accumulated predominantly in the white matter where the metabolite alterations were also the most pronounced. Compared to the controls values, the white matter NAA (marker of neuronal metabolism) was 6% lower and mI (glia cell marker) 20% higher. Hyperglycemia is therefore a potent risk factor for diabetic brain disease and the metabolic brain alterations may appear even before any peripheral microvascular complications are detectable. During acute hyperglycemia (Study III), the increase in cerebral glucose content in the patients with type 1 diabetes was, dependent on brain region, between 1.1 and 2.0 mmol/l. An every-day hyperglycemic episode in a diabetic patient may therefore as much as double brain glucose concentration. While chronic hyperglycemia had led to accumulation of glucose in the white matter, acute hyperglycemia burdened predominantly the gray matter. Acute hyperglycemia also revealed that chronic fluctuation in blood glucose may be associated with alterations in glucose uptake or in metabolism in the thalamus. The cerebellar white matter appeared very differently from the cerebral (Study IV). In the non-diabetic men it contained twice as much glucose as the cerebrum. Diabetes had altered neither its glucose content nor the brain metabolites. The cerebellum seems therefore more resistant to the effects of hyperglycemia than is the cerebrum.

Expected Performance of the ATLAS Experiment - Detector, Trigger and Physics

A detailed study is presented of the expected performance of the ATLAS detector. The reconstruction of tracks, leptons, photons, missing energy and jets is investigated, together with the performance of b-tagging and the trigger. The physics potential for a variety of interesting physics processes, within the Standard Model and beyond, is examined. The study comprises a series of notes based on simulations of the detector and physics processes, with particular emphasis given to the data expected from the first years of operation of the LHC at CERN.

Diasporakysymyksiä kansainvälisessä yhteisössä. Esimerkkinä Venäjän politiikka ja lapsikiistat Suomessa.

Pro gradu -tutkielmassani tarkastelen diasporia ja niihin kohdistuvaa kansainvälistä sääntelyä. Diasporilla tarkoitan valtion rajojen ulkopuolella asuvia ihmisiä, joilla on jokin, esimerkiksi etninen tai kieliside tuohon valtioon. Valtiolla puolestaan voi olla jokin intressi suhteessa diasporaansa. Diasporiin liittyvät kysymykset ovat ajankohtaisia globaalissa maailmassa, jossa ihmisillä on entistä enemmän mahdollisuuksia pitää siteitä yllä useampaan kuin yhteen valtioon. Diasporien ohella kirjallisuudessa puhutaan myös kin-state -problematiikasta, kun viitataan valtioiden harjoittamaan politiikkaan, joka pyrkii vaikuttamaan toisen valtion alueella asuvien tai oleskelevien ihmisten asemaan. Tutkimuksen teoreettinen viitekehys on englantilaisen koulukunnan ajatus kansainvälisestä yhteisöstä ja sille keskeisistä primaari-instituutioista, kuten valtiosuvereniteetti ja kansainvälinen oikeus. Se, miten valtio politiikassaan suhtautuu instituutioihin, kertoo valtion suhteesta kansainväliseen yhteisöön yleensä. Instituutioita voidaan puolestaan tarkastella tiettyjen kapea-alaisempien normien, sekundaari-instituutioiden, valossa. Edellä mainittuna sekundaari-instituutiona esitellään tutkielmassa se kansainvälinen normisto, joka vaikuttaa diasporien asemaan valtioiden välisessä politiikassa. Normisto perustuu Yhdistyneiden kansakuntien, Euroopan neuvoston ja ETYJ:n sopimuksiin ja suosituksiin. Kansainvälisen oikeuden lähtökohta on, ettei valtio voi puuttua diasporakseen katsomiensa ihmisten asemaan toisessa valtiossa edes ääritapauksessa, jossa asuinvaltio epäonnistuu tehtävässään suojella toisen valtion kansalaisia tai vähemmistöjään. Reagointivastuu on kansainvälisellä yhteisöllä – vaikka interventiota ulkomailla olevien kansalaisten suojelemiseksi onkin suoritettu sekä kylmän sodan aikana että sen jälkeen. Interventiot eivät ole diasporapolitiikan ainoa muoto ja monilla valtioilla onkin niin sanottuja diasporalakeja, joiden puitteissa ne voivat vaikuttaa diasporiensa asemaan. Koulutukseen ja kulttuuriin liittyvä tuki on eurooppalaisissa suosituksissa katsottu hyväksyttäväksi, mutta muiden tukimuotojen ulottamista diasporiin tulee harkita ja soveltaa vain erityistapauksissa, jos politiikalla on valtion toimivallan ulkopuolella vaikuttavia seurauksia. Tutkimus näyttää, että diasporakysymysten sääntely on kansainvälisessä yhteisössä vielä osin määrittelemätöntä sekä altista valtioiden tulkinnoille. Esimerkkinä valtioiden tulkinnoista ja niiden harjoittamasta diasporapolitiikasta tarkastellaan Venäjän federaation politiikkaa, sillä Venäjän ulkopolitiikassa esillä pidetty kiinnostus suojella ulkomailla olevia kansalaisia ja maanmiehiä on herättänyt myös kansainvälistä huomiota. Venäjän politiikan tarkastelu luo kontekstin, jossa tutkielman varsinaista kiinnostuksenkohdetta, Suomen venäläisen diasporan asemaa, on mahdollista tarkastella. Tutkimusaineistoa ovat Venäjän johdon lausunnot liittyen viimeaikaisiin Suomen ja Venäjän välisiin nk. lapsikiistoihin. Teoriaohjaava sisällönanalyysi aineistosta esittää, että vaikka vihjeitä politiikassa yleisesti esiintyvästä nk. suojelupuheesta on havaittavissa myös Suomen venäläisen vähemmistön asemaan liittyen, ei Venäjä lapsikiistoissa kuitenkaan suoraan loukannut Suomen suvereniteettia tai kansainvälistä oikeutta. Tiettyjen puhetapojen ja esimerkiksi lapsiasiamies Astahovin toiminnan voidaan kuitenkin tulkita olevan kansainvälisen normiston vastaisia ja siten myös loukkaavan suvereniteettia ja kansainvälisen oikeuden henkeä. Teoreettisen viitekehyksen tehtävä oli ensisijaisesti antaa näkökulmia aineiston tarkasteluun. Venäjän politiikan suhde suvereniteettiin ja kansainväliseen oikeuteen suhteessa tässä tarkasteltuun politiikkakysymykseen kertoo kuitenkin myös Venäjän asemasta kansainvälisen yhteisön jäsenenä. Diasporanormistoon sitoutuminen osoittaa Venäjän sitoutuneen ainakin periaatteellisella tasolla solidaristiseen, yhteisiä normeja jakavaan kansainväliseen yhteisöön – toisaalta Venäjän viime vuosien politiikka kuitenkin osoittaa Venäjän samalla haastavan myös pluralistisille yhteisöille keskeisiä valtiosuvereniteetin ja kansainvälisen oikeuden noudattamisen periaatteita. Tärkeimmät lähteet: Buzan (2004), Brubakers (1995), Hannikainen (2000), Holsti (2004), Gazzini (2005), Gray (2000), Kántor ym. (2004), Sheffer (2003), Shevel (2010 ja 2011), Turner ja Otsuki (2010) ja Zevelev (2001 ja 2008).