Genes outside The Hla Region affecting Susceptibility to Type 1 Diabetes - The Role of Iddm2 and Iddm9 in the Finnish Population

Tyypin 1 diabeteksen perinnöllinen alttius Suomessa - HLA-alueen ulkopuolisten alttiuslokusten IDDM2 ja IDDM9 rooli taudin periytymisessä HLA-alue, joka sijaitsee kromosomissa 6p21.3, vastaa noin puolesta perinnöllisestä alttiudesta sairastua tyypin 1 diabetekseen. Myös HLA-alueen ulkopuolisten lokusten on todettu liittyvän sairausalttiuteen. Näistä kolmen lokuksen on varmistettu olevan todellisia alttiuslokuksia ja lisäksi useiden muiden, vielä varmistamattomien lokusten, on todettu liittyvän sairausalttiuteen. Tässä tutkimuksessa 12:n HLA-alueen ulkopuolisen alttiuslokuksen kytkentä tyypin 1 diabetekseen tutkittiin käyttäen 107:aa suomalaista multiplex-perhettä. Jatkotutkimuksessa analysoitiin IDDM9-alueen kytkentä ja assosiaatio sairauteen laajennetuissa perhemateriaaleissa sekä IDDM2-alueen mahdollinen interaktio HLA-alueen kanssa sairauden muodostumisessa. Lisäksi suoritettiin IDDM2-alueen suojaavien haplotyyppien alatyypitys tarkoituksena tutkia eri haplotyyppien käyttökelpoisuutta sairastumisriskin tarkempaa ennustamista varten. Ensimmäisessä kytkentätutkimuksessa ei löytynyt koko genomin tasolla merkitsevää tai viitteellistä kytkentää tutkituista HLA-alueen ulkopuolisista lokuksista. Voimakkain havaittu nimellisen merkitsevyyden tavoittava kytkentä nähtiin IDDM9-alueen markkerilla D3S3576 (MLS=1.05). Tutkimuksessa ei kyetty varmistamaan tai sulkemaan pois aiempia kytkentähavaintoja tutkituilla lokuksilla, mutta IDDM9-alueen jatkotutkimuksessa havaittu voimakas kytkentä (MLS=3.4) ja merkitsevä assosiaatio (TDT p=0.0002) viittaa vahvasti siihen, että 3q21-alueella sijaitsee todellinen tyypin 1 diabeteksen alttiusgeeni, jolloin alueen kattava assosiaatiotutkimus olisi perusteltu jatkotoimenpide. Sairauteen altistava IDDM2-alueen MspI-2221 genotyyppi CC oli nimellisesti yleisempi matalan tai kohtalaisen HLA-sairastumisriskin diabeetikoilla, verrattuna korkean HLA-riskin potilaisiin (p=0.05). Myös genotyyppijakauman vertailu osoitti merkitsevää eroa ryhmien välillä (p=0.01). VNTR-haplotyyppitutkimus osoitti, että IIIA/IIIA-homotsygootin sairaudelta suojaava vaikutus on merkitsevästi voimakkaampi kuin muiden luokka III:n genotyypeillä. Nämä tulokset viittaavat IDDM2-HLA -vuorovaikutukseen sekä siihen että IDDM2-alueen haplotyyppien välillä esiintyy etiologista heterogeniaa. Tämän johdosta IDDM2-alueen haplotyyppien tarkempi määrittäminen voisi tehostaa tyypin 1 diabeteksen riskiarviointia.

Verbal learning in mild cognitive impairment and alzheimer's disease : behavioural and neural approaches

The main focus of the present thesis was at verbal episodic memory processes that are particularly vulnerable to preclinical and clinical Alzheimer’s disease (AD). Here these processes were studied by a word learning paradigm, cutting across the domains of memory and language learning studies. Moreover, the differentiation between normal aging, mild cognitive impairment (MCI) and AD was studied by the cognitive screening test CERAD. In study I, the aim was to examine how patients with amnestic MCI differ from healthy controls in the different CERAD subtests. Also, the sensitivity and specificity of the CERAD screening test to MCI and AD was examined, as previous studies on the sensitivity and specificity of the CERAD have not included MCI patients. The results indicated that MCI is characterized by an encoding deficit, as shown by the overall worse performance on the CERAD Wordlist learning test compared with controls. As a screening test, CERAD was not very sensitive to MCI. In study II, verbal learning and forgetting in amnestic MCI, AD and healthy elderly controls was investigated with an experimental word learning paradigm, where names of 40 unfamiliar objects (mainly archaic tools) were trained with or without semantic support. The object names were trained during a 4-day long period and a follow-up was conducted one week, 4 weeks and 8 weeks after the training period. Manipulation of semantic support was included in the paradigm because it was hypothesized that semantic support might have some beneficial effects in the present learning task especially for the MCI group, as semantic memory is quite well preserved in MCI in contrast to episodic memory. We found that word learning was significantly impaired in MCI and AD patients, whereas forgetting patterns were similar across groups. Semantic support showed a beneficial effect on object name retrieval in the MCI group 8 weeks after training, indicating that the MCI patients’ preserved semantic memory abilities compensated for their impaired episodic memory. The MCI group performed equally well as the controls in the tasks tapping incidental learning and recognition memory, whereas the AD group showed impairment. Both the MCI and the AD group benefited less from phonological cueing than the controls. Our findings indicate that acquisition is compromised in both MCI and AD, whereas long13 term retention is not affected to the same extent. Incidental learning and recognition memory seem to be well preserved in MCI. In studies III and IV, the neural correlates of naming newly learned objects were examined in healthy elderly subjects and in amnestic MCI patients by means of positron emission tomography (PET) right after the training period. The naming of newly learned objects by healthy elderly subjects recruited a left-lateralized network, including frontotemporal regions and the cerebellum, which was more extensive than the one related to the naming of familiar objects (study III). Semantic support showed no effects on the PET results for the healthy subjects. The observed activation increases may reflect lexicalsemantic and lexical-phonological retrieval, as well as more general associative memory mechanisms. In study IV, compared to the controls, the MCI patients showed increased anterior cingulate activation when naming newly learned objects that had been learned without semantic support. This suggests a recruitment of additional executive and attentional resources in the MCI group.

First-Line Chemotherapy with Anthracycline and Taxane Combination in Metastatic Breast Cancer. Detection of bone metastases with TRACP 5b

Background: In Finland, breast cancer (BC) is the most common cancer among women, and prostate cancer (PC) that among men. At the metastatic stage both cancers remain essentially incurable. The goals of therapy include palliation of symptoms, improvement or maintenance of quality of life (QoL), delay of disease progression, and prolongation of survival. Balancing between efficacy and toxicity is the major challenge. With increasing costs of new treatments, appropriate use of resources is paramount. When new treatment regimes are introduced into clinical practice a comprehensive assessment of clinical benefit, adverse effects and cost is necessary. Both BC and PC show a predilection to metastasize to bone. Bone metastases cause significant morbidity impairing the patients´ QoL. Diagnosis of bone metastases relies mainly on radiological methods, which however lack optimal sensitivity and specificity. New tools are needed for detection and follow-up of bone metastases. Aims: Anthracyclines and taxanes are effective chemotherapeutic agents in the treatment of metastatic breast cancer (MBC) with different mechanisms of action. Therefore, evaluation of the combination of anthracyclines with taxanes was a justifiable approach in the treatment of MBC patients. We assessed the efficacy, toxicity, cost of treatment and QoL of BC patients treated with first-line chemotherapy for metastatic disease with the combination epirubicin and docetaxel. We also evaluated the diagnostic potential of tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptides of type I collagen (ICTP) in the diagnosis of bone metastases in BC and TRACP 5b in PC patients. Results: The combination of epirubicin and docetaxel was effective in this phase II study, but required individual dose adjustment to avoid neutropenic infections, and the use of growth factors to maintain a feasible dose level. The response rate was 54 % (95 % CI 37-71) and the median overall survival (OS) was 26 months. Of the patients, 87 % were treated for infections. The treatment of adverse events required additional use of health resources mainly due to neutropenic infections, thereby raising direct treatment costs by 20 %. Despite adverse events, the global QoL was not significantly compromised during the treatment. Clinically evident acute cardiac toxicity was not observed. The combination of serum TRACP 5b and ICTP was at least equally sensitive and specific in detection of of bone metastases as commonly used total alkaline phosphatise (tALP) in BC patients. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC patients. Conclusions: Treatment with epirubicin and docetaxel showed high efficacy in first-line chemotherapy of MBC. The relatively high incidence of neutropenic infections requiring hospitalization increased the treatment costs. Despite adverse events, the global QoL of the patients was not significantly compromised. The combination of TRACP 5b and ICTP showed similar activity as tALP in detecting bone metastases in MBC. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC.

Application of morphometry, static DNA ploidy analysis, and steroid receptor expression in diagnosis and prognosis of Libyan breast cancer

The aim of this study was to describe the demographic, clinicopathological, biological and morphometric features of Libyan breast cancer patients. The supporting value of nuclear morphometry and static image cytometry in the sensitivity for detecting breast cancer in conventional fine-needle aspiration biopsies were estimated. The findings were compared with findings in breast cancer in Finland and Nigeria. In addation, the value of ER and PR were evaluated. There were 131 histological samples, 41 cytological samples, and demographic and clinicopathological data from 234 Libyan patients. The Libyan breast cancer is dominantly premenopausal and in this feature it is similar to breast cancer in sub-Saharan Africans, but clearly different from breast cancer in Europeans, whose cancers are dominantly postmenopausal in character. At presention most Libyan patients have locally advanced disease, which is associated with poor survival rates. Nuclear morphometry and image DNA cytometry agree with earlier published data in the Finnish population and indicate that nuclear size and DNA analysis of nuclear content can be used to increase the cytological sensitivity and specificity in doubtful breast lesions, particularly when free cell sampling method is used. Combination of the morphometric data with earlier free cell data gave the following diagnostic guidelines: Range of overlap in free cell samples: 55 μm2 -71 μm2. Cut-off values for diagnostic purposes: Mean nuclear area (MNA) >54 μm2 for 100% detection of malignant cases (specificity 84 %), MNA < 72 μm2 for 100% detection of benign cases (sensitivity 91%). Histomorphometry showed a significant correlation between the MNA and most clinicopathological features, with the strongest association observed for histological grade (p <0.0001). MNA seems to be a prognosticator in Libyan breast cancer (Pearson’s test r = - 0.29, p = 0.019), but at lower level of significance than in the European material. A corresponding relationship was not found in shape-related morphometric features. ER and PR staining scores were in correlation with the clinical stage (p= 0.017, and 0.015, respectively), and also associated with lymph node negative patients (p=0.03, p=0.05, respectively). Receptor-positive (HR+) patients had a better survival. The fraction of HR+ cases among Libyan breast cancers is about the same as the fraction of positive cases in European breast cancer. The study suggests that also weak staining (corresponding to as few as 1% positive cells) has prognostic value. The prognostic significance may be associated with the practice to use antihormonal therapy in HR+ cases. The low survival and advanced presentation is associated with active cell proliferation, atypical nuclear morphology and aneuploid nuclear DNA content in Libyan breast cancer patients. The findings support the idea that breast cancer is not one type of disease, but should probably be classified into premenopausal and post menopausal types.

From Molecular Blocks To Bioanalytical Assays: Combining Lanthanide Luminescence and Bioaffinity Binders for Protein Detection

Measuring protein biomarkers from sample matrix, such as plasma, is one of the basic tasks in clinical diagnostics. Bioanalytical assays used for the measuring should be able to measure proteins with high sensitivity and specificity. Furthermore, multiplexing capability would also be advantageous. To ensure the utility of the diagnostic test in point-of-care setting, additional requirements such as short turn-around times, ease-ofuse and low costs need to be met. On the other hand, enhancement of assay sensitivity could enable exploiting novel biomarkers, which are present in very low concentrations and which the current immunoassays are unable to measure. Furthermore, highly sensitive assays could enable the use of minimally invasive sampling. In the development of high-sensitivity assays the label technology and affinity binders are in pivotal role. Additionally, innovative assay designs contribute to the obtained sensitivity and other characteristics of the assay as well as its applicability. The aim of this thesis was to study the impact of assay components on the performance of both homogeneous and heterogeneous assays. Applicability of two different lanthanide-based label technologies, upconverting nanoparticles and switchable lanthanide luminescence, to protein detection was explored. Moreover, the potential of recombinant antibodies and aptamers as alternative affinity binders were evaluated. Additionally, alternative conjugation chemistries for production of the labeled binders were studied. Different assay concepts were also evaluated with respect to their applicability to point-of-care testing, which requires simple yet sensitive methods. The applicability of upconverting nanoparticles to the simultaneous quantitative measurement of multiple analytes using imaging-based detection was demonstrated. Additionally, the required instrumentation was relatively simple and inexpensive compared to other luminescent lanthanide-based labels requiring time-resolved measurement. The developed homogeneous assays exploiting switchable lanthanide luminescence were rapid and simple to perform and thus applicable even to point-ofcare testing. The sensitivities of the homogeneous assays were in the picomolar range, which are still inadequate for some analytes, such as cardiac troponins, requiring ultralow limits of detection. For most analytes, however, the obtained limits of detection were sufficient. The use of recombinant antibody fragments and aptamers as binders allowed site-specific and controlled covalent conjugation to construct labeled binders reproducibly either by using chemical modification or recombinant technology. Luminescent lanthanide labels were shown to be widely applicable for protein detection in various assay setups and to contribute assay sensitivity.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Identification and validation of novel prostate cancer biomarkers

Prostate cancer (PCa) has emerged as the most commonly diagnosed lethal cancer in European men. PCa is a heterogeneous cancer that in the majority of the cases is slow growing: consequently, these patients would not need any medical treatment. Currently, the measurement of prostate-specific antigen (PSA) from blood by immunoassay followed by digital rectal examination and a pathological examination of prostate tissue biopsies are the most widely used methods in the diagnosis of PCa. These methods suffer from a lack of sensitivity and specificity that may cause either missed cancers or overtreatment as a consequence of over-diagnosis. Therefore, more reliable biomarkers are needed for a better discrimination between indolent and potentially aggressive cancers. The aim of this thesis was the identification and validation of novel biomarkers for PCa. The mRNA expression level of 14 genes including AMACR, AR, PCA3, SPINK1, TMPRSS2-ERG, KLK3, ACSM1, CACNA1D, DLX1, LMNB1, PLA2G7, RHOU, SPON2, and TDRD1 was measured by a truly quantitative reverse transcription PCR in different prostate tissue samples from men with and without PCa. For the last eight genes the function of the genes in PCa progression was studied by a specific siRNA knockdown in PC-3 and VCaP cells. The results from radical prostatectomy and cystoprostatectomy samples showed statistically significant overexpression for all the target genes, except for KLK3 in men with PCa compared with men without PCa. Statistically significant difference was also observed in low versus high Gleason grade tumors (for PLA2G7), PSA relapse versus no relapse (for SPON2), and low versus high TNM stages (for CACNA1D and DLX1). Functional studies and siRNA silencing results revealed a cytotoxicity effect for the knock-down of DLX1, PLA2G7, and RHOU, and altered tumor cell invasion for PLA2G7, RHOU, ACSM1, and CACNA1D knock-down in 3D conditions. In addition, effects on tumor cell motility were observed after silencing PLA2G7 and RHOU in 2D monolayer cultures. Altogether, these findings indicate the possibility of utilizing these new markers as diagnostic and prognostic markers, and they may also represent therapeutic targets for PCa.