Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC ESI-MS. It was discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.

Evaluation of regional and distant metastases in head and neck squamous cell carcinoma patients with special reference to the assessment of regional lymph nodes in oral cancer

For optimal treatment planning, a thorough assessment of the metastatic status of mucosal squamous cell carcinoma of the head and neck (HNSCC) is required. Current imaging methods do not allow the recognition of all patients with metastatic disease. Therefore, elective treatment of the cervical lymph nodes is usually given to patients in whom the risk of subclinical metastasis is estimated to exceed 15-20%. The objective of this study was to improve the pre-treatment evaluation of patients diagnosed with HNSCC. Particularly, we aimed at improving the identification of patients who will benefit from elective neck treatment. Computed tomography (CT) of the chest and abdomen was performed prospectively for 100 patients diagnosed with HNSCC. The findings were analysed to clarify the indications for this examination in this patient group. CT of the chest influenced the treatment approach in 3% of patients, while CT of the abdomen did not reveal any significant findings. Our results suggest that CT of the chest and abdomen is not indicated routinely for patients with newly diagnosed HNSCC but can be considered in selected cases. Retrospective analysis of 80 patients treated for early stage squamous cell carcinoma of the oral tongue was performed to investigate the potential benefits of elective neck treatment and to examine whether histopathological features of the primary tumour could be used in the prediction of occult metastases, local recurrence, or/and poor survival. Patients who had received elective neck treatment had significantly fewer cervical recurrences during the follow-up when compared to those who only had close observation of the cervical lymph nodes. Elective neck treatment did not result in survival benefit, however. Of the histopathological parameters examined, depth of infiltration and pT-category (representing tumour diameter) predicted occult cervical metastasis, but only the pT-category predicted local recurrence. Depth of infiltration can be used in the identification of at risk patients but no clear cut-off value separating high-risk and low-risk patients was found. None of the histopathological parameters examined predicted survival. Sentinel lymph node (SLN) biopsy was studied as a means of diagnosing patients with subclinical cervical metastases. SLN biopsy was applied to 46 patients who underwent elective neck dissection for oral squamous cell carcinoma. In addition, SLN biopsy was applied to 13 patients with small oral cavity tumours who were not intended to undergo elective neck dissection because of low risk of occult metastasis. The sensitivity of SLN biopsy for finding subclinical cervical metastases was found to be 67%, when SLN status was compared to the metastatic status of the rest of the neck dissection specimen. Of the patients not planned to have elective neck dissection, SLN biopsy revealed cervical metastasis in 15% of the patients. Our results suggest that SLN biopsy can not yet entirely replace elective neck dissection in the treatment of oral cancer, but it seems beneficial for patients with low risk of metastasis who are not intended for elective neck treatment according to current treatment protocols.

SPECT-aided diagnostics and genetic risk factors in Parkinson's disease

Hypokinesia, rigidity, tremor, and postural instability are the cardinal symptoms of Parkinson s disease (PD). Since these symptoms are not specific to PD the diagnosis may be uncertain in early PD. Etiology and pathogenesis of PD remain unclear. There is no neuroprotective therapy. Genetic findings are expected to reveal metabolic routes in PD pathogenesis and thereby eventually lead to therapeutic innovations. In this thesis, we first aimed to study the usefulness and accuracy of 123I-b-CIT SPECT in the diagnosis of PD in a consecutive clinic-based material including various movement disorders. We subsequently a genetic project to identify genetic risk factors for sporadic PD using a candidate gene approach in a case-control setting including 147 sporadic PD patients and 137 spouse controls. Dopamine transporter imaging by 123I-b-CIT SPECT could distinguish PD from essential tremor, drug-induced parkinsonism, dystonia and psychogenic parkinsonism. However, b-CIT uptake in Parkinson plus syndromes (PSP and multiple system atrophy) and dementia with Lewy bodies was not significantly different from PD. 123I-b-CIT SPECT could not reliably differentiate PD from vascular parkinsonism. 123I-b-CIT SPECT was 100% sensitive and specific in the diagnosis of PD in patients younger than 55 years but less specific in older patients, due to differential distribution of the above conditions in the younger and older age groups. 123I-b-CIT SPECT correlated with symptoms and detected bilateral nigrostriatal defect in patients whose PD was still in unilateral stage. Thus, in addition to as a differential diagnostic aid, 123I-b-CIT SPECT may be used to detect PD early, even pre-symptomatically in at-risk individuals. 123I-b-CIT SPECT was used to aid in the collection of patients to the genetic studies. In the genetic part of this thesis we found an association between PD and a polymorphic CAG-repeat in POLG1 gene encoding the catalytic subunit of mitochondrial polymerase gamma. The CAG-repeat encodes a polyglutamine tract (polyQ), the two most common lengths of which are 10Q (86-90%) and 11Q. In our Finnish material, the rarer non-10Q or non-11Q length variants (6Q-9Q, 12Q-14Q, 4R+9Q) were more frequent in patients than in spouse controls (10% vs. 3.5 %, p=0.003), or population controls (p=0.001). Therefore, we performed a replication study in 652 North American PD patients and 292 controls. Non-10/11Q alleles were more common in the US PD patients compared to the controls but the difference did not reach statistical significance (p=0.07). This larger data suggested our original definition of variant length allele might need reconsideration. Most previous studies on phenotypic effects of POLG1 polyQ have defined 10Q as the only normal allele. Non-10Q alleles were significantly more common in patients compared to the controls (17.3% vs. 12.3 %, p= 0.005). This association between non-10Q length variants and PD remained significant when compared to a larger set of 1541 literature controls (p=0.00005). In conclusion, POLG1 polyQ alleles other than 10Q may predispose to PD. We did not find association between PD and parkin or DJ-1, genes underlying autosomal recessive parkinsonism. The functional Val158Met polymorphism, which affects the catalytic effect of COMT enzyme, and another coding polymorphism in COMT were not associated with PD in our patient material. The APOE e2/3/4 polymorphism modifies risk for Alzheimer s disease and prognosis of for example brain trauma. APOE promoter and enhancer polymorphisms 219G/T and +113G/C, and APOE e3 haplotypes, have also been shown to modify the risk of Alzheimer s disease but not reported in PD. No association was found between PD and APOE e2/3/4 polymorphism, the promoter or enhancer polymorphisms, or the e3 haplotypes.

Search for new physics in the μμ+e/μ+E̸T channel with a low-pT lepton threshold at the Collider Detector at Fermilab

A search for new physics using three-lepton (trilepton) data collected with the CDF II detector and corresponding to an integrated luminosity of 976 pb-1 is presented. The standard model predicts a low rate of trilepton events, which makes some supersymmetric processes, such as chargino-neutralino production, measurable in this channel. The mu+mu+l signature is investigated, where l is an electron or a muon, with the additional requirement of large missing transverse energy. In this analysis, the lepton transverse momenta with respect to the beam direction (pT) are as low as 5 GeV/c, a selection that improves the sensitivity to particles which are light as well as to ones which result in leptonically decaying tau leptons. At the same time, this low-p_T selection presents additional challenges due to the non-negligible heavy-quark background at low lepton momenta. This background is measured with an innovative technique using experimental data. Several dimuon and trilepton control regions are investigated, and good agreement between experimental results and standard-model predictions is observed. In the signal region, we observe one three-muon event and expect 0.4+/-0.1 mu+mu+l events

Egyptin kristillisten kirjureiden jäljillä : Tutkimus 100- ja 200-lukujen Uuden testamentin kanonisten ja apokryfisten käsikirjoitusten ulkoisista piirteistä

Tutkimukseni tarkoituksena on tutkia kaikkein varhaisimpia Uuden testamentin kanonisia ja apokryfisia käsikirjoituksia sekä niiden kirjureita. Huomion keskipisteenä ovat kyseisten käsikirjoitusten ulkoiset piirteet, kuten esimerkiksi kirjoitusmateriaali, muoto, koko ja käsiala. Näiden avulla vertaan kanonisia käsikirjoituksia apokryfisiin ja pyrin selvittämään, erosivatko näiden kopiointimenetelmät toisistaan. Yksi keskeisimmistä kysymyksistä on se, voidaanko ulkoisten piirteiden avulla päätellä jotain käsikirjoitusten asemasta ja arvostuksesta. Tutkimuksen aluksi esittelen tutkimusaineistoni käsikirjoitukset ja tekstit. Päädyin vertaamaan kanonisia evankeliumeita kaikkiin 100- ja 200-luvuilta löydettyihin apokryfisiin käsikirjoituksiin, joihin sisältyy esimerkiksi Tuomaan ja Pietarin evankeliumit sekä Hermaan paimen. Kaikki tämän ajanjakson kristilliset käsikirjoitukset ovat löytyneet Egyptistä, minkä vuoksi tutkimukseni keskittyy Egyptiin ja sen kirjureihin. Ennen varsinaista käsikirjoitusten analyysiä käsittelen käsikirjoitusten löytöpaikkoja, kaanonin sekä antiikin kirjureiden historiaa. Ensimmäiseksi käsittelen käsikirjoitusten materiaalia, eli papyrusta ja pergamenttia. Tässä suhteessa kanoniset käsikirjoitukset eivät eroa apokryfisista, vaan aineistot ovat tämän suhteen identtisiä. Tämän jälkeen huomio kiinnittyy käsikirjoitusten muotoon, eli siihen kirjoitettiinko käsikirjoitukset koodeksiin vai kääröön. Analyysin edetessä paljastuu, että apokryfisissa käsikirjoituksissa käytettiin useammin käärömuoto kuin kanonisissa käsikirjoituksissa. Voidaan esittää, että kristityt kirjoittivat koodeksiin kaikkein arvostetuimmat tekstit, joten käärömuoto voi kertoa käsikirjoituksen sisältävän tekstin alemmasta arvostuksesta. Tutkimuksen seuraavissa osioissa käsitellään koodeksien kokoa, marginaaleja, palstoja sekä käsialan kokoa. Lukujen perusteella voidaan sanoa, että kanoniset koodeksit muodostavat hieman yhtenäisemmän linjan kuin apokryfiset koodeksit. Kanonisten koodeksien kirjureiden toimintatavat ovat siis olleet hieman yhtenäisempiä. Käsikirjoitusten käsialan tarkempi analyysi paljastaa kuitenkin, että niin kanoniset kuin apokryfiset käsikirjoitukset kirjoitettiin suhteellisen hyvällä käsialalla. Tutkimuksen viimeisessä luvussa käy ilmi, että apokryfisten koodeksien epäyhtenäisyys johtuu osittain niiden erilaisista käyttötarkoituksista. Apokryfisia koodekseja valmistettiin enemmän kristittyjen henkilökohtaiseen käyttöön verrattuna kanonisiin koodekseihin. Tästä huolimatta suurin osa käsikirjoituksista oli seurakuntien liturgisessa käytössä. Toisin sanoen niin kanonisia evankeliumeita kuin apokryfisia tekstejä luettiin ääneen seurakuntien kokoontumisissa, mikä kertoo niiden arvosta.