Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.

Since 1750, human emissions of greenhouse gases have created a temperature increase, affecting the whole world. Diatoms, single-celled microscopic algae that lives in waters all over the world, have different environmental preferences such as the water depth at which the thrive, salinity, nutrients, pH-values and more. Diatoms also responds quickly to changing circumstances, which makes the suitable as environmental indicators. The purpose of the study is to investigate how the abundance and composition of diatoms has changed during the last 200 years in lake Ensjön, located just south of Norrköping, and to relate those changes both to climate change and known events and environmental factors in the area.  The survey is conducted by subsampling sediment cores from which each diatom sample was prepared in accordance with the scheme of Battarbee (1984). The diatoms in the samples were counted under a microscope and the results were analyzed and related to the Cyclotella-Aulacoseira-Fragilaria-theory (CAF) and documented historical events in the local environment. The result of the analysis shows that the composition and abundance of diatoms has changed during the last 200 years, and that this change is due to both climate change (temperature and precipitation increase) and the land-use history of Ensjön. The cahnges in composition of Aulacoseira spp. and Cyclotella spp., where Aulacoseira spp. is expected to decrease, and Cyclotella spp. is expected to increase with a rising temperature, match the CAF-theory for the most part. The change in composition also coincides with several documented environmental factors in the area, especially the change in use of fertilizers and herbicides in agriculture. / Människans utsläpp av växthusgaser har sedan år 1750 skapat en temperaturökning som går allt snabbare, och påverkar hela världen. Kiselalger, encelliga mikroskopiska alger som lever i vatten över hela världen, har olika miljöpreferenser såsom bland annat på vilket vattendjup de trivs, optimal halt av salt, näringsvärden, pH-värden, samt reagerar snabbt på förändrade omständigheter, vilket gör dem lämpliga som miljöindikatorer. Syftet med studien är att studera förändringar i kiselalgernas sammansättning under de senaste 200 åren i den lilla sjön Ensjön belägen något söder om Norrköping och att relatera dessa förändringar till dokumenterade händelser och miljöfaktorer i sjöns avrinningsområde samt till de pågående klimatförändringarna.  Undersökningen är utförd genom provtagning av sedimentkärnor från vilka prover preparerades enligt Battarbees (1984) flödesschema. Proverna av kiselalger räknades under mikroskop och sammansättningen analyserades utifrån teorin Cyclotella-Aulacoseira-Fragilaria (CAF) och kända historiska händelser i närmiljön. Analysresultatet visade att kiselalgssammansättningen har förändrats under de senaste 200 åren, och att denna förändring sannolikt kan kopplas både till klimatförändringar (en ökad temperatur) och Ensjöns markanvändningshistoria. Förändringen hos Aulacoseira spp. och Cyclotella spp. följer till stor del CAF-teorin om att det första släktet skulle minska och det andra skulle öka vid en temperaturökning. Vid flera tillfällen sammanfaller förändringar i kiselalgssammansättningen med kända miljöfaktorer i närmiljön, främst i samband med utvecklingen i användningen av mineralgödsel och växtskyddsmedel.

Antalet monoklonala antikroppar som används i läkemedel ökar kraftigt. Dessa läkemedel är dyra och risken för förfalskning är stor. Behovet att utveckla en metod för snabb och precis identifiering av monoklonala antikroppar är därför brådskande. För identifiering utfördes analyser med Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) på nio monoklonala antikroppar. Fokuset var att undersöka huruvida signifikanta fysiokemiska egenskaper och unika aminosyrasekvenser var närvarande och kunde urskiljas. Olika analyser med MALDI-ToF-MS användes till att både separera de monoklonala antikropparna baserat på dess fysiokemiska egenskaper, och annotera aminosyrasekvenser innehållande nyckelfragment. Med metoderna baserade på kapillärelektrofores uppnåddes också separation. CZE föredras framför CGE då mängden data som erhålls från CZE är större och provberedningen är enklare. Sammanfattningsvis utformades ett protokoll för identifieringsprocessen, vilket inleds med MALDI-ToF-MS-analyser av monoklonala antikroppar på reducerad form mot kända referenser. Därefter är en hypotes formulerad utifrån vilka antikroppar som ser mest lika ut. Slutligen analyseras dessa med CZE för fastställning av den monoklonala antikroppens identitet. / The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.

The aim of this project has been to investigate whether there are correlations to be found between gut microbes and serum metabolites, which could be involved in the development of epilepsy. To do so, metabolomics data containing metabolites and metagenomics data containing bacteria have been integrated and used in a pipeline utilizing the software package DIABLO in R Studio. DIABLO stands for Data Integration Analysis for Biomarker discovery using Latent cOmponents and utilizes multi-block pls-da to integrate multiple omics data sets to find potential biomarkers. The results in this project are mainly divided into two groups, the first group being from taking samples at an early time point, where subjects have not yet developed symptoms of epilepsy and the second group being from taking samples at a late time point, where the subjects have developed epilepsy. To find biomarkers in the data used for the integration, two subgroups are of highest interest, namely subgroup PTE, which is the group that develops epilepsy symptoms after an induced trauma to the brain, as well as subgroup TBI which do not develop epilepsy symptoms after an induced trauma to the brain. Results from the early time point suggests that bacteria such as those from Phelethenecus, Christenselellales, Ventrimonas, Ruminococcaceae and Acetatifactor, as well as metabolites such as LPC 17:0, Indole and Indole-3-carboxyaldehyde might be of interest in finding biomarkers previous to the development of epilepsy after induced brain trauma.  Results from the late time point suggests that bacteria such as those from Muribaculaceae and Avidehalobacter, as well as metabolites such as Dioctyl sulfosuccinate, Canrenone, LPC 18:0, Uric acid, Arjunolic acid and Pseudouridine might be of interest in finding underlying mechanisms behind the existing condition of epilepsy. The hope is that findings in this paper might aid in future development of knowledge behind this disease as well as its underlying mechanisms.