Endokannabinoidijärjestelmä säätelee monia kehon fysiologisia vasteita, kuten syömistä ja energia-aineenvaihduntaa, uni-valverytmiä sekä kipua. Vaikka kannabinoideja on käytetty kivunlievitykseen tuhansia vuosia, niiden todellinen farmakologinen potentiaali on vielä kartoittamatta. Tässä tutkielmassa tarkastellaan endokannabinoidijärjestelmän toimintaa ja kuinka kannabinoidireseptorien aktivoiminen lievittää kipua.

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Veren hyytyminen on pitkä reaktioiden sarja, joka lopulta johtaa trombiinin katalysoimana fibriinin muodostumiseen fibrinogeenista, jolloin syntyy verihyytymä, trombi. Aiheettoman trombin syntyminen verisuonen sisään tukkii verisuonia ja paikallisesti haittaa verenkiertoa. Vakavampia seurauksia voi aiheuttaa trombista irtoava osa, joka kulkeutuu verenkierron mukana muualle elimistöön. Trombi voi tukkia sydämen sepelvaltimoita ja saada aikaan sydäninfarktin. Keuhkoihin kulkeutuessaan se aiheuttaa keuhkoembolian. Aivojen verisuonissa syntynyt, tai sinne kulkeutunut trombi voi aikaansaada aivoinfarktin. Antikoagulantteja käytetään lääkkeenä veren liialliseen hyytymiseen. Yleisimmät näistä lääkkeistä ovat varfariini ja hepariini. (Koulu & Tuomisto 2000) Kumariinit löydettiin sattumalta Yhdysvalloissa 1920-luvulla samoihin aikoihin kuin K-vitamiinit. Karjaeläimet söivät pilaantunutta rehua, joka sisälsi paljon dikumarolia, Karl Link tunnisti tämän aineen, ja havaittiin, että vastalääkkeeksi dikumarolin aiheuttamaan hallitsemattomaan verenvuotoon tehosi suuri annos K-vitamiinia. Dikumarolia tutkittiin laboratorioissa ja sen analogeja kehiteltiin useita kymmeniä. Yksi näistä analogeista oli varfariini. Varfariini on ainoa nykyään antikoagulanttina käytetty kumariinijohdannainen. Varfariini ei kuitenkaan suoraan päätynyt lääkeaineeksi, vaan ensin sitä käytettiin rotanmyrkkynä. Varfariinia käytettiin rotanmyrkyissä niin paljon, että se on aiheuttanut massiivisen evoluutiopaineen rottien VKOR-geenille. Vuonna 2009 löydettiin 29 erilaista rotan VKOR:n mutaatiota, jotka aiheuttavat varfariiniresistenssin. Kesti pitkään, ennen kuin varfariinia alettiin käyttää ihmisten hyytymishäiriöiden hoidossa, sillä rotanmyrkyn tärkeintä vaikuttavaa ainetta ei ollut helppo tuoda markkinoille lääkkeenä ihmisille. Nykypäivänä varfariini kuitenkin on yleisin antikoagulantti. Pitkän aikavälin hoidossa se on yleisempi kuin hepariini. Varfariinia alettiin käyttää lääkkeenä 1954, noin 30 vuotta sen löytämisen jälkeen (Van Horn 2013). Nykyään joka sadas Yhdysvaltain ja Iso-Britannian kansalainen käyttää varfariinia säännöllisesti veren hyytymiseen liittyvien sairauksien, kuten keuhkoembolian ja laskimotukoksen sekä sydänkohtauksen ehkäisyyn. Varfariinin yleisyydestä huolimatta sen toimintamekanismi ja kohde-entsyymin rakenne ja toiminta ovat huonosti tunnetut. Parempi ymmärrys vaikutuksesta on tärkeää, sillä varfariinin turva-alue on erittäin kapea. Varfariiniherkkyys myös vaihtelee henkilökohtaisesti, joten yliannostuksen riski on huomattava. (Shen et al. 2017) Varfariini on otettu lääkkeenä käyttöön yli 60 vuotta sitten, mutta vasta nyt on alettu ymmärtää sen sitoutumista VKOR:iin ja inhibition mekanismia tarkemmin. Tuloksien avulla voidaan alkaa suunnitella tulevaisuuden turvallisempia varfariinianalogeja antikoagulanteiksi. Lisäksi yliannostuksen riski pienenee mekanismin selvitessä. (Shen et al. 2017) Tutkielmassa käydään laajasti veren hyytymisjärjestelmä läpi ja perehdytään K-vitamiinisykliin kiinnittäen erityistä huomiota ihmisen K-vitamiiniepoksidireduktaasin toimintamekanismiin. Lopuksi käydään läpi varfariinin inhiboiva vaikutusmekanismi K-vitamiiniepoksireduktaasiin. Tällöin saadaan kattava kuva siitä, missä vaiheessa hyytymisjärjestelmää varfariinin inhibitio vaikuttaa.

Hevosen ruuansulatuskanava on hyvin uniikki. Hevosen maha on pieni verrattuna muihin saman kokoisiin nisäkkäisiin ja vapaasti laiduntava hevonen käyttääkin lähes puolet vuorokaudestaan syömiseen. Hevosen ruuansulatuskanava on kehittynyt hyödyntämään kuitupitoisen ja matalaenergisen ruuan ravintoaineet, joten paljon fermentoituvia hiilihydraatteja sisältävä ruokavalio voi muuttaa mahan olosuhteita ja bakteerikantaa joskus niinkin rajusti, että eläin sairastuu. (Perkins et al. 2012) Mahahaavat ovat hevosten yleisimpiä sairauksia ja niistä kärsii lähteestä riippuen 53–93 % täysikasvuisista kesyhevosista (Begg & O’Sullivan 2003, Perkins et al. 2012). Mahahaavaan liittyy usein ähkyjä, painon laskua ja suorituskyvyn heikkenemistä, mutta monet kilpahevoset sairastavat sitä täysin oireettomana (Perkins et al. 2012). Suurin osa aikuisten hevosten mahahaavoista esiintyy mahan limakalvon pintakerroksessa ruokatorvessa, mahalaukussa, mahanportissa tai pohju-kaissuolessa (Dong et al. 2016, Perkins et al. 2012). Mahan happopitoisuuden nousu ja hapolta suojaavan limakalvon heikkeneminen edistävät usein mahahaavan syntyä. Mahan happopitoisuutta ja siten pH:ta voivat nostaa esimerkiksi kasvanut suolahapon eritys sekä VFA:t, joita syntyy käymisreaktioissa bakteerien hajottaessa väkirehua. (Perkins et al. 2012) Kasvinsyöjillä ruuansulatuskanavan mutualistinen mikrobikanta osallistuu suurelta osin ravintoaineiden muuttamiseen käytettävään muotoon, jolloin kannalla on myös suuri merkitys eläimen terveyden, kasvun ja selviytymisen kannalta. Tyypillisesti kasvinsyöjän ruuansulatuskanavan mikrobikanta koostuu bakteereista, arkeista, alkueläimistä ja fungeista (St-Pierre et al. 2012). Suurinta osaa hevosen mahasta hallitsevat vain tiettyihin lajeihin kuuluvat bakteerit. 92,7 % mahan bakteerikannasta koostuu Firmicutes-, Proteobacteria-, Bacteroidetes- ja Actinobacteria-pääjaksoista (Dong et al. 2016). Terveen hevosen mahan limakalvojen mikrobiomista tiedetään hyvin vähän, eikä mikrobiomin vaikutusta mahan sairauksiin ole kattavasti tutkittu. Helicobacter-lajit ovat riskitekijöitä esimerkiksi ihmisen mahahaavan synnyssä (Shio-tani & Graham 2002). Hevosen mahasta tätä bakteeria ei kuitenkaan monissa tutkimuksissa ole löydetty lainkaan tai löydökset eivät ole korreloineet mahahaavan esiintymisen kanssa (Bezdekova & Futas 2009, Dong et al. 2016, Husted et al. 2010). Muilla mikrobeilla voi sen sijaan olla vaikutusta mahahaavan syntyyn, sillä Randwickin Equine Centerissä vuonna 2003 tehdyssä tutkimuksessa havaittiin mahalaukun limakalvon haavaumien olevan usein vakavampia kuin mahanportin (Begg & O’Sullivan 2003). Tämä on mielenkiintoinen havainto, sillä monet tekijät suojaavat mahalaukun limakalvoa hapolta, ja viittaa siis muiden tekijöiden, esimerkiksi joidenkin mahalaukun bakteerien olevan riskitekijöitä mahahaavan synnyssä.

Cohesin is a ring-shaped Smc (Structural Maintenance of Chromosomes) complex that mediates DNA-DNA interactions between chromosomes (sister chromatid cohesion) and within chromosomes (looping). The mechanisms behind loop formation and cohesion establishment are not known despite cohesin's discovery 20 years ago. In this thesis findings are presented from two projects: one examining the establishment of sister chromatid cohesion and the other the formation of DNA loops by cohesin. <b>Cohesin can remain associated with chromosomes during DNA replication</b> - Sister chromatid cohesion is established at the DNA replication fork. It is not known whether cohesin that is loaded ahead of the fork is used to build cohesion or if it is dislodged and cohesion is built from cohesin rings in the soluble pool. To address this the objective was to determine whether cohesin removal from chromatin is a necessary aspect of DNA replication. Cohesin was fluorescently labelled in a cell line in which cohesin's normal releasing activity was abrogated and cohesin's association with chromatin was followed across S phase. It was found that cohesin could remain in defined subnuclear areas demonstrating that the complex can remain bound to DNA despite replication fork passage. This finding is consistent with the conversion of cohesin that is loaded ahead of the replication fork into its cohesive state. <b>The cohesin loading factor Scc2 hops between cohesin rings</b> - The human genome is organised into a series of megabase scale domains that interact with themselves more than sequences outside of the domain. Interactions inside theses Topologically Associating Domains (TADs) depend on the cohesin complex and are postulated to form by progressive loop enlargement through cohesin's lumen (loop extrustion). If this is the mechanism then cohesin must be able to translocate vast distances. Recent evidence suggests that Smc complexes are motor proteins and their translocation depends on ATP hydrolysis. Cohesin's ATPase is stimulated by Scc2 (Nipbl) however this protein is thought to be involved exclusively during the cohesin loading reaction. Consistently, little colocalisation is detected between cohesin and Scc2 as measured by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq). The aim of this project was to determine whether cohesin and Scc2 interact on DNA after the loading reaction. Using fluorescence recovery after photobleaching (FRAP) and single particle tracking it has been shown that Scc2 turns over rapidly on chromatin indicative of an association independent of cohesin loading, a process that is infrequent due to cohesin's long residence time. It was also found that stabilisation of cohesin on DNA leads to increased Scc2 chromosomal association and cohesin depletion leads to reduced association. These findings provide evidence for a post-loading association between cohesin and Scc2. Intriguingly, Scc2 was also found to spread across chromatin and cohesin vermicelli very slowly consistent with hopping between cohesin rings. Fluorescence analysis of Scc2 and Scc1 indicate that Scc2 is substoichiometric to cohesin. It was therefore proposed that due to an abundance of binding sites for Scc2, a low diffusion coefficient and a high on-rate, that Scc2 hops between cohesin rings performing a function distinct from loading.

abstract: The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future. / Dissertation/Thesis / Ph.D. Biochemistry 2010

abstract: Many natural and synthetic quinones have shown biological and pharmacological activity. Some of them have also shown anticancer activity. Ubiquinone (CoQ10) which is a natural quinone, is a component of the electron transport chain and participates in generation of ATP (adenosine triphosphate). Cellular oxidative stress is key feature of many neurodegenerative diseases such as Friedreich's ataxia, Alzheimer's disease and Parkinson's disease. The increased generation of reactive oxygen species damages cell membranes and leads to cell death. Analogues of ubiquinone in the form of pyrimidinols and pyridinols, were effective in protecting Friedreich's ataxia lymphocytes from oxidative stress- induced cell death. There were some structural features which could be identified that should be useful for the design of the analogues for cellular protection against oxidative stress. There are quinones such as doxorubicin, daunomycin and topopyrones which have anticancer activity. Here I evaluated topopyrone analogues which poison both topoisomerases I and II. The topopyrone analogues were lethal to human breast cancer cells, but these analogues were not as potent as camptothecin. / Dissertation/Thesis / M.S. Biochemistry 2011

abstract: Natural products that target the DNA of cancer cells have been an important source of knowledge and understanding in the development of anticancer chemotherapeutic agents. Bleomycin (BLM) exemplifies this class of DNA damaging agent. The ability of BLM to chelate metal ions and effect oxidative damage of the deoxyribose sugar moiety of DNA has been studied extensively for four decades. Here, the study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of BLM to effect single-stranded was then extensively characterized on both the 3&#8242; and 5&#8242;-arms of the hairpin DNAs. The strongly bound DNAs were found to be efficient substrates for Fe&middot;BLM A5-mediated cleavage. Surprisingly, the most prevalent site of damage by BLM was found to be a 5&#8242;-AT-3&#8242; dinucleotide sequence. This dinucleotide sequence and others generally not cleaved by BLM when examined using arbitrarily chosen DNA substrate were found in examining the library of ten hairpin DNAs. In total, 111 sites of DNA damage were found to be produced by exposure of the hairpin DNA library to Fe&middot;BLM A5. Also, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double stranded DNA damage. Adapting methods previously described by the Povirk laboratory, one hairpin was characterized using this method. The results were in accordance with those previously reported. / Dissertation/Thesis / M.S. Biochemistry 2011

Two superfamilies were used to explore the structure/function relationship as it pertains to enzyme specificity. The structures and mechanisms of phosphatases belonging to the Haloalkanoate Dehalogenase Superfamily (HADSF) and thioesterases of the Hotdog Fold Superfamily (HDFSF) were determined using X-ray crystallography and other biophysical tools in combination with steady-state kinetics and site-directed mutagenesis. Together, the specific structural components of enzymes crucial for substrate recognition, substrate promiscuity, and catalysis were uncovered. In the HADSF, the phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate and mannose 1-phosphate, an essential step in the protein glycosylation pathway. In humans, two isoforms PMM1 and PMM2 catalyze this reaction. Deficiency in PMM2 activity is the major cause of congenital disorders of glycosylation (CDG-1a). However, PMM1 activity is not sufficient to replace PMM2 in protein glycosylation. Instead, PMM1 functions as a glucose-1,6-bisphosphate phosphatase in the presence of IMP and enables the temporary rescue of glycolysis during brain ischemia. Herein, the structure of IMP bound to PMM1 in combination with kinetics revealed a mechanism for the differential substrate preference and the mechanistic switch from a mutase to phosphatase activity. In the HDFSF, the majority of which function as thioesterases of aliphatic or aromatic compounds bound to coenzyme A or acyl carrier protein (ACP), the structural determinants for substrate preference were identified in PA1618, an enzyme with high substrate promiscuity. The structural determinants of the specific thiosterases MA0038 and BVU1957 were also identified. The variation in substrate range observed among these enzymes, from specific to promiscuous, led to the design of a comprehensive thioester screen to identify HDFSF thioesterase substrates. The profiles of substrate specificities from 42 previously uncharacterized thioesterases were determined allowing comparison of the sequence/substrate relationships across a representative selection of thioesterases. Examples drawn from both HADSF and HDFSF enzymes suggested a model relating substrate promiscuity and specificity to regions of protein flexibility. The motion of a domain within a multidomain protein or a flexible loop near the active site was correlated with the occurrence of substrate promiscuity/specificity.

Mesenchymal stem cells (MSCs) are a type of multipotent stem cell capable of differentiating into several cell types, including fat and bone cells. The Hippo pathway effectors TAZ and YAP have been implicated as important regulators of MSC fate, but their roles in this process are poorly understood. The goal of my thesis work was to illuminate the roles of YAP and TAZ in MSC differentiation. I examined how depleting YAP and/or TAZ, or expressing YAP mutants affect the differentiation of C3H10T1/2 cells, which are a multipotent mouse embryo fibroblast cell line capable of forming bone, fat and cartilage. Interestingly, knocking down either YAP or TAZ had different effects on C3H10T1/2 differentiation. YAP knockdown cells that underwent a brown/beige adipogenic protocol showed a significant increase in the amount of lipids produced as compared to control, suggesting that YAP has a role in inhibiting adipogenesis in these cells. YAP knockdown also increased alkaline phosphatase activity in cells subjected to an osteogenic protocol, while simultaneously producing lipid droplets. In contrast, knockdown of TAZ resulted in a decrease in both lipogenesis (in adipogenic differentiation) and alkaline phosphatase activity (in osteogenic differentiation). These observations indicate that depletion of YAP or TAZ leads to defective MSC differentiation, and that without proper YAP and TAZ signaling MSC cells may arrest in an immature state. Bone Morphogenic Proteins (BMPs) are known to play important roles in MSC differentiation, and YAP/TAZ have been implicated in BMP regulation in other systems. I therefore hypothesized that YAP/TAZ may affect MSC differentiation in part by altering BMP signaling. Analysis of activated phosphorylated Smad1 (p-Smad1), a key transcriptional effector of BMP signaling, showed that TAZ and YAP are required to promote p-Smad1 levels. Depletion of TAZ and YAP showed an increase in the expression of Gremlin1 (GREM1), an inhibitor of BMP signaling, and an increase in the expression of BMP4. These observations suggest that TAZ and YAP promote BMP signaling by controlling BMP ligand activity. Given that BMP signaling has important roles early in MSC differentiation, I hypothesized that YAP and TAZ may direct these early processes. To test this, I generated dox-inducible C3H10T1/2 cells capable of expressing wild type YAP or two mutant forms of YAP: a nuclear localized YAP mutant (YAP-5SA), and a transcriptionally inactive YAP mutant (YAP-5SA,S94A). Adipogenic differentiation with these cells showed a reduced level of lipogenesis in the constitutively active nuclear YAP mutant. Analysis of adipogenic markers by qPCR showed a reduction in both PPARγ and UCP1. Osteogenic differentiation experiments in these cells showed that transient expression of all forms of YAP increased the amount of alkaline phosphatase activity, with the 5SA form showing the greatest activity. RNA expression analysis showed that the early marker Runx 2 was highly upregulated in cells overexpressing wild type YAP, while the late markers osteocalcin and osteopontin were greatly reduced in comparison to control. As alkaline phosphatase activity is a mid-stage marker for bone differentiation, the staining results and qPCR data indicate that overexpression of YAP may result in arrest of the differentiation process. A microarray was performed using isolates from 10T1/2 cells transduced with TAZ, YAP or TAZ/YAP siRNA to further evaluate the mechanism through which TAZ and YAP may be acting. The TAZ and YAP double knockdown showed a large change in global gene expression as compared to both the control and the single knockdowns. Among the genes that showed a large change from control was BMP4, the expression of which was reduced more than two-fold over control. Additionally, GREM1 was up-regulated almost two-fold by the double knockdown. Also upregulated was the gene periostin which encodes a protein that enhances BMP incorporation into connective tissues, and may play a role in the mineralization of the extracellular matrix of bone. Based on my observations, along with those of others, I propose that TAZ and YAP are major factors in controlling early cell fate determination of MSCs. There appear to be multiple levels of regulation, and thus the next steps should include clarifying the roles of YAP and TAZ in vivo, and placing their activity chronologically in the differentiation timeline. Such studies will offer insight into stem cell differentiation and may eventually allow for therapeutic opportunities.