Cellulose is the most abundant polymer on earth. It is one of the main components in lignocellulosic biomass, which has great potential as a renewable energy source. To utilize the biomass, for instance in biofuel production, cellulose needs to be degraded. In nature there are microorganisms that are specialized on such degradation, and they produce interesting cellulose hydrolysing enzymes. Understanding the function of these enzymes can hence be one step towards a more sustainable future.  The aim of this project was to find out if the enzyme GtCel45A from Gloeophyllum trabeum could hydrolyse soluble oligosaccharides and produce mono- or disaccharides as products. The study was executed by cultivating Aspergillus nidulans A773 recombinantly expressing GtCel45A followed by a purification process consisting of anion exchange chromatography and size exclusion chromatography. From 1.4 liters of culture, grown for 8 days at 30°C, 9.9 mg of purified GtCel45A was obtained. Activity measurements using p-hydroxybenzoic acid hydrazide (PHBAH) reagent for reducing sugar showed that the enzyme is active against and does hydrolyse barley beta-glucan. However, no hydrolysis of cellohexaose, cellotetraose, cellotriose or cellobiose could be detected, even after 223 minutes of incubation with GtCel45A as shown by carbohydrate analysis with high performance anion exchange chromatography with pulsed amperiometric detection (HPAE-PAD). In addition, a number of crystallization trials were performed, which resulted in formation of crystals that could subsequently be used to solve the structure of the protein.

Lipases are one of the most important biocatalysts for biotechnological applications. They have high importance since lipases have a catalytic activity that is related to their hydrolysis and synthesis reactions to high regioselectivity and enantioselectivity. They also have an activity over a wide range of temperature, pH, and diverse substrates. The aim of this master thesis project was to apply computational calculations to understand the reaction mechanism of the hydrolysis of p-nitrophenyl butyrate performed by the cold-adapted lipase, Bacillus pumilus Lipase A (BplA). Cold-adapted lipases are enzymes that have evolved to perform catalysis in a colder environment. The methods used for performing the computational calculations in this thesis project can be divided into three sections. The structure preparation, molecular dynamic (MD) simulations using NAMD, and quantum mechanics combined with molecular mechanics (QM/MM) using ORCA. Through eight substeps with MD and QM/MM implementations potential energy surfaces (PES), minimum energy path (MEP) and frequency calculations for the enzymatic reaction were provided. The rate-limiting step is the formation of the tetrahedral intermediate, the nucleophilic addition during the deacylation. The largest free energy barrier provided from the results had an activation free energy of 20.3 kcal/mol. The barriers of the deacylation were larger than the one for the acylation process. By understanding the reaction mechanism, one can understand how cold-adapted enzymes could catalyze the reaction to create lower energy barriers at low temperatures.

The ErbB family of receptors are involved in signalling relating to cell proliferation and differentiation through activation of pathways such as PI3K and MAPK. Their overexpression is often found in different cancer types and therefore, their expression is under tight regulation. The ErbB family includes, EGFR, ErbB2, ErbB3, and ErbB4 where all but ErbB3 has a kinase domain, making ErbB3 a pseudokinase. Upon activation of the receptors, they are endocytosed through the formation of a clathrin-coated pit and are degraded in the lysosome. Interestingly, researchers have found that newly synthesised ErbB3 can also be degraded in the proteasome by protein Nrdp1. Suggesting that ErbB3 might work in a ligand-independent manner and needs additional regulatory mechanisms. ACK1 is a non-receptor tyrosine kinase that has a reported effect on EGFR by promoting receptor degradation in the autophagosome. However, their role in EGFR regulation is still debated. Therefore, this information alludes to the fact that ACK1 might influence other ErbB family members as well.   This report aims to investigate whether ACK1 influences ErbB3 levels. Through RNAi mediated knockdown of ACK1 in MCF10A cells, a novel role of ACK1 acting as a regulator of ErbB3 is hinted at. Surprisingly, these results also show that ACK1 seems to act specifically on ErbB3 and not on its family members, EGFR and ErbB2. Moreover, this report shows that ErbB3 expression is linked to cell density.

Ultraviolett (UV) strålning genererar fotodimerer i celler som leder till komplikationer med replikering och transkribering. Dessutom bidrar det också med DNA lesioner och brott. Förståelsen för hur de molekylära mekanismerna fungerar är av högsta vikt för att förhindra uppkomsten och bota hudsjukdomar som orsakats av UV-strålning. Den här uppsatsen fokuserar på hudens komposition och hur den förändras med åldern, av både inre och yttre faktorer samt cellernas egna reparationsmekanismer. Huden består av cellulära och extracellulära matriskomponenter, båda är fint fördelade och organiserade för att bidra med skydd, elasticitet och spänst. Åldrande leder till att hudens förmågor försämras, rynkor uppstår och det sker en förlust av spänst. Detta sker naturligt som konsekvens av inre åldrande, men detta påskyndas av yttre faktorer som miljöföroreningar och UV-strålning. Olika mekanismer i celler reparerar dessa skador, det här arbetet tar upp nukleotidreparation (NER) och hur det reparerar via att klyva bort de mutagena delarna av genomet för att förhindra uppkomst av tumörer. Trots hudens olika reparationssystem förblir vissa celler skadade, personer med ljusa pigment är mer känsliga för mutationer än personer med mörka pigment. För att förhindra och förebygga uppkomst av farliga hudförändringar rekommenderas idag inte bara solskyddsfaktorer, utan även antioxidanter som skyddar mot oxidativ stress som även den orsakas av UV-strålning.

Den snabba utvecklingen av det nya genredigerande verktyget CRISPR-Cas9 har öppnat dörren för behandlingen av svåra, virusorsakade sjukdomar. Herpes simplexvirus typ 1 (HSV-1), en av de mest vanligt förekommande virus i världen, kan etablera sig latent i känselnervceller. Stressfaktorer kan orsaka herpeskeratit, som är en sekundär infektion av ögat vilket leder till inflammation och potentiell ärrbildning. Vid upprepade återfall kan herpeskeratit resultera i synnedsättningar och i allvarligare fall blindhet, och därför anses det som en stor samhällsbörda.  Denna uppsats undersöker förutsättningarna att CRISPR-Cas9 i framtiden ska användas för att effektivisera behandlingen av herpeskeratit. Detta görs först genom att beskriva hur HSV-1 infekterar människokroppen, etablerar latens och skadar hornhinnan. Sedan introduceras utvecklingen av CRISPR-Cas9 som ett molekylärt verktyg där dess fördelar och nackdelar beskrivs. Slutligen sammanfattas de tre olika tillvägagångssätten av forskare som undersöker möjligheten att skapa en CRISPR-Cas9-baserad behandling.  Den första metoden baseras på användningen av CRISPR-Cas9 för att dämpa virusreplikation vilket resulterar i att konsekvenserna blir lindrigare vid eventuella återfall. Detta alternativ har visat sig vara framgångsrikt in vitro och det kringgår en del utmaningar gällande leveranssystemet, men är begränsad i sina användningsområden. Den andra metoden baseras på att redigera känselnervceller så att de konstant utsöndrar CRISPR-Cas9 samt relevanta guide- RNA för dämpningen av virionbildandet likväl som förebyggandet av en primärinfektion. I praktiken blir detta svårt att tillämpa som behandlingsmetod på grund av ett antal tekniska svårigheter. Det tredje alternativet är att klyva det latenta HSV-1 genomet. Detta skulle omöjliggöra en sekundärinfektion. In vitro-experiment har visat att det är möjligt, men svårigheter med leveranssystem och effektiviteten har gjort det problematiskt att dra några relevanta slutsatser om CRISPR-Cas9 är rätt väg eller inte. Andra genredigerande enzymklasser, exempelvis meganukleaser, har haft större framgång i musmodeller. Forskningen gällande CRISPR-Cas9 är vid ett tidigt stadium, men har väckt stort intresse inom forskningsvärlden. Med fortsatt optimeringen av CRISPR-Cas9, och dess framgång i andra virussammanhang är det troligt att CRISPR-Cas9 eventuellt kommer vara en del av lösningen för att bota herpeskeratit.

Bakgrund: Platelet derived growth factor receptorn (PDGF receptorn) är en receptor i kroppen som tillhör typ III av Recetor tyrosine kinas (RTK) familjen. PDGF-β receptorn är en typ av dessa receptorer som enligt en studie klyvs i två delar efter ligandbindning med PDGF-BB ligand: till en extracellulär del och en intracellulär del. Hypotesen är att den extracellulära delen går till lysosomen medan den intracellulära delen går till proteasomen efter klyvningen. Syfte: Att undersöka rollen för proteasomen i ligandinducerad fragmentation av PDGF-β receptorn och se om det finns ko-lokalisation mellan receptorns extracellulära del och proteasomen. Dessutom ska det undersökas hur eventuell ko-lokalisering av PDGF receptorns extracellulära fragment med proteasomet påverkas när proteasomerna i cellerna inhiberas. Metod: Bj-hTERT celler stimulerades med PDGF-BB ligand under fyra olika stimuleringstidspunkter (0, 30, 60 och 90 min) och med hjälp av immunofluorescens undersöktes det om det fanns ko-lokalisering mellan proteasomen och PDGF-β receptorns extracellulära del. I ett annat experiment inhiberades även proteasomens aktivitet med inhibitoren MG132 och ko-lokalisationen mättes och jämfördes med kontrollceller behandlat med DMSO för de fyra stimuleringstidspunkterna. Resultat: För det första ko-lokalisationsexperimentet visades ingen större ko-lokalisering mellan proteasomen och receptorns extracellulära del för alla fyra tidspunkter. För det andra ko-lokalisationsexperimentet visades ingen skillnad i ko-lokalisationen mellan proteasomen och receptorns extracellulära fragment för cellerna vars proteasomaktivitet inhiberats och kontrollcellerna. Det visade sig däremot att proteasomen spelade roll för internaliseringen av receptorns extracellulära del in i cellen.

To maintain a healthy growth rate of living cells, protein synthesis must take place accurately and efficiently. Translation, the core step decoding the genetic information, governs both fidelity and efficiency. For keeping a low error level, translation mainly relies on the codon-anticodon basepairing of mRNA and tRNA, which is known as ‘initial selection’. Apart from that, nature also evolved proofreading to reduce errors in protein synthesis. The current study focuses on initial selection of the correct tRNA.  EF-Tu, Elongation Factor Thermo-unstable, is an essential housekeeping GTPase factor responsible for transferring aa-tRNA to the ribosome. EF-Tu contributes to accuracy of initial selection although the exact mechanism is unknown. Here we have characterized and compared two sequence reconstructed ancestral EF-Tus, which are 1.3 and 3.3 billion years old respectively. Using dipeptide formation assay, we obtained the Michaelis-Menten parameters for Leu-tRNALeu on a near-cognate codon. Comparing the specificity parameter kcat/KM for the near-cognate vs. cognate we determine the accuracy of tRNA selection. My results show lower efficiency but higher accuracy using ancestral EF-Tus supporting the theory of trade-off between efficiency and accuracy. We envisage that during evolution EF-Tu sacrifices some accuracy to achieve higher efficiency as seen with modern EF-Tus.  My results demonstrate that EF-Tu can coordinate both the fidelity and the efficiency of translation.

RNA interference (RNAi) is an important mechanism of gene silencing in Caenorhabditis elegans, in which short RNAs direct sequence-specific silencing of gene expression mediated by Argonaute proteins. RNAi in C. elegans can be sorted into exogenous RNAi, where the short RNAs originate from foreign RNA sequences, and endogenous RNAi, where the short RNAs originate from RNA sequences in the genome. One endogenous RNAi pathway is the ERGO-1 pathway, which is active in the germline and in embryos. Mutants deficient in the ERGO-1 pathway show an increased response to exogenous RNAi, which is thought to be due to competition between exogenous and endogenous silencing pathways.  FUBL-1 is an RNA-binding protein found in C. elegans, which has three predicted functional isoforms: isoforms a, b and c. Prior research by the Hinas group has indicated that FUBL-1 may play a role in the ERGO-1 pathway of RNAi. FUBL-1 deletion mutants show an increased response to exogenous RNAi similar to ERGO-1 mutants, and also an upregulation of ERGO-1 target genes. They have also found that FUBL-1 is broadly expressed in somatic tissues, but germline expression has not been confirmed. The function of the three different isoforms has not yet been examined. The aim of this study was to further investigate the function of FUBL-1 by assessing the function of the different isoforms through RT-qPCR of C. elegans with mutations affecting the different isoforms, to use immunofluorescence staining to see whether FUBL-1 is expressed in the germline, and to identify FUBL-1 RNA targets through CLIP-seq. Preliminary RT-qPCR results indicated that the upregulation of ERGO-1 targets is less in isoform mutants than in FUBL-1 deletion mutants, indicating partial redundancy of the isoforms. Immunofluorescence staining showed that FUBL-1 is expressed in the germline nuclei. Growth protocols have been optimized and crosslinking has been performed on worms, but the full CLIP-seq protocol has not been performed yet.

Density functional theory is employed to study the reaction mechanisms of different epoxide-transforming enzymes. Calculations are based on quantum chemical active site models, which are build from X-ray crystal structures. The models are used to study conversion of various epoxides into their corresponding diols or substituted alcohols. Epoxide-transforming enzymes from three different families are studied. The human soluble epoxide hydrolase (sEH) belongs to the α/β-hydrolase fold family. sEH employs a covalent mechanism to hydrolyze various epoxides into vicinal diols. The Rhodococcus erythrobacter limonene epoxide hydrolase (LEH) constitutes a novel epoxide hydrolase, which is considered the founding member of a new family of enzymes. LEH mediates transformation of limone-1,2-epoxide into the corresponding vicinal diol by employing a general acid/general base-mediated mechanism. The Agrobacterium radiobacter AD1 haloalcohol dehalogenase HheC is related to the short-chain dehydrogenase/reductases. HheC is able to convert epoxides using various nucleophiles such as azide, cyanide, and nitrite. Reaction mechanisms of these three enzymes are analyzed in depth and the role of different active site residues is studied through in silico mutations. Steric and electronic factors influencing the regioselectivity of epoxide opening are identified. The computed energetics help to explain preferred reaction pathways and experimentally observed regioselectivities. Our results confirm the usefulness of the employed computational methodology for investigating enzymatic reactions. / QC 20101108

The present thesis is based upon two papers concerning the core-valence double onization of molecular oxygen and mutual neutralization of H+ and H- ions at low collision energies. The former of these processes has been studied for the first time using a magnetic bottle time-of-ight electron coincidence spectrometer in combination with ab initio electronic structure calculations. The core-valence photoelectron spectra have been interpreted by comparing with the calculated double ionization energies, as well as the conventional valence band spectrum. Based on this comparison, some general features of the process are discussed and assignments for several of the dicationic states proposed. The latter process has been studied by means of a molecular close coupling approach in which both the nuclei and the electrons have been treated at a quantum mechanical level of theory. Accurate ab initio potential energy curves and non-adiabatic couplings have been used to calculate the neutralization cross section in the collision energy region 0.001 to 100 eV. Special emphasis has been put on the energy region below a few eV from which the low temperature rate coe_cient is evaluated. In this region, the calculated neutralization cross section is in good agreement with several other theoretical studies, but is a factor of two to three lower than the only published experimental data. / QC 20101123