Prostate cancer is one of the most common cancer types worldwide. However, current diagnostic approaches and treatments are invasive and unspecific. Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer and can act as a target for therapeutic or diagnostic agents. Previous attempts to develop an affibody with affinity towards PSMA have been unsuccessful, therefore this thesis aimed at making the affibody selections against PSMA more efficient. In this thesis HEK293 cells expressing a modified version of PSMA containing a 3C protease cleavage site were generated, to enable extraction of the extracellular domain of PSMA during the selections. However, further analyses must be performed to determine if the extracellular domain can be successfully cleaved off. To develop an affibody that can be used both in vitro and in vivo, selections will be carried out against recombinant PSMA as well. The recombinant PSMA was previously produced incorporating an Avi tag for site-specific biotinylation and immobilization for the selections. To biotinylate the recombinant PSMA, the enzyme BirA that catalyzes the biotinylation of the Avi tag, was produced. A protein yield of 8.95 mg/liter culture was obtained and the site-specific biotinylation was highly efficient. To evaluate the proposed affibody selection strategy the next step is to determine if cleavage of the PSMA expressed on the HEK293 cells is possible, optimize the cleavage conditions and to start initial selections using the generated HEK293 cells and the produced BirA enzyme. / Prostatacancer är en av de mest förekommande cancertyperna över hela världen. Nuvarande diagnostiska metoder och terapeutiska behandlingar är dock invasiva och ospecifika. Prostataspecifikt membranantigen (PSMA) är en idealisk biomarkör för prostatacancer och kan agera som en målmolekyl för terapeutiska eller diagnostiska ändamål. Tidigare försök att utveckla en affibody med affinitet mot PSMA har inte lyckats, därför var målet med detta examensarbete att effektivisera selekteringen av affibodies mot PSMA. I detta projekt har HEK293 celler som uttrycker en modifierad version av PSMA, innehållande ett 3C-proteas- klyvningsställe, genererats för att möjliggöra extraktion av den extracellulära domänen av PSMA under selekteringen. Ytterligare analyser måste dock utföras för att avgöra om den extracellulära domänen kan klyvas av. För att utveckla en affibody som kan användas både in vitro och in vivo kommer selekteringen att utföras även mot rekombinant PSMA. Rekombinant PSMA har producerats tidigare med en Avi tag för specifik biotinylering och immobilisering under selekteringen. För att biotinylera det rekombinanta PSMA producerades enzymet BirA, som katalyserar biotinyleringen av en Avi tag. Ett proteinutbyte av 8,95 mg/liter kultur erhölls och den specifika biotinyleringen var effektiv. För att utvärdera den föreslagna strategin för selektering av affibodies är nästa steg att avgöra om klyvning av PSMA uttryckt av HEK293 cellerna är möjlig, optimera klyvningsförhållandena och starta initiala selektioner med de genererade HEK293-cellerna och det producerade BirA-enzymet.

The time-course of interaction formation or breaking can be studied using LigandTracer, and the data obtained from an experiment can be analyzed using a model of ligand-target binding kinetics. There are different kinetic models, and the choice of model is currently motivated by knowledge about the interaction, which is problematic when the knowledge about the interaction is unsatisfactory. In this project, a Bayesian model selection procedure was implemented to motivate the model choice using the data obtained from studying a biological system. The model selection procedure was implemented for four kinetic models, the 1:1 model, the 1:2 model, the bivalent model and a new version of the bivalent model.Bayesian inference was performed on the data using each of the models to obtain the posterior distributions of the parameters. Afterwards, the Bayes factor was approximated from numerical calculations of the marginal likelihood. Four numerical methods were implemented to approximate the marginal likelihood, the Naïve Monte Carlo estimator, the method of Harmonic Means of the likelihood, Importance Sampling and Sequential Monte Carlo. When tested on simulated data, the method of Importance Sampling seemed to yield the most reliable prediction of the most likely model. The model selection procedure was then tested on experimental data which was expected to be from a 1:1 interaction and the result of the model selection procedure did not agree with the expectation on the experimental test dataset. Therefore no reliable conclusion could be made when the model selection procedure was used to analyze the interaction between the anti-CD20 antibody Rituximab and Daudi cells. / Interaktioner kan analyseras med hjälp av LigandTracer. Data från ett LigandTracer experiment kan sedan analyseras med avseende på en kinetisk modell. Det finns olika kinetiska modeller, och modellvalet motiveras vanligen utifrån tidigare kunskap om interaktionen, vilket är problematiskt när den tillgängliga informationen om en interaktion är otillräcklig. I det här projektet implementerades en Bayesiansk metod för att motivera valet av modell utifrån data från ett LigandTracer experiment. Modellvalsmetoden implementerades för fyra kinetiska modeller, 1:1 modellen, 1:2 modellen, den bivalenta modellen och en ny version av den bivalenta modellen. Bayesiansk inferens användes för att få fram aposteriorifördelningarna för de olika modellernas parametrar utifrån den givna datan. Sedan beräknades Bayes faktor utifrån numeriska approximationer av marginalsannolikeheten. Fyra numeriska metoder implementerades för att approximera marginalsannolikheten; Naïve Monte Carlo estimator, det harmoniska medelvärdet av likelihood-funktionen, Importance Sampling och Sekventiell Monte Carlo. När modellvalsmetoden testades på simulerad data gav metoden Importance Sampling den mest tillförlitliga förutsägelsen om vilken modell som generade datan. Metoden testades också på experimentell data som förväntades följa en 1:1 interaktion och resultatet avvek från det förväntade resultatet. Följaktligen kunde ingen slutsas dras av resultet från modelvalsmetoden när den sedan används för att analysera interaktionen mellan anti-CD antikroppen Rituximab och Daudi-celler.

Chronic avian malaria and low-intensity parasite infections can have a negative influence on reproduction and survival which can reduce the fitness of the host. Therefore, the effect on host due to avian malaria infection can affect a bird’s lifespan. Consequently, avian parasites can influence evolution, physiology, and host morphology. Among birds, there are several blood parasites used as model organisms for the study of parasite interactions with hosts, of which three genera of a haemosporidian parasite called avian malaria parasite. It is possible nowadays, to investigate and detect if the organism is infected with a blood parasite using fast and accurate molecular techniques.     In this study, I investigated how cellular respiration, an important trait regulating energy demands in animals, is affected by the infection of malaria in pied and collared flycatchers and if sex and age have a cross-relation influence on the cellular respiration rate. I used recently collected respiratory data and molecular techniques to evaluate the infection’s influence on cellular respiration. 138 samples were extracted and the nested-Polymerase chain reaction (PCR) was used to assess the presence of the parasite. Data was collected and interpreted and statistical analyses were performed.     I found that, females and juvenile flycatchers have higher cellular respiration rates and that there is a significant difference in respiration rate between the sexes. Also, I found that Leucocytozoon, but not Haemoproteus and/or Plasmodium infection show significant effect on flycatcher cellular respiration rate. However, parasite infection shows no significant effect on the total number of red blood cells count. Therefore, our study supports the idea that parasites can influence the organism’s metabolic activity. The study also shows how the identification of factors underlying the effect on cellular respiration rate can get us closer to understanding the important links between infection, age, sex and metabolic activity and performance

Aktivt slam är en biologisk process som används i stor utsträckning för rening av avloppsvatten, där bakterier som odlas under luftiga förhållanden avlägsnar organiska ämnen och näringsämnen från förorenat vatten. För industriell verksamhet modelleras denna process för att representera och förutsäga biologiska fenomen. De modeller som används för närvarande (främst modellen för aktiverat slam) ger acceptabla resultat men har vissa begränsningar, t.ex. lång kalibreringstid och beroende av många modellparametrar som är svåra att förutsäga. Under detta projekt som genomfördes på företaget SUEZ bedömdes det om sådana modeller kunde förbättras genom att lägga till ett termokemiskt perspektiv i deras arkitektur, antingen genom att minska antalet parametrar eller genom att teoretiskt förutsäga deras värden. Det termokemiska bidraget bedömdes på två olika nivåer: de stökiometriska förhållanden som beskriver systemet och de biologiska processernas kinetik. På grundval av en omfattande litteraturstudie valdes två metoder ut för att föra in termodynamiska överväganden i dessa delar av modellen. Termodynamiska beräkningar som krävs för att förbättra specifikationen av de förorenande molekyler som finns i vattenmatrisen. Arkitekturen i den ursprungliga modellen för aktiverat slam ändrades för att möjliggöra en differentiering mellan substrat av olika slag. När det gäller den stökiometriska studien visade det sig att den dynamiska förutsättningen av termodynamiska begränsningar för reaktionerna inte var av något större intresse med tanke på de stora mängder energi som finns tillgängliga. Användningen av olika organiska substrat gav dock intressanta resultat, eftersom det gjorde det möjligt att modellera de biologiska beteendena på ett mer detaljerat sätt och att förutsäga tillväxten av specifika typer av mikroorganismer. Den kinetiska studien gav måttliga resultat, med adekvata simuleringar men utan någon ny större förbättring av modellen. Den mest lovande utvecklingen av denna studie tycks vara en modell som omfattar en mängd olika organiska substrat med motsvarande biomassapopulationer, för att kunna rikta in sig på den specifika förekomsten av intressanta bakterier. / Activated sludge is a biological process that is widely used for wastewater treatment, where bacteria grown in aerated conditions remove organic substances and nutrients from polluted waters. For industrial operations, this process is modeled to represent and predict biological phenomena. Currently used models (mainly, the Activated Sludge Model) give acceptable results but suffer from some limitations such as long calibration time and dependency on an many model parameters that are difficult to predict. During this project conducted at the company SUEZ, it was assessed whether such models could be improved by adding a thermochemical perspective to their architecture, either by reducing the number of parameters involved or by predicting theoretically their values. The contribution of thermochemistry was assessed on two different levels: the stoichiometric relations that describe the system, and the kinetics of the biological processes. Based on an extensive literature study, two methods were selected to inject thermodynamical considerations to these parts of the model. Thermodynamic calculations required to improve the specification of the pollutant molecules present in the water matrix. The architecture of the initial Activated Sludge Model was modified to allow for a differentiation between substrates of different nature. Regarding the stoichiometric study, it appeared that the dynamic prevision of thermodynamical constraints over the reactions did not show a major interest considering the high amounts of energy available. However, the use of different organic substrates gave interesting results because it allowed to model more finely the biological behaviors and to predict the growth of specific types of microorganisms. The kinetic study gave mitigated results, with adequate simulations but no new major improvement to the model. The most promising development of this study appears to be a model involving a multiplicity of organic substrates, with corresponding biomass populations, to target the specific occurrence of bacteria of interest.

Argonaute proteins play substantial roles in post-transcriptional regulation of gene expression within RNA interference (RNAi) pathways, making them crucial subjects for research, aimed at understanding their interactions with small non-coding RNAs (ncRNAs) and other RNAi components. This study focuses on investigating these properties of Argonaute proteins, particularly Argonaute protein A (AgnA), in the social amoeba Dictyostelium discoideum that is renowned for its broad genetic toolbox and unique life cycle. While previous studies have examined the disruption of three Argonaute genes (agnB, agnC, agnE) and their effect on mRNA levels and small ncRNA expression, this study extends to agnA gene, which remains less studied. Key questions surrounding the influence of AgnA on the cellular processes such as the cell growth rate, development, gene expression, as well as potential targets and small ncRNA binding, remain unanswered. A well-established approach that could provide the necessary answers is the disruption of the gene through traditional homologous recombination, by insertion of a drug-resistance cassette flanked by homology arms complementary to the target locus. However, the emerging CRISPR/Cas9 gene editing tool on contrary offers straightforward protocols for disruption of gene expression through efficient induction of genomic knockouts, point mutations and deletions. In this study, both approaches were applied in parallel to knockout the agnA gene, enabling comparison of knockout efficiency and further study of the growth rate, development and gene expression in the knockout strains. Moreover, important information regarding the growth patterns of both wild-type and agnE knockout strains were also elucidated, complementing the previous growth rate analyses. The obtained data from this research could provide valuable insights for future studies ofthe RNAi machinery components and particularly the function of Argonaute proteins in D. discoideum.

Åldrande befolkningar är en global utmaning särskilt med ökande incidens av åldersrelateradesjukdomar, särskilt Alzheimers sjukdom (AD). AD är den mest förekommande formen avdemens. Sjukdomen, kännetecknas av kognitiv försämring, med varierande grad avminnesförlust och funktionsnedsättning. Alzheimers patologi involverar ackumulering av β-amyloidpeptider och hyperfosforylerat tauprotein,vilka bidrar till sjukdomens progression. Östrogen samt östrogenreceptorerna påverkarmånga kroppsfunktioner och är särskilt viktiga för neuroprotektion och reglering av genuttryck.Trots kunskapsframsteg är mekanismerna bakom könsskillnader i AD och östrogenets effekterinte helt förstådda, vilket understryker behovet av vidare forskning för att utveckla effektivabehandlingar och förstå sjukdomens underliggande biologi. Syftet med denna studie är att djupare förstå östrogens roll i Alzheimers sjukdom, samt attundersöka utveckling, respektive förbättring av AD beroende på östrogens nivåer i kroppen.För att uppnå syftet, gjordes en systematisk litteratursökning på internationella databaser.Artiklarna som inkluderas i studien granskades och dess relevans bedömdes med noggrannhetvia urvals steg. Endast relevanta artiklar som uppfyller inklusionskraven inkluderades för attbesvara syftet och frågeställningen. Det visades att behandling med östradiol förbättrade minnet hos AD-musmodeller. E2-behandlade möss uppvisade lägre nivåer av β-amyloid plack och minskad tau-fosforyleringjämfört med obehandlade AD-möss. Långvarig hormonbehandling, särskilt hos kvinnor sompåbörjar behandlingen vid en yngre ålder, kan öka risken för Alzheimers sjukdom genom ettöverskott av östrogen, vilket belyser östrogenets komplexa roll i sjukdomens patogenes ochbehandling. Östrogenreceptorerna spelar en roll i regleringen av tau-fosforylering, där ERαöveruttryck ökade tau-fosforyleringen och ERβ överuttryck minskade den. Effekten avöstrogenbehandling på APOE-genotypen i möss visade genotypberoende effekt. Östrogenerbjuder en potentiellt skyddande roll mot Alzheimers sjukdom men effekterna är beroende avindividuella och genetiska faktorer. Resultaten indikerar att östrogen har en potentiellskyddande effekt mot AD genom att modulera β-amyloid och tau-fosforylering samt genom attfrämja neurogenes. Dessa fynd understryker östrogens betydelse som ett potentiellt terapeutisktmål för AD, särskilt med tanke på de genetiska och hormonella faktorer som bidrar tillsjukdomsutvecklingen. Resultaten understryker vikten av försiktighet vidhormonersättningsterapi och behovet av ytterligare forskning för att förstå de genetiska ochhormonella faktorernas inverkan på risken för Alzheimers.

Parkinson’s Disease (PD) is the second most common neurodegenerative disease (NDD) affecting approximately 1 - 2% of the population older than 65 and it is characterized by both motor and non-motor symptoms such as rest tremors, stooping posture, and rigidity. The neuromolecular basis of PD is quite complex and that is why there is a need for in vitro systems that can be utilized for studies of PD and NDDs in general. Human-derived cell lines are a good candidate for in vitro systems since they are easy to manipulate and are a less costly alternative to post-mortem human tissue sections or animal models. In this study, I optimize the Lund human mesencephalic (LUHMES) cell line differentiation protocol by determining that the optimal seeding density of cells is 37 500 cells/ml and that the differentiation media can contain quadruple the recommended concentration of tetracycline hydrochloride. Additionally, I use the differentiated LUHMES cells to conduct an exploratory study by treating the cells with cerebrospinal fluid (CSF) from PD patients and CSF from healthy individuals to investigate the neuromodulatory effects of the CSF on the neuronal cell culture. Cell viability assay showed neurotoxicity 24 hours post-treatment for the control CSF and 48 hours post-treatment for both control and PD CSF. Immunohistochemistry showed differential expression of proteins of interest that reflect hallmarks of neurodegenerative diseases. Further studies are needed to reach conclusive results.

Breast cancer is the most common form of cancer in women worldwide and the second leading cause of cancer death. It is a heterogeneous disease and is subdivided into different subtypes, all with different treatment responses and survival outcomes. Luminal breast cancers are characterised by the expression of oestrogen receptor and generally have a good prognosis. More aggressive tumours are marked by the presence of growth stimulating receptor tyrosine kinase HER2 (HER2-like breast cancer) or the absence of oestrogen receptor, progesterone receptor, and HER2 (triple-negative breast cancer,TNBC). The latter is the most aggressive form and is difficult to treat due to lack of treatment targets. This thesis aimed to explore possible prognostic and predictive biomarkers in different subtypes and study their role in breast cancer. To this aid, breast cancer tumours of pre- and post-menopausal patients enrolled in two cohorts were analysed for gene copy numbers and expression of proteins involved in cell proliferation. Gene copy numbers of receptor tyrosine kinases MET and EGFR, Met’s ligand HGF, and protein tyrosine phosphatase PTPN2 were determined by droplet digital PCR or quantitative PCR in both cohorts. Met, phosphorylated Met (pMet), HGF, and PTPN2 protein expression levels were analysed with immunohistochemical staining in the pre-menopausal cohort. Moreover,the role of the aforementioned proteins was investigated in breast cancer cell lines. Amplification of MET, HGF, and EGFR in breast tissues was found to be low (5-8%). These three genes, all located on chromosome 7, were found to be strongly correlated with eachother and to be associated with shortened distant recurrence-free survival. High protein expression of Met, pMet, and HGF was found in 33%, 53%, and 49% of the breast tumours. MET and EGFR were found to be more often amplified in TNBC disease, correlating with worse survival. Moreover, stromal expression of HGF was associated with shorter survival in TNBC. EGF stimulation in TNBC cell line MDA-MB-468 led to inhibited cell proliferation and migration. Partial knockdown of EGFR caused TNBC cells to proliferate and migrate more upon EGF treatment, mirroring EGFR inhibitor resistance. Knockdown of Met had in part the opposite effects, indicating that Met inhibitors might be useful in the treatment of TNBC. The increase in proliferation and migration upon EGFR depletion could be counteracted with simultaneous knockdown of EGFR and Met, indicating that dual inhibition of these proteins might be a future treatment option in TNBC. Copy loss of PTPN2 was reported in 15% of the cases in both pre- and post-menopausal cohorts. Low cytoplasmic PTPN2 protein expression was found in half of the cases. Loss of PTPN2 gene or protein was associated with a shorter distant recurrence-free survival in Luminal A and HER2-positive tumours, not in TNBC, suggesting a subtype-related prognostic value of PTPN2. Subtype relevance of PTPN2 was further implied by in vitro analyses. Whereas PTPN2 knockdown had no observed effect on TNBC cell lines, knockdown in the Luminal A cell line MCF7 inhibited Met phosphorylation and promoted phosphorylation of Akt, a key regulator of cellular proliferation and survival. The cell growth and survival regulating RAS/MAPK pathway remained unaffected. Knockdown in the HER2-positive cell line SKBR3 led to increased Met phosphorylation and decreased RAS/MAPK-related Erk phosphorylation as well as EGF-mediated transcription factor STAT3 phosphorylation. These results indicate that the role of PTPN2 in breast cancer is subtype-related and needs to be further investigated for future treatment options.

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

<p>Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.</p><p>The thermodynamic integration technique is used to calculate p<i>K</i>_a values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated p<i>K</i>_a values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine p<i>K</i>_a shifts in wild-type and mutant epoxide hydrolase. The calculated p<i>K</i>_a values support a model that can explain some of the pH dependent properties of this enzyme.</p><p> Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.</p><p>Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.</p>