Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques.  This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.

Syftet med detta projekt var att undersöka hur vanligt använda cancerläkemedel påverkar mRNA-uttryck och kromatinorganisation i humana erytroleukemiceller. Som modell användes K562-celler från en patient i blastocystkris (2), för att utvärdera leukemicellernas svar på cancerläkemedel vinblastin och doxorubicin. Vinblastin och doxorubicin valdes på grund av deras distinkta mekanismer i cancercellen: medan doxorubicin interkaleras i DNA, hämmar topoisomeras II-aktivitet vilket orsakar celldöd, riktar vinblastin sig mot mikrotubuli för att stoppa mitotisk delning och proliferation. Uttryck av mRNA undersöktes i celler vid 0-timmar, 6-timmar och 24-timmar drogbehandling, samt efter en veckas återhämtning från 24-timmars drogbehandling. Kromatintillgänglighet med ATAC-seq undersöktes i K562-celler vid 0- timmar, 1-timmar, 6-timmar, 24-timmar och 24-timmar + en veckas återhämtning. Därefter utfördes DNA (ATAC-seq) och RNA (mRNA-seq) extraktion och biblioteksberedning på tre biologiska replikat, och öppna DNA-regioner samt mRNA expression undersöktes via sekvensering. Resultaten visade en stark korrelation mellan de biologiska replikaten, vilket indikerar att resultaten var upprepbara. Differentiellt uttryck av mRNA vid doxorubicin- och vinblastinbehandlingar utfördes genom att jämföra mRNA-nivåerna i läkemedelsbehandlade prover med obehandlade (0-timmar). Uppreglerade och nedreglerade gener identifierades och MA-grafer genererades för att visuellt analysera de differentiellt uttryckta generna vid olika tidpunkter efter läkemedelsbehandling och en veckas återhämtning. För att hitta anrikningar av funktionella genkategorier bland de läkemedelsinducerade eller -undertryckta generna, utfördes genontologianalyser. Slutligen användes verktyget Integrative Genomics Viewer (IGV) för att visuellt utforska mRNA-nivåerna och deras differentiella uttrycksmönster under läkemedelsbehandlingar. För ATAC-seq utfördes inte detaljerad dataanalys på grund av tidsbegränsning, men genomets öppenhet undersöktes visuellt genom IGV. Sammantaget inducerade doxorubicinbehandling en långsamt men långvarig förändring av genuttrycket, vilket involverade flera olika biologiska processer. Doxorubicinbehandlade K562-celler ändrade genuttryck att stöda kemoresistens snarare än att inducera apoptos eller celldöd. Behandlingen hade en långvarig inverkan på mRNA-nivåer som sträckte över återhämtningsveckan. Den totala uttrycksförändringen i återhämtningsproverna var förknippad med återhämtning av tumörigena egenskaper och återställning av mekanismener som stöder cellernas tillväxt. Vinblastine förorsakade snabb ökning av mRNA involverade i cytoskelettet. Vid 24-timmars vinblastinbehandling upplevde tumörcellerna stress på grund av grovt elongerad struktur, och de inducerade gener som stöder tumörbildning. En ökning av totala mRNA-nivåer detekterades i vinblastinbehandlade K562-leukemiceller, vilket var särskilt tydligt under återhämtningen. Resultaten visade att cellerna som överlevde vinblastinbehandling fokuserade på att återställa sin strukturella form. Sammantaget visade resultaten att monoterapi inte fungerar effektivt mot leukemiceller eftersom K562-leukemiceller inte bara överlevde läkemedelsbehandlingarna utan också inducerade mRNA som är involverade i resistens mot läkemedelsbehandlingar. / The primary objective of this project is to investigate how commonly used cancer drugs affect mRNA expression and chromatin organization in human erythroleukemia cells. As a model, K562 cells derived from a patient in blastocyst crisis (2) were utilized, evaluating the leukemia cells’ cellular responses to cancer medicines vinblastine and doxorubicin. Vinblastine and doxorubicin were chosen due to the distinct pathways they target in the cell: while doxorubicin intercalates into DNA and inhibits topoisomerase II activity, which eventually cause cell death, vinblastine targets microtubules to stops mitotic division and excessive proliferation. Expression of mRNA was investigated in cells harvested at 0h, 6h, 24h and 24h + one week recovery. Chromatin accessibility with ATAC-seq was investigated in K562 cells harvested at 0h, 1h, 6h, 24h and 24h + one week recovery. Then DNA (ATAC-seq) and RNA (mRNA-seq) extraction and library preparation were performed on three replicates, and the genome-wide results was investigated via sequencing. The results showed a strong correlation between the biological replicates, indicating that the experimental conditions were sustained in these biological variables. Differential Expression of mRNA upon doxorubicin and vinblastine treatments was performed by comparing the mRNA levels in drug-treated samples to non-treated (0h) upregulated and down regulated genes were identified and MA plots generated to visually analyze the differentially expressed genes at different time points after drug treatment and one week recovery. To find enrichments of functional gene categories among the drug-induced or -repressed genes, gene ontology analyses were performed. Finally, the Integrative genomics viewer (IGV) tool was used to visually explore the mRNA levels and their differential expression pattern during drug treatments. For ATAC-seq, detailed data analysis was not performed due to limitation of time, and data was only visually explored through IGV. Taken together, doxorubicin treatment showed slow initial response within 6h followed by an extensive change in gene expression in 24h, involving several different biological processes. The response was more inclined towards chemoresistance rather than inducing apoptosis or cell death. There was a sustained increase in mRNA levels of doxorubicin treated leukemia cells during recovery week. The overall expression change in the recovery samples was majorly linked with not only gaining back the tumourigenic properties and restoring the mechanism which were affected by doxorubicin action but, based on changes in mRNA expression, it looks like doxorubicin treatment made the tumour cells more aggressive. The initial, 6h, response to vinblastine increases mRNAs involved in cytoskeleton. Upon 24h vinblastine treatment the tumour cells experienced stress due to shear force and structural deformity, and they induced genes supporting tumourigenesis. An increase in total mRNA levels was detected in vinblastine-treated K562 leukemia cells, which was particularly evident during recovery. The results indicated that the cells that survived vinblastine treatment focused on recovering its structural form. Overall, the results indicated that monotherapy does not effectively work against leukemia cells as K562 leukemia cells not only survived the drug treatments but also induced mRNAs involved in resistance against drug treatment.

POPULAR SCIENTIFIC SUMMARY Systemic lupus erythematosus (SLE) is not your average disorder. It behaves like a mischievous troublemaker, wreaking havoc throughout the body, causing inflammation that affects multiple organs. SLE presents a puzzle that keeps health care professionals worldwide intrigued, searching for answers amidst its complex of immunologic manifestations and clinical symptoms. While we’ve made progress in understanding SLE, its specific cause remains a mystery. What we do know is that SLE triggers a fascinating interplay between genetic, hormonal, and environmental factors in susceptible individuals. Macrophages, specialized white blood cells, can be likened to moody actors on a stage wearing different masks and wielding functional props. Among them are M1 macrophages, fiery troublemakers who provoke pro-inflammatory responses, and M2 macrophages, peacemakers striving for balance by generating anti-inflammatory responses. Then there is NRF2, the vigilante, normally held by its captor, KEAP1. However, when cells stress NRF2 manages to break free from KEAP1 and spring into action, embarking on a crucial journey into the cell nucleus where DNA is stored. Once inside, NRF2 binds specific regions of the DNA, promoting genes associated with protective activities, including antioxidative responses and detoxification processes, thereby shielding cells from further harm. Now, let us envision a therapeutic strategy that utilizes this; if we can deliberately unleashNRF2 on command, triggering a powerful cascade of antioxidative responses throughout the body,such a treatment would offer tremendous promise and serve as a paradigm for patients sufferingfrom chronic inflammation. But the question remains: Is it possible? In this study, we investigated the effects of certain chemicals on macrophages in a controlledlab environment. Our goal was to explore their potential for therapeutic purposes. Excitingly, wediscovered that these chemicals can indeed influence macrophages to produce a stronger antiinflammatory and antioxidant response. These findings could be promising for developing futuretreatments, especially in patients diagnosed with conditions such as SLE. / ABSTRACT Systemic lupus erythematosus (SLE) is a multifaceted, chronic autoimmune disorder that leads to inflammation and affects various organs. A wide range of immunologic manifestations and clinical symptoms characterizes SLE. While the specific cause remains unknown, it is thought to result from a combination of genetic susceptibility and the intricate interplay between environmental and hormonal factors. A significant subset of SLE patients also experience renal manifestation, lupus nephritis (LN), characterized by distinct inflammatory responses in which macrophages play a role. Macrophages exhibit different functional characteristics depending on their environment, and generally display two contrasting phenotypes; M1, which elicits proinflammatory responses, and M2, which generates anti-inflammatory responses Homeostasis is vital, yet environmental stress is inevitable. NRF2, a transcription factor known for its involvement in oxidative stress response, plays a pivotal role. Under basal conditions, NRF2 resides in the cytoplasm and is targeted for degradation by the protein KEAP1. However, during cellular stress, the NRF2-KEAP1 complex dissociates, allowing NRF2 to translocate into the nucleus where it binds specific regulatory regions of genes that promote cytoprotective activities. The NRF2 pathway has gained attention as a potential target for therapeutic strategies in inflammatory conditions, including SLE. This study aimed to assess the effects of certain chemical NRF2 activators and a KEAP1 inhibitor on an in vitro model of M1 and M2 macrophage polarization. The objective was to investigate whether these compounds could enhance antioxidative response. To evaluate this, key genes and proteins involved in antioxidative pathways were analyzed. Gene expression was assessed using quantitative real-time PCR (qPCR), and protein presence was determined through immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). The findings of this study indicate that stimulation of macrophage subgroups with the selected compounds promotes a shift towards anti-inflammatory and antioxidative response. / <p>Rektor tilldelade Leo Svahn stipendie Österby för <em>välartade obemedlade studier</em>.</p>

Motile cilia play critical roles in the human body, including expelling mucus from the lungs and facilitating the transport of oocytes and sperm through the fallopian tubes. Understanding the complex structure and motility of cilia, as well as the diseases associated with them, is of big importance. This study investigates the proteins expressed in ciliated cells from both respiratory and reproductive tissues using multiplex immunofluorescence. We determined the subcellular localization of 134 proteins in the fallopian tube, endometrium, cervix, nasopharynx, and bronchus, focusing on five subcellular regions: the cilia tip, transition zone, basal body, cytoplasm, and nucleus. This analysis was conducted using an automated image analysis method developed specifically for this project. Our findings revealed a high correlation in protein expression across all tissues, although several proteins exhibited distinct expression patterns between different tissues. Notably, the fallopian tube showed a higher correlation with the nasopharynx and bronchus than with the endometrium and cervix. Within these proteins, six gene clusters were identified, with the two largest clusters being strongly associated with ciliary structure. This study enhances our understanding of motile ciliary structures and ciliated cells, identifying key proteins for further research into cilia motion, function, and related diseases.

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.

High Performance Liquid Chromatography (HPLC) is a widely used tech-nique both for detecting and purifying substances in academy and in the industry. In order to facilitate the use of, and knowledge in HPLC, character-ization of stationary phases is of utmost importance. Tailor made characteri-zation methods and workflows are steadily increasing the speed and accura-cy in which new separation systems and methods are developed. In the field fundamental separation science and of preparative chromatography there is always the need for faster and more accurate methods of adsorption isotherm determination. Some of that demand are met with the steadily increase of computational power, but the practical aspects on models and methods must also be further developed. These nonlinear characterization methods will not only give models capable of describing the adsorption isotherm but also actual values of local adsorption energies and monolayer saturation capacity of an individual interaction sites etc.The studies presented in this thesis use modern alkali stable stationary phas-es as a model phase, which will give an insight in hybrid materials and their separation mechanism. This thesis will include an update and expansion in using the Elution by Characteristic Points (ECP) method for determination of adsorption isotherms. The precision is even further increased due to the ability to use slope data as well as an increase in usability by assigning a set of guidance rules to be applied when determine adsorption isotherms having inflection points. This thesis will further provide the reader with information about stationary phase characterization and the power of using existing tech-niques; combine them with each other, and also what the expansion of meth-ods can revile in terms of precision and increased usability. A more holistic view of what benefits that comes with combining a non-linear characteriza-tion of a stationary phase with more common linear characterization meth-ods are presented.

Vid blodgivning donerar blodgivare blod frivilligt. Blodet kan sedan användas inom sjukvården för exempelvis blodtransfusion, vilket kräver blodprodukter kompatibla med patienten. Förekomst av leukocyter i blodprodukter medför en ökad risk för febrila transfusionsreaktioner hos transfunderade patienter. Därför krävs det att vid framställning leukocytreducera blodprodukter och utföra kvalitetskontroll. Med analysen B-leukocytpartikelkoncentration (LPK) kan totalantalet leukocyter i helblod beräknas. Flödescytometri är en metod som kan analysera optiska och fluorescerande egenskaper hos exempelvis celler i en suspension, vilket kan användas för att kvantifiera cellantal. BD Leucocount™-Kit (BD Biosciences) är avsett för flödescytometrisk analys av antalet kvarvarande leukocyter i leukocytreducerade blodprodukter. Vid framställning av blodprodukter ska helblodspåsen vila vid rumstemperatur i minst 3 timmar efter blodgivning. I Falun används antingen ett dagsprogram där produktion sker samma dag som blodgivningen, eller ett övernattningsprogram där produktion sker dagen därpå. Prover från 505 kontrollerade erytrocytenheter, samlade i Falun, har påvisat en skillnad i leukocytkoncentration beroende på vilket program som använts. Anledningen till att erytrocytenheternas leukocytinnehåll skiljer sig är inte känt. Syftet med denna studie är därav att undersöka om vilotiden har någon effekt på leukocytkoncentrationen i helblodspåsar. LPK varierade mellan helblodspåsarna. Ett ökande leukocytantal observerades över tid i majoriteten av helblodspåsar, inklusive medelvärde. Däremot kunde inte hypotesprövning påvisa statistisk signifikans. Hypotesen om att leukocytantalet ökar över tid går emot grundläggande hematologi. Utifrån resultaten i denna studie kan inte hypotesen bevisas. Vidare studier bör genomföras. / During blood donation, blood donors donate blood voluntarily. The blood can then be used in healthcare for, for example, blood transfusions, which requires blood products compatible with the patient. The presence of leukocytes in blood products increases the risk of febrile transfusion reactions in transfused patients. Therefore, leukocyte-reduction in blood products is necessary during production. Each blood center must perform quality control on produced blood products. With the analysis B-leukocyte particle concentration (LPK), the total number of leukocytes in whole blood can be calculated. Flow cytometry is a method that can analyze the optical and fluorescent properties of, for example, cells in a suspension, which can be used to quantify cell numbers. The BD Leucocount™-Kit (BD Biosciences) is intended for flow cytometric analysis of the number of leukocytes remaining in leukocyte-reduced blood products. When producing blood products, the whole blood bag should rest at room temperature for at least three hours after the donation. In Falun, either a day program is used where production takes place on the same day as the blood was donated, or an overnight program where production takes place the next day. Samples from 505 controlled erythrocyte units, collected in Falun, have shown a difference in leukocyte concentration depending on the program used. The reason why the leukocyte content of erythrocyte units differs is not known. The purpose of this study is therefore to investigate whether the resting period has any effect on the leukocyte concentration in whole blood bags. The LPK varied between the whole blood bags. An increasing leukocyte count was observed over time in most of the whole blood bags. However, hypothesis testing did not show statistical significance. The hypothesis that leukocyte counts increase goes against basic hematology. Based on the results of this study, the hypothesis cannot be proven. Further studies should be conducted. / <p>Vårdförbundet tilldelade Leo Svahn stipendium 2021 för <em>bästa kandidatuppsats inom biomedicinsk laboratorievetenskap</em>.</p>

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.

Alzheimer's disease (AD) was first described more than 100 years ago and is today the most common cause of dementia. It is one of the progressive neurodegenerative diseases that affect 47 million people around the world between the ages of 60 and 90. One of the contributing factors to AD is extracellular amyloid – β (Aβ) plaques that form as a result of protein aggregation. These Aβ proteins are neurotoxic, leading to degeneration of brain neurons and loss of cognitive abilities. Because AD largely affects society, researchers are constantly working to find a cure, which currently does not exist. The purpose of this study was to use Drosophila melanogaster as a living organism model for the expression of two types of Aβ proteins related to AD, Arctic (Glu22Gly) and TandemAβ, and to study the survival of these AD flies when Bovine serum albumin (BSA) was added to the fly food. The hypothesis was that BSA would be effective in slowing down and/or preventing formation of toxic Aβ-aggregates. The focus was therefore to investigate whether the AD flies would live longer if they were allowed to eat Bovine serum albumin and whether the soluble/insoluble Aβ levels in these flies would decrease in comparison to the control AD flies that were not allowed to eat BSA. The effect of BSA on toxicity was evaluated using survival assay on male flies and the levels of soluble/insoluble Aβ were evaluated using Meso Scale Discovery (MSD) on female flies. In both experiments, the following six groups of flies were examined: myow1118 ± BSA; myoArctic ± BSA; myoTandemAβ ± BSA. Conclusions from the studies are that the survival of AD flies could not be extended by adding 0.61 mM BSA to the food, rather the data showed a weak but significant toxic effect in the presence of BSA in the AD flies. However, MSD data showed a reduction of insoluble Aβ aggregates and an equilibrium shift from insoluble Aβ aggregates to soluble Aβ aggregates in the presence of BSA in the AD flies. Equilibrium shifts were particularly detectable in Myo-TandemAβ flies fed with BSA. In Myo-Arctic flies fed with BSA only reduction of insoluble Aβ could be detected. This shows that it is not the amount of Aβ aggregates that is decisive for toxicity, but rather the presence of specific aggregates that have toxic properties. If BSA shows good results in further studies, it could be used in the future to improve AD symptoms in patients.