Neuroblastoma is a form of embryonal neuroendocrine tumor that arises from neural crest progenitor cells, which can differentiate into multiple cell types. It is caused by genetic abnormalities such as MYCN oncogene amplification and 11q deletion, both of which can deregulate neuroblastoma suppressor genes located on chromosome 11q. MiRNAs are noncoding RNAs with an average length of 22 nucleotides. By binding to the 3′ untranslated regions of target messenger RNA, the RISC represses protein synthesis. The aim of this experimental project was to determine the effects of MYCN amplification or 11q deletion on the expression level of the miRNA, hsa-miR-34b-3p in neuroblastoma. The total RNA was extracted from the neuroblastoma cell lines, NB69 without MYCN amplification and 11q deletion, Kelly with 11q deletion and SK-NBE(2) with MYCN amplification. cDNAs were generated from the miRNA, hsa-miR-34b-3p located on chromosome 11q and the reference gene, RNU6B. The cDNAs were amplified and quantified by qPCR. The qPCR data were analysed using the comparative Ct method, Kruskal-Wallis test with multiple comparisons to determine whether or not hsa-miR-34b-3p was significantly differentially expressed in Kelly and SK-N-BE(2) compared to NB69. The results showed that hsa-miR-34b-3pwas significantly downregulated in the cell lines, Kelly and SK-N-BE(2) compared to NB69. In conclusion, the findings of this study showed that hsa-miR-34b-3p is downregulated in Kelly and SK-N-BE(2) compared to NB69, and call for further research to investigate its clinical potential in the therapy of neuroblastoma.

When examining data and statistics one thing is very clear. Smoking tobacco is causing a lot of different problems both in an individual’s health and in the society. Even though there is abundant evidence regarding its hazardous consequences, tobacco holds still a lot of users under its influence. Most of tobacco users do want to quit but they cannot make it last and relapse on their old habits. Tobacco is consumed everywhere in the world but effective products for quitting it, are still not that many available. Thus, it is made clear that there is a need for a product that will help counterattack this addiction. Deversify is an Uppsala-based start-up that wants to introduce to the market a tool for facilitating permanent smoking cessation. Their solution is a product called Aspirable. Aspirable is a breathalyzer that records the level of carbon monoxide (CO) in the exhaled breath, and it is accompanied with an app that will make use not only the recordings of the CO levels for a factual check but also help the user identify their habits to facilitate quitting. Regardless of the size of ones’ business, one thing is the same; it is better to first analyse if an idea is an endeavor worth trying. This project has utilized a case study for Aspirable, in order to explore in depth, the product and most importantly its market. Information available on the web was used to construct a picture on the smoking cessation market. Different aspects were examined inside theoretical frameworks such as the 7 dimensions of Aaker and Porter’s 5 forces. We made comparisons between Greece and Sweden to explore if another geographical market would be a better start for launching the product. In addition, we looked into what kind of consumer would be the best. Among those options, were governments, businesses, and individuals. The recommendation would be to aim at entering the Swedish market by approaching clinical trials so as to gain more scientific proof for the effectiveness of the product and also help indirectly another research be more reliable. Meanwhile, it would be best to avoid the public health system due to complicated regulations, segmented market due to each county having its own budget and decision-making processes.

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3. / <p>QC 20170330</p>

The newly introduced regulation (EU) 2017/746 aims to make In Vitro Diagnostic Medical Devices (IVDMD), which include companion diagnostic tests (CDx), a widespread method of authorising medicinal products in the European market. However, European SmPCs (Summary of Product Characteristics) currently do not explicitly refer to the term or classify tests associated with medicinal products as CDx. This paper is the first to examine and classify tests for medicinal products currently authorised in Sweden as being potential CDx, under the definitions of the new regulatory paradigm. The aim is to serve as the foundation for future research. 141 medicinal products with potential associated CDx are identified in the database of the Swedish Medical Products Agency (MPA). These products are then classified under the major ATC (Anatomical Therapeutic Chemical) therapeutic areas to search for commonalities and patterns in their usage and are later examined in conjunction with the techniques they use. The results reveal that a majority are concentrated in the Antineoplastic and immunomodulating agents and Antiinfectives for systemic use therapeutic areas. The methods used by these tests reveal diversity among the test technique usage, including instances where multiple techniques comprise a single CDx product, which may focus on detecting several biomarkers.

Genomic alterations may initiate cancer development as the consequence of endogenous or exogenous DNA damaging factors. Defects in DNA repair mechanisms may also facilitate cancer progression as well as accumulation of mutations which favor cancer cell survival. However, DNA repair pathways in cancer cells can be considered as their Achilles heel which are possible targets in order to compromise their survival. For instance, it has been demonstrated recently that inhibition of a protein called MTH1 via RNA interference (RNAi) or chemical inhibitors can stop tumor growth and triggers cell death by increasing the load of oxidative DNA damage. MTH1 is a hydrolase which converts 8-oxo-dGTP into 8-oxo-dGMP in order to prevent incorporation of oxidatively damaged nucleotides into DNA. In addition, DNA glycosylases which recognize and remove mismatched or damaged nucleotide pairs in DNA can also participate in repair of 8-oxo-dG, such as MUTYH repairing A:8-oxo-dG pair. The goal of the current study was to investigate the importance of MUTYH activity upon MTH1 depletion. The current study tried to answer whether simultaneous knock-down of MTH1 and MUTYH sensitizes cancer cells to oxidative stress and increases cell death. Both enzymes were simultaneously depleted in T cell acute lymphoblastic leukemia cells using RNAi. Then, we analyzed the efficiency of gene and protein knock-down by quantitative real-time-PCR and western blotting, respectively. Induction of cell death was also assessed by flow cytometric analysis of cell cycle. Afterwards, the effect of the treatments on DNA repair pathways was studied by analysis of gene expression of several DNA glycosylases and DNA polymerases using qRT-PCR. The results showed that concurrent depletion of both enzymes led to synergistic induction of cell death. Down-regulation of NEIL1 DNA glycosylase as well as POLQ and POLH DNA polymerases mRNAs adapted their DNA repair pathways to cope with induced damages under these conditions. Finally, the results of this study suggest that dual suppression of MTH1 and MUTYH may provide a new approach to reduce survival of T cell ALL.

Maternell immunisering är ett sjukdomstillstånd där moderns försvarsmekanism förstör fostrets röda blodkroppar och orsakar anemi hos fostret. Den primära behandlingsmetoden är intrauterina blodtransfusioner. Undersökning av foster under graviditeten utförs i stor utsträckning med hjälp av ultraljud. Ultraljudsundersökning begränsas av att fostret rör på sig och har en hög hjärtfrekvens. Målet med arbetet var att undersöka deformationen av myokardiet hos två grupper av foster. Den ena bestod av nio foster som genomgår intrauterina blodtransfusioner, den andra bestod av nio normala foster i motsvarande graviditetsvecka. Mätningar utfördes i programmet EchoPAC med 2D STE metoden, en tvådimensionell metod där speckle från myokardiet markeras och observeras över tid för att studera förändringen i hjärtats segment, hjärtdeformationen. Detta gjordes som en pilotundersökning primärt i syfte att bedöma genomförbarheten samt möjliga svårigheter med undersökningarna samt undersöka om metoden skulle kunna påvisa skillnader i deformationen mellan foster som behandlas med intrauterina blodtransfusioner och normala foster. Metoden visades vara genomförbar. Resultaten visar att deformationen i hjärtat hos foster som genomgår intrauterina blodtransfusioner är en aning högre än hos normala foster. Skillnaden i hjärtdeformationen mellan grupperna har visat sig vara signifikant. / Maternal immunization is a condition in which the mother's defense mechanism destroys the red blood cells in the fetus, which causes anemia. The primary treatment method is intrauterine blood transfusions. Ultrasound is one of the most common tools used to assess fetuses. Examination with ultrasound is limited by the fetal movements and a high fetal heart rate. The aim of this project was to pilot an investigation of myocardial strain within two groups of fetuses. One group consisted of nine fetuses undergoing intrauterine blood transfusions, the other consisted of nine normal fetuses in the corresponding week of pregnancy. Measurements were performed in the program EchoPAC with the 2D STE method, a two-dimensional method where speckles from the myocardium are marked and observed over time to study the change in the heart segment, the strain. This was done primarily to investigate if measurements were feasible and what problems could occur, and secondarily whether the method could show a difference in strain between fetuses treated with intrauterine blood transfusions and normal fetuses. The results show that the measurements were feasible. Results show that the strain of the heart in fetuses undergoing intrauterine blood transfusions is higher than in normal fetuses. The difference in the strain between the groups has shown to be significant

Det finns idag flera tusen genetiska sjukdomar som inte kan botas med hjälp av dagens läkemedelsbehandlingar. Detta är något forskarna försöker finna en lösning på. Två nya potenta genredigeringsverktyg har utvecklats och tros kunna bota och behandla många av de idag kända genetiska sjukdomarna. Detta är clustered regularly interspaced short palindromic repeats med CRISPR-associerade proteiner, CRISPR/Cas9 och prime editing. Tekniker som utvecklats från det adaptiva immunförsvaret hos prokaryoter. Både CRISPR/Cas9 och prime editing är RNA-guidade system med DNA som mål, de är även möjliga att programmera. Syftet med denna litteratursökning var att: 1) Jämföra teknikerna CRISPR/Cas9 och prime editing, 2) Undersöka vilka idag pågående kliniska prövningar som finns där någon av teknikerna används vid behandling av sjukdom. 3) Undersöka vilka sjukdomstillstånd som tros kunna botas och/eller behandlas med hjälp av någon av teknikerna samt 4) undersöka hur forskare ser på de etiska aspekterna av dessa tekniker. Information har hämtats under arbetets gång, främst från PubMed, Google och clinicaltrials.gov. Det finns idag 16 pågående studier där CRISPR/Cas9 används som behandlingsmetod. För prime editing finns det inga pågående studier. Sjukdomarna som forskarna hoppas kunna behandla med hjälp av metoderna är många, men de har kommit längst i utvecklingen av läkemedel för cancer, blodsjukdomar och ögonsjukdomar. De etiska diskussionerna har varit många och den stora frågan som diskuteras är hur tekniken skall regleras för att inte utnyttjas till sådant som potentiellt kan vara skadligt. Detta är två tekniker med hopp om nya behandlingsmetoder för genetiska sjukdomar, dock är de endast i början av sin utveckling och mer forskning och förfining av metoderna krävs innan de kan tillämpas kliniskt. / Today, there are thousands of genetic diseases that cannot be cured with the help of today's drug treatments. This is something the researchers are trying to find a solution to. Two new potent gene editing tools have been developed and are believed to be able to treat or cure many of today's genetic diseases. These are Clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins, CRISPR/Cas9 and prime editing. Techniques developed from the adaptive immune system of prokaryotes. Both CRISPR/Cas9 and prime editing are RNA-guided DNA-targeted systems that are programmable. The purpose of this literature search was to: 1) compare the CRISPR/Cas9 and prime editing techniques, 2) investigate the current clinical trials in which any of the techniques are used to treat disease. 3) investigate which diseases that are believed to be cured and/or treated by using one of the techniques, and 4) investigare how researchers view the ethical aspects of these techniques. Information was gathered during a period between January to May 2020, mainly from PubMed, Google and clinicaltrials.gov. There are currently 16 ongoing studies using CRISPR/Cas9 as a treatment method. For prime editing there are no ongoing studies. The diseases that the researchers hope to be able to treat using the methods are many, but they have come the farthest in the development of a drug for cancer, blood diseases and eye diseases. There have been many discussions about the ethical side, but the big question being discussed is how the technology should be regulated so that it may not be used to harm instead of treat. These two techniques give hope of new treatment methods of genetic diseases, however, they are in the early stages of their development and more research and refinement of the methods is required before they can be applied clinically.

Neuromodulation, such as spinal cord stimulation (SCS) and deep brain stimulation (DBS), has been shown to modulate pathophysiological brain activity and provide symptomatic therapy for several neurological disorders, including Parkinson’s Disease. The effectiveness of this therapy could likely be further improved by neuromodulation that is adaptive, delivering stimulation more selectively, by monitoring a biomarker in recorded brain signals, which indicates the presence of a pathological state. In the treatment of Parkinson’s Disease, the most commonly proposed solutions for adaptive neuromodulation are relying on excessive beta-band oscillatory activity as a biomarker, which is however often highly variable between patients during movement and in conjunction with neuromodulatory treatment, such as levodopa. These limitations hinder broader use of this biomarker and prompts further research for alternative solutions. In this work, we instead present the use of a novel feature of evoked electrophysiological activity, which utilizes the inter-trial phase coherence between stimulation pulses, to classify parkinsonian brain states in 6-OHDA lesioned rats. We developed a method, which relates to the rate of decay in inter-tral phase coherence, evoked by single SCS or DBS pulses, that is able to statistically separate experimental conditions recorded from a dopaminergic depleted hemisphere from conditions a non-depleted hemisphere, while also being able to separate conditions with levodopa treatment from conditions without treatment. For animals undergoing SCS we can classify phase decay measurements from pharmacologically treated or untreated parkinsonian states, using a Bayesian model, with a high accuracy and strong classifier performance for a single channel (AUC 0.85 – 0.99) in the motor cortex and striatum. In ongoing experiments, similar implementation of adaptive DBS is being evaluated. Our results support the implementation of our feature in a protocol aimed at performing closed-loop neuromodulation in the 6-OHDA rat model of Parkinon’s Disease, that can serve as the basis for further studies. / Neuromodulering, såsom ryggmärgsstimulering (SCS) och djup hjärnstimulering (DBS), har visat sig kunna modulera patofysiologisk hjärnaktivitet och ge symtomatisk behandling av flera neurologiska sjukdomar, inklusive Parkinsons sjukdom. Effekten av denna behandling skulle sannolikt kunna förbättras ytterligare genom neuromodulering som är adaptiv och ger stimulering mer selektivt, genom övervakning av en biomarkör i registrerade hjärnsignaler, som indikerar förekomsten av ett patologiskt tillstånd. Vid behandling av Parkinsons sjukdom förlitar sig de vanligaste lösningarna för adaptiv neuromodulering på överdriven beta-bands oscillatorisk aktivitet som en biomarkör som dock ofta är mycket varierande mellan patienter, under rörelse och i samband med behandling så som levodopa. Dessa begränsningar hindrar en bredare användning av denna biomarkör och ytterligare forskning krävs för att hitta alternativa lösningar. I detta arbete presenterar vi istället en ny egenskap hos väckt elektrofysiologisk aktivitet, som utnyttjar faskoherens mellan stimuleringspulser för att klassificera parkinsonistiska hjärntillstånd hos 6-OHDA-lesionerade råttor. Vi har utvecklat en metod som relaterar till avklingningshastigheten i faskoherens, framkallad av enstaka SCS- eller DBS-pulser, som kan statistiskt särskilja de experimentella tillstånden i en dopaminergiskt utarmad hemisfär från liknande tillstånd, fast i en icke utarmad hemisfär. Den kan även statistiskt särskilja tillstånd med levodopabehandling från tillstånd utan behandling. För djur som genomgår SCS kan vi klassificera fasförfallsmätningar från farmakologiskt behandlade eller obehandlade parkinsontillstånd, med hjälp av en Bayesiansk modell, med hög noggrannhet och stark klassificeringsprestanda för en enda kanal (AUC 0,85 - 0,99) i motorcortex och striatum. I pågående experiment utvärderas en liknande implementering av adaptiv DBS. Våra resultat stöder implementeringen av vår funktion i ett protokoll som syftar till att utföra sluten neuromodulering i 6-OHDA-råttmodellen för Parkinons sjukdom, som kan tjäna som grund för ytterligare studier.