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.

In recent years, medical treatments against respiratory tract infections (RTIs) based on trypsin from Atlantic cod targeting the surface proteins on viral particles have been approved. However, the stability and compatibility in the human body of human trypsin would be expected to be greater than that from cod. In this study, the process of producing human trypsin recombinantly in E. coli in a fed batch bioreactor was optimised using a Design of Experiment (DOE) method with four 2-level factors and two responses. The factors considered were temperature, carbon source, induction time point and feeding rate, and the two responses were yield and purity. The study was done in 8 consecutive runs in the bioreactor, with controlled changes made to the factors for each run. The responses were measured and analysed for significance and optimisation of factors. The optimal settings for the highest yield with a purity of at least 80% were found to be at 18 °C, glucose based feed at a restricting feed rate and induction at a low OD600. Only the induction time has a significant effect on purity, and no factor had a stand-alone significant effect on yield. These findings provide a baseline for further studies on purification and further production of trypsin recombinantly in E. coli at a larger industrial scale. The highest yield achieved was 7.9 g trypsin per litre media, almost 80 times higher than comparable methods using shake flasks. However, the sample with over 80% purity was 6.3 g trypsin per litre media.

Analysis of biological samples plays a crucial role in disease diagnostics and monitoring, as well as in research. For the analysis, detection and quantification is of the essence, and can be performed with immunoassays. These immunoassays exploit antibodies, and their characteristic specificity, to target analytes. Furthermore, immunoassays can be either singleplex or multiplex, meaning they assess one single, or multiple analytes in parallel. As of today, singleplex methods such as ELISA dominate the market, favoured for their precision and reliability. However, multiplexing exhibit reductions in time, costs, and materials. Nevertheless, one common challenge is the detection of small amounts of analyte, possibly discovering and monitoring earlier stages of disease. For this purpose, Cavidi AB has developed a signal amplification technology, namely the binding oligo ladder detection (BOLD). Currently, this technique is applicable to the singleplex market, while the implementation on the multiplexing remains. Here,  we present a literature review of a selection of multiplexing techniques, and a thorough investigation of their possible compatibility with Cavidi AB’s signal enhancement technology BOLD. After undergoing a comprehensive analysis to evaluate various multiplexing technologies and theircompatibility with BOLD, a spectrum of compatibility levels were revealed. Some of the multiplexing technologies, exemplified by Luminex’s xMAP technology, demonstrated inherent compatibility. Contrarily, other platforms, including Olink’s PEA technology, exhibited incompatibility. Moreover, certain technologies, such as Standard Biotools’ CyTOF technology, were found to possess potential compatibility if modification to either BOLD or to the respective multiplexing technology were to be implemented. Furthermore, the need for signal amplification was identified to vary amongst the versatile technologies and the different analytes. This stems from the fact that the lowest detectable concentration level fluctuates between different analytes and technologies. Therefore, Cavidi AB should target companies with a generally high limit of detection (LOD). In addition, because of the fluctuations in LOD between analytes, specifying to which analytes an integration of BOLD would be beneficial, is recommended.

Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine.  Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment.  In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection. In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA. In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA).  We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer. In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold. In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.

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>

Data analysis can be a time consuming part of an experimental method, especially when the method is used frequently and large amounts of data are produced each time. In this study, an application software was developed to improve work flow and data management for respiration rate measurements using an optical oxygen sensor. The application was used to analyze data files from the oxygen sensor without the need to manually enter and analyze the data in a spreadsheet application. The software was written in the Python programming language and utilized available scientific computing packages as well as a graphical user interface framework to provide user friendly access to all functions. Any number of files with experimental data were imported into the program and a linear regression analysis was done for each file and viewed to verify the quality of the data. Tables and summarizing graphs were used to display the key information and statistical results. The final results were exported for use in other applications. Data processing that used to take an hour to complete was done with the new application in five to ten minutes and the risk of introducing human errors in the data was simultaneously reduced. User tests indicated that learning the basics of the program was easy. This study shows the usefulness of a bioinformatics approach and the tools provided by Python and its related software to solve problems that arise with managing large volumes of numerical data. / Älvburet organiskt kol och bakteriers syre respiration

Diss. Uppsala : Uppsala universitet, 2005.

There is an ongoing worldwide obesity epidemic. As a consequence, prevalence of obesity- related diseases and conditions are rapidly increasing. One of these related conditions is type 2 diabetes (T2D), which alone caused 1.5 million deaths in 2012. Thus, it is of upmost importance to develop a more complete understanding of these interrelated diseases. At the heart of all these diseases lies the adipose tissue. This tissue is a major endocrine organ, and one of the key secreted cytokines is adiponectin. Adiponectin interplays with the complex insulin signaling network, and adiponectin levels are inversely related with increased adiposity. The presence of these complex dependencies argues for the usage of mathematical modelling. In the work of Brännmark et al, a model of short-term adiponectin release has been validated. However, this model did not include adrenergic signaling, which is the canonical pathway for in situ regulation of adiponectin secretion. To fill this gap, herein, a mechanistic model describing adrenaline-induced short-term adiponectin exocytosis in white adipocytes has been constructed. The newly constructed model is capable of describing experimental data depicting adiponectin release due to adrenergic stimulation as well as data for different mediator combinations. By implementing adrenergic receptor components, the transition to a more physiological model has been initiated. By finding the smallest possible model capable of describing data, one can argue that the model depicts, to some degree, the fundamental mechanisms for short-term adiponectin secretion. Thus, this work has contributed to solidifying the framework of the mechanisms behind short-term adiponectin secretion from white adipocytes. The result of the model work upholds the role of adrenergic signaling as a central regulatory mechanism for adiponectin release. The constructed model could be used as a fundament for creating a model describing adiponectin release under diabetic conditions.

The heterogeneity of salivary gland neoplasms have made classification and prognostication of these tumours sometimes difficult, and the in­troduction of techniques, such as enzyme and carbohydrate histochemis­try and electron microscopy have only to a certain extent increased our knowledge in these respects. In the present study immunohistochemical methods have been used to identify intermediate filament proteins (IFP) in normal fetal and adult parotid glands, as well as in salivary neo­plasms. The intermediate filaments (IF) make up the cytoskeleton in eucaryotic cells. Epithelial tissue contains IF composed of different cytokeratins (CK 1-19) whilst mesenchymal tissue generally contains IF composed of vimentin, and the IFP pattern is very stable even during cell transformation. It would thus be possible to further clarify the histogenesis of salivary neoplasms by identifying IFP, in addition the IFP pattern would probably be useful in tumour typing. Furthermore, ultrastructural cytochemical studies, microspectorphotometry on nuclear DNA as well as enzyme secretory studies of certain tumour types were carried out, in order to further characterize the biology of salivary neoplasms. The immunohistochemical investigations showed that in normal parotid tissue, the different cell types differed in IFP expression: acinar cells express mainly CK 18 and myoepithelial cells mainly CK 17 and 19, whilst duct cells contained a broad range of CK. Vimentin could in ad­dition to CK be detected in myoepithelial cells and basal cells of ex­cretory ducts. Fetal parotid cells showed a similar CK pattern as mature duct cells. In addition, vimentin could be found in some basal cells of the terminal tubules of the fetal glands. Salivary neoplasms could be divided into three types with regard to their IFP pattern:  Acinic cell carcinomas showed a CK-pattern similar to normal acinar cells but a co-expression of CK and vimentin was present in some cells.  Adenoid cystic carcinomas, mixed tumours and basal cell adenomas showed a CK-pattern of normal duct or myoepithelial cells. The peri­pheral cells were also vimentin positive. 3. Mucoepidermoid carcinomas and adenocarcinomas had a similar CK-pattern as duct cells, and no tu­mour cells contained vimentin. This indicates that typing of IFP may be useful for subgrouping of salivary neoplasms. By stereological measurements, the cells of acinic cell carcinomas were found to be very similar to normal parotid acinar cells. Furthermore, they contained amylase and after stimulation by norepiphrine a secre­tory response was induced, with a rise in intracellular cAMP as well as a release of amylase. By single cell measurements of nuclear DNA con­tent, no difference was found between acinic cell carcinomas with de­finite metastasis and those without recurrence, both in paraffin sec­tions and cytological smears. / digitalisering@umu.se

Targeted radionuclide therapy is becoming a widely used cancer treatment strategy. By radiolabeling receptor-specific peptides, cancer cells overexpressing the receptor can be selectively targeted, and the cytotoxic radionuclide can be delivered to the target cell or tissue for therapeutic or diagnostic purposes. Bombesin analogues have been previously developed and utilized to target the gastrin-releasing peptide receptor (GRPR), a receptor commonly overexpressed in prostate cancer cells. The RM26 analogue derived from the native bombesin is an antagonistic ligand of GRPR and a possible candidate for targeted radiotherapy. Prolonging the half-life of the molecule is an important aspect of developing a new protein therapeutic. Using albumin binding domain (ABD) for this purpose is an emerging strategy in recent years. ABD is able to bind to serum albumin and thus remains in the blood circulation for a long period of time. It is also a scaffold for protein engineering efforts and by coupling receptor-specific ligands to ABD, the target-specific binding along with extended in vivo halflife can be achieved. In this project, an RM26 analogue with a PEG linker and ABD with a DOTA chelator for future radiolabeling were synthesized with solid phase peptide synthesis (SPPS), conjugated, purified by RP-HPLC and analyzed by mass spectrometry. The binding properties of the conjugate were evaluated by SPR-based biosensory studies, and further experiments are planned for the testing the product and its potential application in radionuclide therapy. / Riktad radioterapi är en allt vanligare metod för behandling av cancer. Genom att radioinmärka receptor-specifika peptider kan dessa selektivt levereras till tumörceller som uttrycker receptorn. Radioterapi kan användas för diagnostik eller terapi, beroende på kopplad radionuklid. Bombesinanaloger har utvecklats och använts för att selektivt binda gastrinfrisättande peptidreceptor (gastrin-releasing peptide receptor, GRPR), en receptor som ofta är överuttryckt i prostatacancer. Bombesinanalogen RM26, som har sitt ursprung från nativt bombesin, är en antagonist till GRPR och kan möjligen användas för riktad radioterapi av prostatacancer. Vid utvecklingen av nya proteinläkemedel är halveringstiden i serum en viktig aspekt. En nyligen utvecklad strategi för att förlänga halveringstiden i serum är fusion av det  tumörspecifika proteinet till en albumin-bindande domän (ABD). ABD binder till albumin, ochsåledes kan fusionsproteinet bevaras i blodcirkulationen under en längre tid. I detta projekt, har både RM26 med en PEG-linker, och ABD med en DOTA kelator syntetiserats med fastfaspeptidsyntes (solid phase peptide synthesis, SPPS). RM26-PEG och DOTA-ABD har därefter konjugerats, renats med RP-HPLC och analyserats med massspektrometri. Bindning till albumin har utvärderats med ytplasmonresonans (surface plasmon resonance, SPR). Vidare studier planeras för att utvärdera peptid-proteinkonjugatet och dess potential för riktad radioterapi.