Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system. 

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

The polysaccharide xyloglucan as a wet-end additive improves paper properties. In the present study, paper strength improvement was analysed for dry handsheets made from chemical, mechanical and recycled pulps coated with xyloglucan in a spray application. Results are compared with sheets made from the same pulps treated with xyloglucan in the wet-end. Kraft pulp handsheets of bleached hardwood and softwood showed significant improvements of tensile, tear and Z-strength by xyloglucan spray treatment versus wet-end application, whereas handsheets of de-inked and thermomechanical pulp were improved only slightly. In both wet-end and spray applications, the effect of xyloglucan addition was intimately related to the presence of non-cellulosic components on the fibre surface. Further strength improvements were obtained for chemical pulps by addition of borax to the spray solution, which were likely to be due to the formation of borate-mediated xyloglucan cross-links. Spray coating of xyloglucan, with or without borax, thus represents a potential new application of this polysaccharide to increase paper dry strength.

In this study, phenyl (1,4,6-triphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methanone was obtained by using the Biginelli reaction method. The structure of this compound was analyzed using elemental analysis, IR, 1H, and 13C NMR. The quantum chemical calculations (QCC) of this compound were performed density functional theory (DFT) method, 6–31 G (d, p) base set, and B3LYP functions with the Gaussian09W software package. Literature shows that pyrimidine-derived compounds have very active biological properties. For this reason, the biologically active properties of the synthesized compound were also examined. To determine embryotoxic, genotoxic, and cytotoxic effects of compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase (LDH) release, micronucleus (MN) and 8-OH-dG assays were carried out. On the other hand, pharmacokinetic and toxicity properties (ADMET) were predicted in silico via SwissADME and Protox-II web tools. In silico estimates of this compound used in the study showed that the compound has the covetable physicochemical properties for bioavailability. In conclusion, the obtained results of our study clearly showed that this compound exerted strong toxicity potential.

Advancements in medical diagnostics have allowed us to understand the underlying mechanism and treat the root cause for many diseases which had been causing morbidity and mortality up until this point in human history. Furthermore, many of the standard diagnostic procedures have now been transformed to provide answers at or near the point-of-care. However, the effects of these positive developments have not trickled down to the parts of our society which are considered underdeveloped and lack the necessary infrastructure and facilities. Diagnostics in such resource limited settings still lag behind and fail to provide the requisite healthcare. 

In order to translate the diagnostic solutions designed for central laboratories to resource limited settings, there are certain challenges that need to be addressed, such as portability, reduction in cost and ease-of-use, while keeping the sensitivity and specificity at the required level. The work presented in this thesis focuses on addressing some of these issues by using microfluidics to develop diagnostic platforms that are suitable to be used in resource limited settings. 

In paper I, a very low-cost and simple centrifugal microfluidic platform was developed to be used in settings which do not have a reliable supply of electricity. The platform uses a smartphone as a source of power and the sensors of the phone for speed control.

In paper II, a portable and low-cost diagnostic platform was developed for multiplexed detection of biomarkers based on centrifugal microfluidics. The platform uses colorimetric detection and a simple readout method which does not require a spectrophotometer for quantification.

In paper III, a platform was developed for COVID-19 diagnostics which combines centrifugal microfluidics with a novel bead-based strategy for signal enhancement. The platform uses fluorescent detection with a smartphone readout and has the capability to process up to 20 samples at the same time.

In paper IV, as a follow up of paper III, a more advanced platform was developed for COVID-19 diagnostics which allows the operator to carry out nucleic acid amplification in a completely automated manner, from adding the sample to getting the final result.

In paper V, an alternative method for detection of SARS-CoV-2 was developed using electrochemical biosensing. This work combines the electrochemical technique with a flexible printed circuit board for a rapid, amplification-free and label-free detection of target SARS-CoV-2 sequences.

COVID-19 pandemic made it evident that the world is unprepared for effectively tackling a pandemic resulting from an infectious disease. The conventional diagnostic methods for detection of infectious diseases were limited to centralized laboratories. As the burden of testing increased with the spread of the disease, the centralized testing facilities were strained for resources and personnel. These problems were further exacerbated in low- and middle-income countries where the health and transport infrastructure are not very well developed. To overcome this reliance on centralized testing and to facilitate decentralized testing, focus was shifted towards development of novel point-of-care diagnostic methods. We report the development of a fully automated centrifugal microfluidic platform that uses loop mediated isothermal amplification (LAMP) combined with computer vision-based readout for COVID-19 detection. The integrated platform allows sample to answer analysis at the push of a single button and can process 26 samples in 40 minutes. The platform performs a completely automated assay protocol involving heating, rotation and detection without the need for user intervention. A limit of detection of approximately 100 RNA copies in 10 µL reaction was achieved using RNA fragments spiked in water and similar results were obtained for artificial saliva samples. 

Cytokines play a very important role in our immune system by acting as mediators to put up a coordinated defense against foreign elements in our body. Elevated levels of cytokines in the body can signal to an ongoing response of the immune system to some abnormality. Thus, the quantification of a panel of cytokines can provide valuable information regarding the diagnosis of specific diseases and state of overall health of an individual. Conventional Enzyme Linked Immunosorbent Assay (ELISA) is the gold-standard for quantification of cytokines, however the need for trained personnel and expensive equipment limits its application to centralized laboratories only. In this context, there is a lack of simple, low-cost and portable devices which can allow for quantification of panels of cytokines at point-of-care and/or resource limited settings.

Here, we report the development of a versatile, low-cost and portable bead-based centrifugal microfluidic platform allowing for multiplexed detection of cytokines with minimal hands-on time and an integrated colorimetric signal readout without the need for any external equipment. As a model, multiplexed colorimetric quantification of three target cytokines i.e., Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-2 (IL-2) was achieved in less than 30 min with limits of detection in ng/mL range. The developed platform was further evaluated using spiked-in plasma samples to test for matrix interference. The ease of use, low-cost and portability of the developed platform highlight its potential to serve as a sample-to-answer solution for detection of cytokine panels in resource limited settings.

The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.

The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

A comprehensive characterization of blood proteome profiles in cancer patients can contribute to a better understanding of the disease etiology, resulting in earlier diagnosis, risk stratification and better monitoring of the different cancer subtypes. Here, we describe the use of next generation protein profiling to explore the proteome signature in blood across patients representing many of the major cancer types. Plasma profiles of 1463 proteins from more than 1400 cancer patients are measured in minute amounts of blood collected at the time of diagnosis and before treatment. An open access Disease Blood Atlas resource allows the exploration of the individual protein profiles in blood collected from the individual cancer patients. We also present studies in which classification models based on machine learning have been used for the identification of a set of proteins associated with each of the analyzed cancers. The implication for cancer precision medicine of next generation plasma profiling is discussed.

This project was conducted at Sangair. The company is currently developing a medical device aimed at treating bacteremia with ozone. Bacteremia is a condition that occurs when bacteria enters the bloodstream, and can trigger sepsis and septic shock, potentially leading to death if untreated. Antibiotics have traditionally been the way to treat bacteremia, but the looming threat of antibiotic resistance worldwide threatens this way of treatment, and research into novel approaches to eradicate the bacteria needs to be done. As part of Sangairs development, an investigation was done to see how ozone interacts with bacteria. The project aimed to 1) develop a method to detect bacterial lysis and 2) use the method to detect if ozone had a lysing effect on the bacterial model organism Escherechia coli. To test the method, the BL21 strain of  E. coli was transformed with the pUC-19 plasmid to produce the reporter enzyme Beta-galactosidase. The Beta-galactosidase activity was then measured in a supernatant of sonicated bacteria, which confirmed the suitability of the method to detect bacteria cell lysis. To be able to see if ozone had a lysing effect, while still being able to measure Beta-galactosidase, it was found that the optimal setup for this was using an ozone concentration of 6 g/m³, a gas flow of 5 ml/min and a liquid flow of 25 ml/min. The results acquired with this setup indicated that ozone had a lysing effect on E. coli but more studies are needed to verify this. It was further investigated if Beta-galactosidase detection could be improved by addition of bovine serum albumin (BSA) to quench residual ozone in the samples, but the results showed that it did not have any effect on the Beta-galactosidase enzymatic activity. As a final experiment, endotoxins that were released upon treatment were also measured, and it was found that when bacteria are treated with ozone, lipopolysaccharides (LPS) and peptidoglycans are released, further confirming lysis of the bacterial cells. 

This thesis presents a comprehensive longitudinal proteomic analysis of HIV progression and treatment response. The study aims to elucidate the molecular mechanisms underlying disease progression and therapeutic outcomes, identify biomarkers of disease progression and therapeutic response, and explore the proteomic profiles of elite controllers. The cohort comprises 97 HIV-1 infected individuals, categorized into four subgroups. Proteomic profiling was performed using the Olink® Explore Proximity Extension Assay, targeting 1463 proteins. Key findings include significant upregulation of chemokines and inflammation markers in HIV samples compared to healthy controls at baseline, with notable proteins being CXCL13, LAG3, and CD6. ART led to significant downregulation of these proteins, indicating reduced immune activation. However, residual immune activation was observed in follow-up samples. Comparative analysis of ART regimens (INSTI vs. NNRTI) and backbone drugs (Tenofovir vs. Abacavir) revealed minimal differences in proteomic profiles. Elite controllers displayed unique proteomic profiles with less immune activation, resembling healthy controls. Correlation analysis identified a few proteins associated with clinical parameters, such as CD4/CD8 ratio and CD4 counts, suggesting their potential as biomarkers to predict immune reconstitution. The study highlights the dynamic proteomic changes in HIV patients, offering insights into disease mechanisms and potential therapeutic targets. Future research should focus on expanding the cohort, integrating multi-omics approaches, and utilizing targeted proteomic measurements to enhance the understanding of HIV progression and treatment response.

Autoimmune diseases are complex, chronic, inflammatory conditions characterized by dysregulation of the immune system, resulting in inflammation and damage to various tissues, cells and organs. These diseases significantly impact individuals’ quality of life and often contribute to increased mortality risk in the presence of comorbidities. However, due to the diverse array of symptoms associated with different autoimmune diseases, accurate diagnosis, prognosis, and treatment evaluation pose significant challenges. Thus, there is a pressing need for the discovery of novel biomarkers. 

In this study, a comprehensive analysis of 944 plasma samples using the OlinkR Explore platform was conducted, generating data on 1463 unique proteins. Based on the expression data, associated proteins were identified for six selected autoimmune diseases, namely multiple sclerosis, myositis, rheumatoid arthritis, systemic sclerosis, Sjögren’s syndrome, and systemic lupus erythematosus, as well as some of their defined subgroups. These are prospective biomarkers and have the potential to aid in early diagnosis, therapeutic intervention, subgroup identification, disease differentiation, and disease prognosis. Notably, some of these proteins have not been previously associated with the specific diseases in the existing literature, especially not in plasma samples, thereby offering intriguing new perspectives for biomarker development. However, it is of great importance to conduct robust validation studies in independent cohorts to confirm the outcomes of this study. 

In summary, our findings highlight the potential utility of these proteomic plasma biomarkers in improving the early detection, subgroup characterization, and disease differentiation of autoimmune diseases. The identification of these proteins will hopefully stimulate further investigation in the field of biomarker research and potential advancements in personalized medicine. 

Autoimmune diseases arise when the immune system is dysregulated and elicits an immune response against our healthy cells and tissues. This misguided response primarily entails activating self-reactive B and T cells, with B cells generating autoantibodies detectable in the bloodstream. These autoantibodies are valuable as biomarkers, and there is a critical need for disease-specific and prognostic autoantibody markers to improve the diagnosis, stratification, and management of individuals with these diseases. Although many potential autoantibody markers have been published in the literature, there remains a gap between these candidates and their implementation in clinical practice due to a lack of studies validating their clinical significance and existing uncertainty about their disease specificity. Additionally, the specificity of currently used clinical markers needs to be better defined.

In the present study, a targeted antigen suspension bead array was employed to profile immunoglobulin G (IgG) reactivity in sera from patients with two rare autoimmune disorders associated with diagnostic challenges and adverse outcomes, namely, systemic sclerosis (SSc) and ANCA-associated vasculitis (AAV). Using a cross-disease design, the aim was to study the disease specificity of clinically used and previously published potential autoantibody biomarkers. Additionally, the prevalence of these markers in a subgroup of SSc patients manifesting lung fibrosis was explored. Our findings suggest that autoimmune responses to clinically used markers, namely TOP1, CENPA, CENPC, MPO, and PR3, are specific to the respective disease, at least in our experimental context, but also that many markershave a shared presence across diseases, highlighting the necessity for a cross-disease approach in the discovery of clinical diagnostic biomarkers. Furthermore, a novel candidate, anti-PIP4K2B, showed promising results as a diagnostic marker for "seronegative" SSc patients in our study, especially when interpreted with clinical symptoms. Several markers that may predict the development of lung fibrosis and pulmonary arterial hypertension (PAH) in SSc patients were also identified in the study; however, future studies in larger independent cohorts are needed to confirm their clinical value. Expanding the scope of the analysis in the future and including a broader range of diseases is necessary to elucidate the disease specificity of autoantibody biomarkers further and to address challenges related to delayed accurate clinical diagnosis and improve the management of autoimmune diseases.

The underlying biology of Schizophrenia (SCZ) remain elusive despite its profound impact on patients. Due to SCZs estimated 79% heritability, a key to understand the condition is to analyze the genetic 22q11.2 deletion which increases the risk of SCZ by 28-fold. This study investigates the effects of the 22q11.2 deletion on cell type co-localization and abundance in mice in the prefrontal cortex using the Xenium platform, a Spatially-Resolved Transcriptomics (SRT) method. The investigation was performed on nine coronal sections, including four mice with the 22q11.2 deletion and five wild-type (WT) controls. Cell types were annotated using the scMap algorithm and a reference single cell RNA sequencing (scRNA-seq) data set using the 345 genes in the Xenium panel. The DIMPLE algorithm was employed to analyze cell type co-localization, utilizing isotropic Gaussian kernel smoothing and Jensen-Shannon distance calculations. Spatstat was used to measure cell type abundance using calculated intensities.

Results did not show significant differences in cell type abundance or spatial distribution between the 22q11.2 deletion and WT groups in this data set of 32,298 cells in the prefrontal cortex. While specific differences might exist between different layers of the prefrontal cortex, or with a larger sample size to reduce individual variability, this analysis did not cover such scenarios. There are limitations to this study since it is only a comparison of 32 298 cells and therefore future research should focus on analyzing larger sample sizes as well as investigating potential layer-specific differences to further elucidate the cellular consequences of the 22q11.2 deletion. In addition, it is important to consider that cellular function and activity might still differ. Cellular activity, including synaptic transmission, signal transduction, and metabolic processes, plays a crucial role in brain function and could be affected by the 22q11.2 deletion. Differential gene analysis can be utilized to further explain differences between 22q11.2 and WT.

Cell proliferation rates vary significantly among different cell types. Terminally differentiated cells rarely divide, focusing on their specialized functions, while stem cells actively proliferate to maintain tissue homeostasis. This study aims to culture and analyze two human blood cancer cell lines: erythroleukemia K562 cells and Hodgkin's lymphoma L428 cells. The investigation focuses on their proliferation rates and transcriptional activities under various conditions and treatments. The project also includes the establishment of a novel, streamline protocol for extracting and quantifying DNA, nascent RNA, mRNA and protein from one single sample.

The study also addresses the challenge of correlating cell state with transcriptional activity, noting that current sequencing normalization techniques renders total levels of transcription incomparable between cell lines. By quantifying proliferation rates and transcriptional activity, a potential connection between proliferation rate and transcriptional activity was found. Higher proliferation rate corresponded to samples with larger amounts of RNA and higher transcription of nascent RNA, indicating an association that facilitates the production of sufficient cellular components necessary for the two daughter cells. 

Additionally, the study investigates the two cell lines’ responsiveness to differentiation-inducing hemin, known to induce differentiation toward the erythrocyte lineage for myeloid cells such as the K562 cell line. The findings from this study show that, despite belonging to different parts of the hematopoietic lineage, both the K562 cells and the L428 cells are responsive to hemin-induced differentiation toward the erythrocyte lineage. This is demonstrated by the fact that their proliferation rates are equally affected upon hemin treatments, and because both K562 and L428 cells turn red as a reflection of the induced production of hemoglobin, which is a recognized effect of hemin-induced erythrocytic differentiation.

We present a simple and rapid method for measuring the acoustic energy density in microchannel acoustophoresis based on light-intensity measurements of a suspension of particles. The method relies on the assumption that each particle in the suspension undergoes single-particle acoustophoresis. It is validated by the single-particle tracking method, and we show by proper re-scaling that the re-scaled light intensity plotted versus re-scaled time falls on a universal curve. The method allows for analysis of moderate-resolution images in the concentration range encountered in typical experiments, and it is an attractive alternative to particle tracking and particle image velocimetry for quantifying acoustophoretic performance in microchannels.

De novo creation of protein coding genes involves the formation of short ORFs from noncoding regions; some of these ORFs might then become fixed in the population These orphan proteins need to, at the bare minimum, not cause serious harm to the organism, meaning that they should for instance not aggregate. Therefore, although the creation of short ORFs could be truly random, the fixation should be subjected to some selective pressure. The selective forces acting on orphan proteins have been elusive, and contradictory results have been reported. In Drosophila young proteins are more disordered than ancient ones, while the opposite trend is present in yeast. To the best of our knowledge no valid explanation for this difference has been proposed. To solve this riddle we studied structural properties and age of proteins in 187 eukaryotic organisms. We find that, with the exception of length, there are only small differences in the properties between proteins of different ages. However, when we take the GC content into account we noted that it could explain the opposite trends observed for orphans in yeast (low GC) and Drosophila (high GC). GC content is correlated with codons coding for disorder promoting amino acids. This leads us to propose that intrinsic disorder is not a strong determining factor for fixation of orphan proteins. Instead these proteins largely resemble random proteins given a particular GC level. During evolution the properties of a protein change faster than the GC level causing the relationship between disorder and GC to gradually weaken.

The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.

Cellulosic materials are the most abundant renewable resource in the world; plant cell walls are natural composite materials containing crystalline cellulose embedded in a matrix of hemicelluloses, structural proteins, and lignin. Xyloglucans are an important group of hemicelluloses, which coat and cross-link crystalline cellulose in the plant cell wall. In this thesis, structure-function relationships of a range of xyloglucan-active enzymes were examined.

A paradigm for efficient enzymatic biomass utilization is the cellulosome of the anaerobic bacterium Clostridium thermocellum. The cellulosome is a high molecular weight complex of proteins with diverse enzyme activities, including the inverting xyloglucan endo-hydrolase CtXGH74A. The protein structure of CtXGH74A was solved in complex with xyloglucan oligosaccharides (XGOs) which stabilized disordered loops of the apo-structure. Further detailed kinetic and product analyses were used to conclusively demonstrate that CtXGH74A is an endo-xyloglucase that produces Glc4-based XGOs as limit digestion products.

In comparison, the retaining glycoside hydrolase family 16 (GH16) contains hydrolytic endo-xyloglucanases as well as xyloglucan transglycosylases (XETs) from plants. To elucidate the determinants of the transglycosylase/hydrolysis ratio in GH16 xyloglucan-active enzymes, a strict transglycosylase, PttXET16-34 from hybrid aspen, was compared structurally and kinetically with the closely related hydrolytic enzyme NXG1 from nasturtium. A key loop extension was identified in NXG1, truncation of which yielded a mutant enzyme that exhibited an increased transglycosylase rate and reduced hydrolytic activity. Kinetic studies were facilitated by the development of new, sensitive assays using well-defined XGOs and a series of chromogenic XGO aryl-glycosides.

A detailed understanding of GH16 xyloglucan enzymology has paved the way for the development of a novel chemo-enzymatic approach for biomimetic fiber surface modification, in which the transglycosylating activity of PttXET16-34 was employed. Aminoalditol derivates of XGOs were used as key intermediates to incorporate novel chemical functionality into xyloglucan, including chromophores, reactive groups, protein ligands, and initiators for polymerization reactions. The resulting modified xyloglucans were subsequently bound to a range of cellulose materials to radically alter surface properties. As such, the technology provides a novel, versatile toolkit for fiber surface modification.

The horse is a highly athletic species, partly due to the thousands of years of selective breeding that has occurred since its domestication. Further, for the past 300 years, racehorses have been under an intense selection of traits associated with superior racing performance, which has further enhanced the athletic performance. Understanding the genetics behind this athletic performance is of importance for both the horse racing industry as well as for increasing the knowledge of certain conditions and diseases in horses as well as other species. A recent study identified a single nucleotide polymorphism (SNP) having significant associations with racing performance. In this degree project, the genomic region containing this SNP (22:46717860C>T) was further investigated through the mapping of its genomic interactions using targeted chromosome conformation capture with unique molecular identifiers (UMI-4C). Our hypothesis was that this genomic region interacts with and regulates a gene called EDN3. Previously prepared 3C DNA templates from horse lung tissue were sonicated, adapter-ligated, PCR amplified using primers targeting six different loci of interest, and sequenced. No direct interactions were found between the SNP and the EDN3 gene. However, multiple potential interactions were shared between them, suggesting that they interact with the same loci and might, therefore, indirectly interact with each other. Further, potential interactions were found between the SNP and loci within the genes GNAS and ZNF831, as well as between the SNP and the promoter of the gene LOC729296. However, the data obtained was scarce, and to be able to draw a clear conclusion regarding the interaction partners of the genomic region containing the SNP 22:46717860C>T, more data is required.

The deep terrestrial subsurface remains an environment where there is limited understanding of the extant microbial metabolisms. At Olkiluoto, Finland, a deep geological repository is under construction for the final storage of spent nuclear fuel. It is therefore critical to evaluate the potential impact microbial metabolism, including sulfide generation, could have upon the safety of the repository. We investigated a deep groundwater where sulfate is present, but groundwater geochemistry suggests limited microbial sulfate-reducing activity. Examination of the microbial community at the genome-level revealed microorganisms with the metabolic capacity for both oxidative and reductive sulfur transformations. Deltaproteobacteria are shown to have the genetic capacity for sulfate reduction and possibly sulfur disproportionation, while Rhizobiaceae, Rhodocyclaceae, Sideroxydans, and Sulfurimonas oxidize reduced sulfur compounds. Further examination of the proteome confirmed an active sulfur cycle, serving for microbial energy generation and growth. Our results reveal that this sulfide-poor groundwater harbors an active microbial community of sulfate-reducing and sulfide-oxidizing bacteria, together mediating a sulfur cycle that remained undetected by geochemical monitoring alone. The ability of sulfide-oxidizing bacteria to limit the accumulation of sulfide was further demonstrated in groundwater incubations and highlights a potential sink for sulfide that could be beneficial for geological repository safety.

Denitrification is a key metabolic process in the global nitrogen cycle and is performed by taxonomically diverse microorganisms. Despite the widespread importance of this metabolism, challenges remain in identifying denitrifying populations and predicting their metabolic end-products based on their genotype. Here, genome-resolved metagenomics was used to explore the denitrification genotype of Bacillota enriched in nitrate-amended high temperature incubations with confirmed N2O and N2 production. A set of 12 hidden Markov models (HMMs) was created to target the diversity of denitrification genes in members of the phylum Bacillota. Genomic potential for complete denitrification was found in five metagenome-assembled genomes from nitrate-amended enrichments, including two novel members of the Brevibacillaceae family. Genomes of complete denitrifiers encode N2O reductase gene clusters with clade II-type nosZ and often include multiple variants of the nitric oxide reductase gene. The HMM set applied to all genomes of Bacillota from the Genome Taxonomy Database identified 17 genera inferred to contain complete denitrifiers based on their gene content. Among complete denitrifiers it was common for three distinct nitric oxide reductases to be present (qNOR, bNOR, and sNOR) that may reflect the metabolic adaptability of Bacillota in environments with variable redox conditions.

In the development of monoclonal antibody biosimilar products to be used as therapeutics, the Fc-receptor binding properties must be comparable to those of the innovator product. Hence, it is in the interest of biosimilar companies such as Xbrane Biopharma AB to de- velop analytical methods that support the assessment of this quality attribute throughout the cell line development and cell culture process development. In this study an antibody acting as an inhibitor to the, on tumor cells, overexpressed protein CD38, and with affinity for Fc gamma receptors located on immune effector cells will be of primary interest. An in- teraction involved in initiating antibody-mediated cell-dependent cytotoxicity, a mechanism of action utilized in therapeutic monoclonal antibodies. This study was therefore carried out, with the purpose to assess if the FcγRIIIa-IgG complex is suitable to be investigated with Bio-Layer Interferometry, more specifically, an Octet RED96E instrument. Thus, a methodology was created to provide useful kinetic parameters, such as the dissociation con- stant. The originator drug with differences in regards to shelf-life was utilized for a full affinity determination. In addition, a full affinity determination was also done with the an- tibody when deglycosylated.  Firstly, a decision was made regarding which bioassay format to use and then the optimal ligand concentration for the specific affinity determination was determined based on the obtained data. Moreover, the dissociation constant was obtained with both steady state analysis and full kinetics results. They were both reasonable when comparing to literature. The dissociation constant was very similar for the antibody despite variability in shelf-life. In conflict with literature data, deglycosylation of the antibody did not show a clear increase of the dissociation constant. Hence, did not indicate a negative impact on the affinity. However, the methodology requires further experiments. This in- cludes validation, comparison to other similar methods in regards to both economic and scientific aspects, analysis of the specific biosimilar not only the originator and evaluate the used data processing. Furthermore, it is necessary to further assess at what stage of the process development the attribute should be tested to adjust and evaluate the method based on those conditions.

The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.

Background Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified. Results A fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all. Conclusions The fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.

F1F0 ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the alpha, beta, gamma and e subunits of F-1 ATPases and could form an F-1-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F-1-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F-1-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F-1-like structure is associated with a hypothetical X-0 sector located in the membrane of mycoplasma cells.

Estrogen regulates a variety of important physiological functions in both males and females, where the regulation of female reproduction and the development of sexual organs are typical examples. The effects of estrogen are predominantly exerted via signaling through the two nuclear receptors estrogen receptor α (ERα) and β (ERβ), or the membrane G protein-coupled estrogen receptor 1 (GPER1). While estrogen signaling is important for human health, dysregulation of signaling can have adverse effects and impact the development and progression of a wide range of diseases including reproductive disorders and cancer.

ERβ has been shown to be highly important for ovarian function by regulating folliculogenesis and ovulation but has also been implied to protect against the development of colorectal cancer (CRC) by mediating the effects of estrogen. Despite the known role of ERβ, there is a lack of mechanistic understanding regarding how ERβ acts under both normal conditions and during disease. The overall aim of this thesis was to characterize the function and molecular mechanism of endogenous ERβ and to decipher its role in the normal ovary as well as its impact on colitis and CRC development. To further understand the role of estrogen signaling in the colon, we also aimed to identify sex differences during CRC development. 

In paper I we characterized the full cistrome of endogenous ovarian ERβ in the mouse and explored its transcriptional impact. We confirmed a direct role for ERβ in the regulation of essential ovarian functions and identified a novel crosstalk with the nuclear receptor LRH-1.  

In paper II we induced colitis-associated CRC (CAC) in intestinal epithelial-specific ERβ knockout mice and identified a protective effect by intestinal ERβ against tumor development in both male and female mice. We further characterized sex-dependent effects and proposed an underlying mechanism involving the regulation of TNFα/NFκB signaling. 

In paper III we expanded the investigation of sex-dependent changes during chemically-induced colitis in wildtype mice and identified a sex-specific response related to inflammatory response. We further found that male mice have an enhanced response to induced colitis.

In paper IV the transcriptome of colitis-induced tumors and their immune cell infiltration was explored in wildtype and intestinal epithelial-specific ERβ knockout mice of both sexes. This showed that sex differences in the transcriptome appear to be dependent on the expression of ERβ. Also, the identified ERβ-dependent changes in the tumor transcriptome of female mice were specifically related to immune response. We corroborated an impact of ERβ on the infiltration of immune cells, especially a reduction of regulatory T cell and NK cell recruitment. 

In summary, this thesis provides new mechanistic understanding of the transcriptional role of ERβ in the normal ovary and in the colon microenvironment. This includes the discovery of crosstalk with LRH-1 in the ovary and NFκB in the colon. Our characterization provides a foundation to develop targeted therapies for improved fertility and chemoprevention in CRC. This thesis also highlights the importance of including both sexes in colitis and CRC research to advance our knowledge and improve treatment development.

Chronic inflammation promotes the development of colorectal cancer (CRC), as evidenced by patients with inflammatory bowel disease (IBD), and sex disparities are evident in CRC. The tumor microenvironment (TME) is composed of stromal cells and infiltrating immune cells that directly affect processes including antitumor immunity. We have previously shown that intestinal estrogen receptor beta (ERβ) protects against colitis and colitis-induced cancer (CAC) by modulating inflammatory signaling and that males are more sensitive to the induction of colitis and cancer. However, sex differences between tumors and the impact of ERβ the tumor immune microenvironment have not been investigated. In this study, we have analyzed colon samples from AOM/DSS-treated wild-type and ERβKO^Vil mice (that lack intestinal ERβ) and profiled the differences in the transcriptome and immune response to CAC on the basis of sex and ERβ expression. RNA-sequencing revealed differences in gene expression and enriched biological processes depending on sex and genotype, and the immune response to cancer appears altered between tumors from female WT and ERβKO^Vil mice. Immunostaining subsequently showed that tumors from ERβKO^Vil mice display significantly increased CD68+ macrophage infiltration, decreased CD3+ T cell infiltration, and, strikingly, impaired NK cell infiltration. Here, for the first time, we show that intestinal ERβ modulates the tumor immune microenvironment during CAC and that lack of intestinal ERβ appears to promote the formation of an immunosuppressive TME. Our findings indicate that activation of ERβ could be used to treat CRC, possibly together with immunotherapies, and provide a foundation for future studies investigating ERβ and immunity. 

Granulosa cell tumors (GCTs) are rare ovarian tumors comprising an adult and a juvenile subtype. They have a generally good prognosis, but the survival rate drastically declines in patients with late-stage or recurring tumors. Due to the rarity of GCTs, the tumor type is largely understudied and lacks a specific treatment strategy. Estrogen receptor beta (ERβ/ESR2) has been found to be highly expressed in GCTs, which could be of therapeutic importance since it can be targeted with small molecules. However, its role in GCTs is not known. In this review, we summarize the current knowledge about the action of ERβ in the ovary and discuss its prospective role in GCTs.

Background:  Estrogen receptor beta (ERβ, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized.

Results: We here performed ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERβ knockout ovaries. By integrating the cistrome and transcriptome, we identify its direct target genes and enriched biological functions in the ovary. This demonstrates a strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERβ loss. Moreover, we identify a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1). ERβ and LRH-1 extensively bind to the same chromatin locations in granulosa cells and we corroborate simultaneous co-binding using ChIP re-ChIP, at the ERβ-repressed gene Greb1. At other shared sites (by ERβ-upregulated genes Cyp11a1 and Fkbp5), they do not bind simultaneously. Transactivation assay experimentation further show that ERβ and LRH-1 can inhibit their respective transcriptional activity at classical response elements.

Conclusions: We characterize genome-wide ERβ chromatin binding and gene regulations which reveal extensive crosstalk between ERβ and LRH-1. We experimentally corroborate co-binding to target genes and impact on transactivation. Our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.

Pancreatic islets are composed of endocrine cells that secrete hormones essential for blood-glucose homeostasis. Prior research has revealed that the gene expression and functionality of human islet cells is heterogenous. However, it is not currently understood how the heterogeneity correlates to normal islet cell function and dysfunction in diabetes pathogenesis. Subsequently, an international collaborative project has been initiated to elucidate what constitutes islet cell heterogeneity from a transcriptional, proteomic, and functional perspective in both health and disease. In this study, a highly multiplex tissue image assay was developed to allow for the study of the localization and distribution of proteins previously identified to correlate with functional activity and heterogeneity in islet cells using the CO-Detection by indEXing (CODEX) platform. In total, 22 proteins were studied simultaneously of which 10 were specifically expressed in islet cells. These included generic pancreatic markers such as C-peptide (C-pep) marking insulin-secreting β-cells, glucagon (GCG) and somatostatin (SST), but also less well-characterized proteins such as Shisa like 2B (FAM159B) and Neural proliferation, differentiation and control 1 (NPDC1). The multiplex tissue imaging allowed for single-cell analysis of protein expression in human islet cells showing that most islet specific proteins were heterogeneously expressed. The observations made in this study serves as a validation to that the human islet microenvironment is highly complex due to islet cell heterogeneity. Additionally, the study demonstrated that multiplex tissue imaging has the potential to reveal novel cell types and interactions.

This Article reports the influence of the protein network structure on the mechanical properties of foams produced from commercial wheat gluten using freeze-drying. Foams were produced from alkaline aqueous solutions at various gluten concentrations with or without glycerol, modified with bacterial cellulose nanosized fibers, or both. The results showed that 20 wt % glycerol was sufficient for plasticization, yielding foams with low modulus and high strain recovery. It was found that when fibers were mixed into the foams, a small but insignificant increase in elastic modulus was achieved, and the foam structure became more homogeneous. SEM indicated that the compatibility between the fibers and the matrix was good, with fibers acting as bridges in the cell walls. IR spectroscopy and SE-HPLC revealed a relatively low degree of aggregation, which was highest in the presence of glycerol. Confocal laser scanning microscopy revealed distinct differences in HMW-glutenin subunits and gliadin distributions for all of the different samples.

The culture of cells in 3D creates a more physiologically relevant cell environment than conventional 2D cultures. Interactions of cells with the extracellular matrix induce important cellular signalling that regulates cell adhesion, migration, proliferation, differentiation, and survival. This is crucial for modelling cell development and disease. This thesis aims to develop and analyse improved 3D cell culture methods for stem cell-derived pancreatic islets (SC-islets) and breast cancer cell lines using a functionalized recombinant spider silk. Spider silk is a natural protein-based material with remarkable mechanical properties of high strength and elasticity. It is also biodegradable and cytocompatible. FN-silk, the recombinant spider silk protein utilized in this thesis, is functionalized with a cell binding motif (RGD) from fibronectin, to improve cell adhesion. Notably, FN-silk self-assembles at the liquid-air interface into a fibrillar structure, making it favourable as support for cell culture. In this thesis, bioinformatic methods were used to discover how the FN-silk supported environment affects the gene expression of cells and the cellular heterogeneity of SC-islets during a differentiation process. Additionally, bioinformatical analysis of the effect of 3D cell culture of breast cancer cell lines in FN-silk networks was performed. The first part of the thesis addresses serval challenges in pancreatic islet transplantation, by presenting an optimized protocol for pancreatic differentiation from human pluripotent stem cells, improving in vitro cultivation, and developing a cryopreservation method for SC-islets. The differentiation protocol presented in Paper 1 resulted in pure endocrine cell populations, avoiding unwanted proliferating and non-endocrine cells. It was also demonstrated that these SC-islets matured in vivo, and could effectively reverse diabetes in a diabetic mouse model. Single-cell RNA sequencing analysis provided new insights into the cellular composition and gene expression of the SC-islets before and after transplantation. In Paper 2, an innovative method for 3D in vitro cultivation of SC-islets using FN-silk networks mimicking the extracellular matrix was established. The FN-silk networks provided structural support for in vitro cultivation and handling during in vivo transplantation. The viability and functionality of free and FN-silk incorporated SC-islets were evaluated and compared. Single-cell RNA sequencing analyses confirmed maintenance of cellular composition, with a slightly improved beta cell maturation for SC-islets supported by FN-silk. In Paper 3, a novel strategy for cryopreservation of SC-islets was explored. The twisted vitrification method, previously employed for 2D cultures, was adapted for 3D cultures by utilizing integration into FN-silk networks to facilitate handling during the vitrification process. The second part of the thesis aimed to develop a method for 3D culture of breast cancer cells to better replicate the complexity of the tumour microenvironment. In Paper 4, FN-silk networks were used to generate a 3D environment for breast cancer cells where crucial cell-ECM interactions can be established. Proliferation rates and key marker expression of the cells cultured in 2D versus in the FN-silk network environment were investigated. Bioinformatic analysis of bulk RNA sequencing data was used to compare breast cancer cells in conventional 2D cell cultures with those cultured in 3D with the support of FNsilk. In conclusion, the work conducted in this thesis presents significant advancements in the development and analyses of 3D cell cultures of both SCislets and breast cancer cell lines, potentially enhancing therapeutic applications, disease modeling, and drug testing.

Cryopreservation is crucial for making cell therapies more accessible by enabling long-term storage and facilitating transplantation logistics. However, preserving cells in 3D models implies significant challenges, including inhomogeneous penetration of cryoprotectants and non-uniform heat transfer during freezing and thawing. Vitrification is a novel cryopreservation approach that improves the survival of living cells by providing optimal cooling and rewarming rates to eliminate ice crystal-induced mechanical injury. Nevertheless, sterility issues can arise from direct contact with liquid nitrogen (LN2). The twisted vitrification method addresses this by separating the cells and LN2 into two different compartments, ensuring a sterile vitrification process.In this study, we optimized the cryopreservation of stem cell-derived pancreatic islets (SC-islets) by combining twisted vitrification with the supporting biomaterial FN-silk. We compared the viability of free SC-islets to those supported by a FN-silk network during cryopreservation using either conventional slow freezing or twisted vitrification. Our results showed a higher post-recovery for SC-islets incorporated in FN-silk using twisted vitrification, outperforming both conventional slow freezing and twisted vitrification of free SC-islets. Additionally, insulin and glucagon expression were maintained after vitrification within the FN-silk network.

Type 1 diabetes is a life-threatening disease characterized by lifelong insulin dependency and a reduced quality of life. Pancreatic islet transplantation is a promising treatment but is limited by donor shortages and significant islet loss during the procedures. The usage of pancreatic islet-like aggregates derived from pluripotent stem cells (SC-islets) could solve the problem of shortage of islets. Incorporating these SC-islets into a supporting scaffold could protect them during handling.FN-silk, a recombinantly produced spider silk protein functionalized with a cell adhesion motif from fibronectin, can generate 3D networks that mimic the extracellular matrix (ECM). We herein demonstrate a reproducible method for integrating SC-islets well-distributed within stable 3D networks of FN-silk. These SC-islets showed high viability and maintained functionality, with increased glucagon expression and improved beta cell maturation as compared to free SC-islets. Additionally, the support of FN-silk networks enables cultivation of SC-islets under conditions needed for scale-up. Moreover, the integration of the SC-islets into sheets of FN-silk networks facilitates handling during transplantation into the anterior chamber of the eye (ACE) of rabbits. Such transplanted FN-silk incorporated SC-islets were found vascularized six weeks post-transplantation, with sustained insulin and glucagon expression. 

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

(1 -> 3)-beta-D-Glucans are major components of the cell walls of Oomycetes and as such they play an essential role in the morphogenesis and growth of these microorganisms. Despite the biological importance of (1 -> 3)-beta-D-glucans, their mechanisms of biosynthesis are poorly understood. Previous studies on (1 -> 3)-beta-D-glucan synthases from Saprolegnia monoica have shown that three protein bands of an apparent molecular weight of 34, 48 and 50 kDa co-purify with enzyme activity. However, none of the corresponding proteins have been identified. Here we have identified, purified, sequenced and characterized a protein from the 34 kDa band and clearly shown that it has all the biochemical properties of proteins from the annexin family. In addition, we have unequivocally demonstrated that the purified protein is an activator of (1 -> 3)-beta-D-glucan synthase. This represents a new type of function for proteins belonging to the annexin family. Two other proteins from the 48 and 50 kDa bands were identified as ATP synthase subunits, which most likely arise from contaminations by mitochondria during membrane preparation. The results, which are discussed in relation with the possible regulation mechanisms of (1 -> 3)-beta-D-glucan synthases, represent a first step towards a better understanding of cell wall polysaccharide biosynthesis in Oomycetes.

Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area.

Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity.

In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant.

In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.

This study models two alternative downstream processes based on magnetic separation with the objective of understanding the economic feasibility of these processes compared to the traditional mAb process. The key focus lies in the economic understanding of the cell harvest and capture steps in the models. Here, the models revealed that integrating cell removal and product capture in a single operation is the main factor driving the unified productivity between USP and the magnetic bead-based processes. This results in significant economic benefits, such as savings in both the cost of goods per gram of mAb and fixed costs, as well as increasing annual facility output. The predicted savings potential approaches 38% for COGs, 17% for capital investment, and 40% for annual facility output. For mammalian cell-based manufacturing, the magnetic separation-based DSP provides a highly valuable option due to its integration of several individual unit operations compared to the traditional process both in reducing process time and cost and accommodating higher demands.

The use of adeno-associated viruses (AAVs) is a prominent delivery method for gene therapy.  The potentials of AAV-gene therapy are nonetheless hindered by production challenges of these viral vectors. One of the main production methods of AAVs is the triple transfection of plasmids into mammalian cells. Triple transfection is expensive, may lead to unequal ratios of plasmids and is an all over tedious production method.  One of the plasmids transfected is the helper vector containing helper genes VA-RNA III, E4orf6 and E2A.  In order to minimize the number of plasmids needed to be transfected, these genes could be stably integrated into the mammalian cells. A setback is however the toxic properties of the helper genes. To circumvent  this, induction systems could be applied to the genes, silencing them during cell proliferation, and producing viruses only upon induction of a small molecule. Here, both toxic effects of individual helper genes and inducibility of a suitable induction system were evaluated. Furthermore, the study initiated the formation   of an inducible helper vector based on the set properties of the helper genes.

Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer.

The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided.

Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD.

In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. 

Adeno-associated virus (AAV) based gene therapy is the therapeutic treatment of introducing geneticmaterial into a cell, with the help of a non-pathogenic vector. However, there are still challenges of the technique to be considered for optimization. Empty capsids are being formed during production andoff-target infection and insufficient targeting leads to high costs and immunogenicity for the patient. Fusing the affinity protein Affibody, with affinity towards a certain cell surface receptor, to the AAV can increase targeting. In this project, the length of the linker between the Affibody and thecapsid is being investigated through genetic modifications and evaluated in terms of bindingand transduction capacity. Eight different Affibody-AAVs with different linker lengths weresuccessfully produced. All constructs were able to bind the its specific receptor in vitro. One construct showed highest transduction efficiency when tested on receptor-expressing ExpiCHO-cell lines. Ion exchange chromatography was also explored to separate full AAV capsids from empty ones, which could in a therapeutic setting increase the therapeutic efficiency of AAVs. An initial attempt was achieved, with a successful binding of AAVs to the column. A separation of AAVs was also achieved, however with unknown fractions of empty versus full capsids. These results make up the basis for further development and optimization to reach a two-capsid population separation. Overall, this project results in efforts with promising outcomes to enhance specificity and effectiveness of AAV-based gene therapy for therapeutic applications.

A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of -galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C-glycosides. Inhibition studies provided important structural information for the two groups, and 1-thiobenzyl--D-galactopyranoside demonstrated the best inhibitory effects.

Natural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.