Cystic fibrosis, a genetic disorder affecting multiple organs in the body, including the lungs, remains a significant threat to patients due to inadequate treatment options. Treatment includes aerosolized deoxyribonuclease I which bolsters pulmonary function and improves affected patient condition. However, taking the liquid formulations requires prolonged inhalation times and nebulization equipment. Conversely, dry powder inhalers are handheld devices, delivering fine particles deep into the lung on a single inhalation. Dry formulations may be enhanced through the use of mesoporous silica particles which have an optimal size for inhalation, are light in weight and have a large surface area. Loading deoxyribonuclease I into mesoporous silica particles could potentially improve drug delivery to cystic fibrosis patients with reduced administration frequency when taken with dry powder inhalers. The incorporation of buffers into this system is crucial for ensuring efficient drug loading and stability at the biointerface during dry powder preparation. Thus, the objective of this project was to ascertain the most suitable buffer composition for loading deoxyribonuclease I into mesoporous silica particles. Protein size and activity were evaluated in different buffers prior to adsorption. Subsequently, dry formulations were prepared by freeze drying, and studied by thermogravimetric analysis and dynamic vapour sorption. Cumulative release analysis in simulated lung fluid was performed, followed by released protein enzymatic activity evaluation. Findings indicated the necessity of incorporating Ca2+ into buffers to increase protein loading efficiency and stability in dry formulations. Highest level of adsorption, and adequate remaining deoxyribonuclease I activity was observed in formulations prepared with calcium doped mesoporous silica particles in pH 5.0 50 mM sodium acetate buffer with added 5 mM CaCl2.

One of the lesser explored research areas is the influence factors such as personal care products, cosmetics, and everyday routines have on skin microbiota. This study investigated the effects of propylene glycol, a widely used ingredient in cosmetics and self-care products, on a staphylococcal system ubiquitously distributed on human skin. The system, comprised of Staphylococcus hominis, Staphylococcus epidermidis, and Staphylococcus aureus, comes from a healthy donor, devoid of any skin afflictions. Because Staphylococcus aureus is part of this microbial community, it was of great interest to contribute to the understanding of the manner in which its growth is kept in check. To fulfill this task, a new methodology was developed. Purposely intended to be facile and easily scalable in laboratories around the world, it can be used for the study of microbial systems of variable dimensions alone or with the complementary use of other methods. Results indicate that the effects of propylene glycol are complex, as it acts on the skin, the resident microbiota, and at the microbiota-skin interface. Commensals such as Staphylococcus hominis and Staphylococcus epidermidis seem to have synergy of action with propylene glycol, increasing each other’s power in reducing the number of viable colonies of Staphylococcus aureus. Lastly, results seem to also reveal the incompletely understood role of Staphylococcus hominis on human skin. While Staphylococcus epidermidis and Staphylococcus aureus ravenously compete with each other, it is the contribution of Staphylococcus hominis that seems to limit the latter’s overgrowing. This speaks volumes of the extent, complexities, and unknowns of microbial interactions.

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

The mastoid, located in the temporal bone, houses an air cell system whose cells have a variation in size that can go far below current conventional clinical CT scanner resolution. Therefore, the mastoid air cell system is only partially represented in a CT scan. Where the conventional clinical CT scanner lacks level of minute details, micro-CT scanning provides an overwhelming amount of ne details. The temporal bone being one of the most complex in the human body, visualization of micro-CT scanning of this boneawakens the curiosity of the experimenter, especially with the correct visualization settings. This thesis first presents a statistical analysis determining the surface area to volume ratio of the mastoid air cell system of human temporal bone, from micro-CT scanning using methods previously applied for conventional clinical CT scans. The study compared current results with previous studies, with successive downsampling the data down to a resolution found in conventional clinical CT scanning. The results from the statistical analysis showed that all the small mastoid air cells, that cannot be detected in conventional clinical CT scans, do heavily contribute to the estimation of the surface area, and in consequence to the estimation of the surface area to volume ratio by a factor of about 2.6. Such a result further strengthens the idea of the mastoid to play an active role in pressure regulation and gas exchange. Discovery of micro-channels through specific use of a non-traditional transfer function was then reported, where a qualitative and a quantitative pre-analysis were performed and reported. To gain more knowledge about these micro-channels, a local structure tensor analysis was applied where structures are described in terms of planar, tubular, or isotropic structures. The results from this structural tensor analysis suggest these microchannels to potentially be part of a more complex framework, which hypothetically would provide a separate blood supply for the mucosa lining the mastoid air cell system. The knowledge gained from analysing the micro-channels as locally providing blood to the mucosa, led to the consideration of how inflammation of the mucosa could impact the pneumatization of the mastoid air cell system. Though very primitive, a 3D shape analysis of the mastoid air cell system was carried out. The mastoid air cell system was first represented in a compact form through a medial axis, from which medial balls could be used. The medial balls, representative of how large the mastoid air cells can be locally, were used in two complementary clustering methods, one based on the size diameter of the medial balls and one based on their location within the mastoid air cell system. From both quantitative and qualitative statistics, it was possible to map the clusters based on pre-defined regions already described in the literature, which opened the door for new hypotheses concerning the effect of mucosal inflammation on the mastoid pneumatization. Last but not least, discovery of other structures, previously unreported in the literature, were also visually observed and briefly discussed in this thesis. Further analysis of these unknown structures is needed.

The human skeleton is a living and dynamic tissue that constantly is being renewed in a process called bone remodeling. Old bone is resorbed by osteoclasts and new bone is formed by osteoblasts. Bone is a composite material made up by mineral crystals in the form of hydroxyapatite (calcium and phosphate) that provides the hardness of bone, and collagen fibrils that provides elasticity and flexibility. Alkaline phosphatase (ALP) is a family of enzymes that is present in most species and catalyzes the hydrolysis of various phosphomonoesters at alkaline pH. Despite the generalized use of ALP as a biochemical marker of bone formation, the precise function of bone ALP (BALP) is only now becoming clear. Three circulating human BALP isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. Paper I. Three endogenous phosphocompounds, (i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP) and phosphoethanolamine (PEA)), have been suggested to serve as  physiological substrates for BALP. The BALP isoforms display different catalytic properties towards PPi and PLP, which is attributed to their distinct N-linked glycosylation patterns. The catalytic activity, using PEA as substrate, was barely detectable for all BALP isoforms indicating that PEA is not a physiological substrate for BALP. Paper II. Mouse serum ALP is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP. We characterized the circulating and tissue-derived mouse ALP isozymes and isoforms from mixed strains of wild-type and knockout mice. All four BALP isoforms (B/I, B1x, B1, and B2) were identified in mouse serum and bone tissues, in good correspondence with those found in human bones. All mouse tissues, except liver, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Paper III. The objective of this study was to investigate the binding properties of human collagen type I to human BALP, including the two BALP isoforms B1 and B2, together with ALP from human liver, human placenta and E. coli. A surface plasmon resonance-based analysis showed that BALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. The B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform, indicating that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I. Paper IV. Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and frequently used as a marker of bone resorption. Intriguingly, recent studies show that TRAP is also expressed in osteoblasts and osteocytes. TRAP displays enzymatic activity towards the endogenous substrates for BALP, i.e., PPi and PLP. Both TRAP and BALP can alleviate the inhibitory effect of osteopontin on mineralization by dephosphorylation, which suggests a novel role for TRAP in skeletal mineralization.

Diabetes is a rapidly increasing health problem. In a global perspective,approximately 415 million people suffered from diabetes in 2015 and this number ispredicted to increase to 640 million by 2040. To tackle this pandemic there is a needfor better analytical tools by which we can increase our understanding of the disease.One discipline that has already provided much needed insight to diabetes etiology isoptical molecular imaging. Using various forms of light it is possible to create animage of the analysed sample that can provide information about molecularmechanistic aspects of the disease and to follow spatial and temporal dynamics. The overall aim of this thesis is to improve and adapt existing andnovel optical imaging approaches for their specific use in diabetes research. Hereby,we have focused on three techniques: (I) Optical projection tomography (OPT),which can be described as the optical equivalent of x-ray computed tomography(CT), and two vibrational microspectroscopic (VMS) techniques, which records theunique vibrational signatures of molecules building up the sample: (II) Fouriertransforminfrared vibrational microspectroscopy (FT-IR) and (III) Ramanvibrational microspectroscopy (Raman). The computational tools and hardware applications presented here generallyimprove OPT data quality, processing speed, sample size and channel capacity.Jointly, these developments enable OPT as a routine tool in diabetes research,facilitating aspects of e.g. pancreatic β-cell generation, proliferation,reprogramming, destruction and preservation to be studied throughout the pancreaticvolume and in large cohorts of experimental animals. Further, a novel application ofmultivariate analysis of VMS data derived from pancreatic tissues is introduced.This approach enables detection of novel biochemical alterations in the pancreasduring diabetes disease progression and can be used to confirm previously reportedbiochemical alterations, but at an earlier stage. Finally, our studies indicate thatRaman imaging is applicable to in vivo studies of grafted islets of Langerhans,allowing for longitudinal studies of pancreatic islet biochemistry.viIn summary, presented here are new and improved methods by which opticalimaging techniques can be utilised to study 3D-spatial, quantitative andmolecular/biochemical alterations of the normal and diseased pancreas.

Background: Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder. ALS has been traditionally believed to be primarily a motor neuron disease. However, accumulating data indicate that loss of contact between the axons and the muscle fibres occurs early; long before the death of motor neurons and that muscle fibres may initiate motor neuron degeneration. Thus, the view of ALS is changing focus from motor neurons alone to also include the muscle fibres and the neuromuscular junctions (NMJs). While skeletal muscles are affected in ALS, oculomotor disturbances are not dominant features of this disease and extraocular muscles (EOMs) are far less affected than limb muscles. Why oculomotor neurons and EOMs are capable to be more resistant in the pathogenetic process of ALS is still unknown. The overall goal of this thesis is to explore the pathophysiology of ALS from a muscle perspective and in particular study the expression and distribution of key neurotrophic factors (NTFs) and Wnt proteins in EOMs and limb muscles from ALS donors and from SOD1G93A transgenic mice. Comparisons were made with age-matched controls to distinguish between changes related to ALS and to ageing. Results: Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4) were present in EOMs and limb muscles at both mRNA and protein levels in control mice. The mRNA levels of BDNF, NT-3 and NT-4 were significantly lower in EOMs than in limb muscles of early and/or late control mice, indicating an intrinsic difference in NTFs expression between EOMs and limb muscles. qRT-PCR analysis showed significantly upregulated mRNA levels of NT-3 and GDNF in EOMs but significantly downregulated mRNA levels of NT-4 in limb muscles from SOD1G93A transgenic mice at early stage. The NTFs were detected immunohistochemically in NMJs, nerve axons and muscle fibres. The expression of BDNF, GDNF and NT-4 on NMJs of limb muscles, but not of EOMs, was significantly decreased in terminal stage ALS animals as compared to the limb muscles of the age-matched controls. In contrast, NTFs expression in intramuscular nerve axons did not present significant changes in either muscle group of early or late ALS mice. NTFs, especially BDNF and NT-4 were upregulated in some small-sized muscle fibres in limb muscles of late stage ALS mice. All the four Wnt isoforms, Wnt1, Wnt3a, Wnt5a and Wnt7a were detected in most axon profiles in all human EOMs with ALS, whereas significantly fewer axon profiles were positive in the human limb muscles except for Wnt5a. Similar differential patterns were found in myofibres, except for Wnt7a, where its expression was elevated within sarcolemma of limb muscle fibres. β-catenin, a marker of the canonical Wnt pathway was activated in a subset of myofibres in the EOMs and limb muscle in all ALS patients. In the SOD1G93A mouse, all four Wnt isoforms were significantly decreased in the NMJs at the terminal stage compared to age matched controls. Conclusions: There were clear differences in NTF and Wnt expression patterns between EOM and limb muscle, suggesting that they may play a role in the distinct susceptibility of these two muscle groups to ALS. In particular, the early upregulation of GDNF and NT-3 in the EOMs might play a role in the preservation of the EOMs in ALS. Further studies are needed to determine whether these proteins and the pathways they control may be have a future potential as protecting agents for other muscles.

In recent years a significant research focus has been on the development of biomimicking three-dimensional substrates for cell culturing. Hydrogels mimicking the extracellular matrix is a well-suited scaffold for this purpose and there are many different ways these can be cross-linked to retain their shape. The group of Molecular Materials at IFM, Linköping University, is focusing on the development of physical hydrogels hybridized through peptide-peptide interactions but all peptides used for this today are created using rational design and on top of this very large, making them time-consuming and expensive to fabricate. The aim of this project was to evaluate if One Bead One Compound (OBOC) libraries could be used as an alternative to rational design in the finding of cyclic peptide binding partners used in the hybridization of hydrogels. The results were not very promising though since only seven peptides passed all screening steps and of these only two could be sequenced. Of these two, only one was water soluble enough to enable binding interactions analysis but was then found to be a false hit. Nevertheless, it should be noticed that only a fraction of all possible combinations was screened and the results cannot exclude OBOC libraries as an approach in the quest of finding new cyclic peptide binding partners.

ABSTRACT The demand of healthier products is increasing, and more people are more interested of what they eat. Statistics show that the consumption of snacks is rising. Hyperglycemia leads to an increased risk for complications in type II diabetes mellitus. Increased levels of postprandial plasma glucose may also lead to equal or maybe more harmful effects than fasting hyperglycemia. When the levels of postprandial plasma glucose are decreased, the development of cardiovascular complications is delayed, why it is important to lower the snacks consumption especially snacks that brings hunger quickly after they are eaten. Because of these factors, healthier products were developed in this study. The aim was to develop a wafer chocolate product that gives higher satiating effect and healthier blood glucose levels compared to one of Cloetta’s chocolate products. Two raw materials were used, a new carbohydrate and a new fat. The new carbohydrate is a healthier sugar alternative than sucrose, since it leads to lower and prolonged increase in blood glucose and insulin levels. The new fat is based on natural oil that is believed to be healthy, mainly due to its satiating effect. The effects of these two materials on blood glucose response and satiety were examined in two products. Furthermore, the products were made of fat reduced milk chocolate in which sucrose in the chocolate mass was 100 % replaced with the new carbohydrate, dietary fibre and fruit concentrate. Only one of the products contained the new fat. The products, together with Cloetta’s chocolate product were consumed by 17 healthy subjects. Blood glucose response and satiating effect after product intake were examined during a period of 3 days. When blood glucose response was analyzed, a slight indication that the products were relatively healthier than placebo, due to placebo’s unhealthy fluctuations, was found. No clear differences regarding blood sugar maxima were found. Placebo showed, as expected, the highest blood glucose maxima and the largest incremental area under curve, but the maxima of the new fat-lacking product was less than half as high as that of the new fatcontaining product and the area was smaller too, which was not expected. The results regarding the hunger levels were not as expected either since the new fat-lacking product was most satiating while the new fatcontaining product was the least satiating. Despite that, 57 % of the subjects reported they would by such products in the future. Several biases may have played a role in the results, for example whether or not subjects followed the criteria (e.g. lunch time, exercise), stress, worry, individual energy requirement and how serious and focused the subjects were. However, for further research, increasing the new fat content to 3 g, a bigger sized product, different filling, more subjects and more repeats of same measurements is recommended. / SVENSK SAMMANFATTNING Efterfrågan på hälsosammare produkter ökar, och fler människor blir mer intresserade av vad de äter. Statistik visar att konsumtionen av mellanmål ökar. Hyperglykemi leder till en ökad risk för komplikationer i typ II-diabetiker. Ökade nivåer av postprandiell plasmaglukos kan leda till lika eller mer skadliga effekter än fastande hyperglykemi. När nivåerna av postprandiell plasmaglukos är lägre reduceras utvecklingen av kardiovaskulära komplikationer, därför är det viktigt att minska småätandet, speciellt av mellanmål som leder till hunger snart efter att de har ätits. På grund av dessa faktorer har hälsosammare produkter utvecklats i denna studie. Syftet var att utveckla en kexchokladprodukt som har högre mättnadseffekt samt hälsosammare blodsockernivåer jämfört med en av Cloettas chokladprodukter. Två produkter som alternativ till Cloettas chokladprodukter utvecklades. I dessa användes en ny kolhydrat och ett nytt fett. Den nya kolhydraten är ett hälsosammare sockeralternativ än sukros, då den leder till en lägre och förlängd ökning av blodglukos- och insulinnivåer. Det nya fettet är baserat på en naturlig olja som är hälsosam på grund av dess mättande effekt. Effekten av dessa två ämnen undersöktes. Vidare så gjordes de två produkterna av fettreducerad mjölkchoklad i vilken sukros i chokladmassan var 100 % ersatt med den nya kolhydraten, kostfiber samt fruktkoncentrat. endast en av produkterna innehöll det nya fettet. Produkterna, tillsammans med Cloettas chokladprodukt (placebo) konsumerades av 17 friska personer. Blodsockerresponsen och den mättande effekten efter produktintaget undersöktes under 3 timmars period per dag i totalt 3 dagar. När blodglukosrespons analyserades hittades en svag indikation på att produkterna var relativt hälsosammare än placebo, på grund av de ohälsosamma fluktuationerna. Inga klara skillnader med avseende på blodsockermaxima hittades. Placebo visade, som väntat, det högsta blodglukosmaximum och den största arean under kurvan, men maximum för produkten utan det nya fettet var mindre än hälften så högt som det för produkten med det nya fettet och även arean under kurvan var mindre, vilket inte var förväntat. De upplevda hungernivåerna var inte heller som förväntat då produkten som saknar det nya fettet mättade flest personer medan produkten innehållande nya fettet mättade minst antal personer. Trots det så kunde 57 % av deltagarna tänka sig köpa sådana produkter i framtiden. Flera faktorer kan ha påverkat resultatet, till exempel huruvida försökspersonerna följde kriterierna (t.ex. lunchtid, träning), stress, oro, individuella energibehov samt hur allvarliga och fokuserade personerna var när de angav hungernivåerna. För vidare studier rekommenderas ett högre innehåll av det nya fettet (3 g), en större produkt, annorlunda fruktbaserad fyllning och fler deltagare och flera upprepningar av samma mätningar.

All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.