Chitosans are cationic polymers desirable for use in nucleic acid delivery due to its biocompability, biodegradable and low toxic nature. The P-glycoprotein (P-gp) pump is a member of membrane transporters in brain endothelial cells which recognizes xenobiotics, extruding them from the cells. P-gp is a highly clinically relevant target as it also limits accessibility of drugs from the blood to the brain.The overall aim of this work was to investigate the feasibility of using chitosan as a delivery vehicle for siRNA-mediated knockdown of P-glycoprotein (P-gp) efflux pump in an in vitro model of blood brain barrier. The human lung cancer cells (H1299) stably expressing GFP was used for screening and optimization of the delivery system and conditions. Lung cancer is often represented among multidrug resistant cell lines and is therefore also a suitable target for knockdown of transporters. The main objective was to implement this knowledge to efficiently downregulate the P-gp pump residing in the rat brain endothelial cell line RBE4. The approach was to first establish suitable physiochemical characteristics of the chitosans like DPn and the N/P ratios of chitosan/siRNA complexes by studying exogenous GFP and endogenous GAPDH expression and uptake of fluorescently labeled siRNA by flow cytometry. Cytotoxicity profiling of the chitosans and polyplexes was preformed with AlamarBlue, PI-staining, Cell counting and BCA assay. We further investigated the effect of chitosan mediated mdr1a-siRNA on drug efflux in the RBE4 cells using two functional assays; Rhodamine 123 efflux by flow cytometry and visualization of the anticancer drug Doxorubicin by CLSM.We demonstrated that chitosan can be used as an efficent delivery vehicle for siRNA in both H1299 and RBE4 cells with negligible cytotoxicity in vitro. The formulated chitosan/siRNA polyplexes was found to dependent on DPn and N/P ratios in order to exert maximal effect on both uptake and knockdown. Specifically, we managed to downregulate the P-gp expression in RBE4 cells using chitosans with a high DPn complexed with mdr1a-siRNA in a low N/P ratio. Together these results elucidate the potential of chitosan mediated siRNA downregulation of the P-gp pump in RBE4 cells and further development for in vivo applications.

digitalisering@umu

<p>This thesis covers studies on endogenous proteolytic enzymes and their impact on fish muscle proteins and texture. The studies have been performed using Atlantic salmon (<i>Salmo salar</i>) and cod (<i>Gadus morhua</i>) subjected to different treatments and storage conditions.</p><p>The textural properties were very different in the two species. Salmon fillets were significantly softer and less resilient than cod fillets, and the properties changed somewhat differently during storage experiments. Different proteolytic enzymes have been reported to participate in muscle softening. Some of these enzymes were investigated, and specific proteolytic activities were detected throughout the storage periods. Collagenase-like enzymes seem to be the most important for cod muscle texture. Microorganisms and/or microbial enzymes seem not to be important for changes in salmon muscle texture. Results suggest that the cathepsin B-like enzymes are important for salmon texture. The activities of the proteolytic enzymes may be greatly affected by the muscle pH, and by the treatment(s) the fish are subjected to. In any case, changes caused by differences in proteolytic activities may need some time to be detectable or have significant impact on fish quality.</p><p>When cod fillets are stored in ice, it is highly recommended to keep the temperature low. Even a relatively mild temperature abuse was sufficient to result in less favorable textural characteristics, and make the fillets seem older than their days of storage.</p><p>Salmon fillets are often subjected to cold-smoking. The smoking temperature was important for the solubility properties of the muscle proteins, and for their composition, but did not affect the proteolytic activity. The effects of the processing parameters were most important early in the product’s shelf life, as the differences caused by the different smoking temperatures were reduced by further storage of the smoked samples.</p> / Paper II and III are reprinted with kind permission of Elsevier, sciencedirect.com

This thesis covers studies on endogenous proteolytic enzymes and their impact on fish muscle proteins and texture. The studies have been performed using Atlantic salmon (Salmo salar) and cod (Gadus morhua) subjected to different treatments and storage conditions. The textural properties were very different in the two species. Salmon fillets were significantly softer and less resilient than cod fillets, and the properties changed somewhat differently during storage experiments. Different proteolytic enzymes have been reported to participate in muscle softening. Some of these enzymes were investigated, and specific proteolytic activities were detected throughout the storage periods. Collagenase-like enzymes seem to be the most important for cod muscle texture. Microorganisms and/or microbial enzymes seem not to be important for changes in salmon muscle texture. Results suggest that the cathepsin B-like enzymes are important for salmon texture. The activities of the proteolytic enzymes may be greatly affected by the muscle pH, and by the treatment(s) the fish are subjected to. In any case, changes caused by differences in proteolytic activities may need some time to be detectable or have significant impact on fish quality. When cod fillets are stored in ice, it is highly recommended to keep the temperature low. Even a relatively mild temperature abuse was sufficient to result in less favorable textural characteristics, and make the fillets seem older than their days of storage. Salmon fillets are often subjected to cold-smoking. The smoking temperature was important for the solubility properties of the muscle proteins, and for their composition, but did not affect the proteolytic activity. The effects of the processing parameters were most important early in the product’s shelf life, as the differences caused by the different smoking temperatures were reduced by further storage of the smoked samples. / Paper II and III are reprinted with kind permission of Elsevier, sciencedirect.com

The increase of obesity that we have experienced during the last decades and its association with insulin resistance, type 2 diabetes and other metabolic diseases has resulted in an enormous interest for understanding the mechanisms underlying these disorders. Tissue inflammation triggered by food with a high glycemic index has been suggested to be an important mediator in the development of insulin resistance. Despite great research efforts lately, more research is needed in order to understand how nutrients interact with the genetic factors that control and triggers the inflammatory responses. The composition of macronutrients in a diet influences the levels of insulin secretion in the body. Besides controlling the blood glucose concentration, insulin also regulates a range of inflammatory processes. Inflammation is largely dependent on some small cell-signaling molecules called cytokines, as these activate a wide range of inflammatory-related genes. The objective of this study is to explore the regulatory effects of insulin and cytokines on the transcription of the following selected genes related to inflammation; D5D, D6D, SCD and FOXO3A. In addition, expression of TRAIL, BTG1 and TWIST1 is studied as they all are target genes for FOXO3A, and related to inflammatory processes and/or glucose metabolism. Quantitative-PCR was used to study mRNA expression of relevant genes in THP-1 cells treated with insulin and cytokines. As the investigation was performed on THP-1 monocytes, it was necessary to optimize the in vitro conditions in order to obtain a maximal response from the insulin and cytokine treatments. The concentration of insulin was an important factor in this study, because the regulation of FOXO3A and desaturases (D5D, D6D and SCD) mRNA expression seemed to be dose-dependent. The treatment period was also critical, as a set of time-course experiments revealed that FOXO3A and the desaturases were regulated by insulin and cytokines at different time-points. In this study, THP-1 cells treated with insulin and/or cytokines revealed significant regulations of the relevant genes. Gene expression of D5D, D6D and SCD was significantly up-regulated in response to insulin. Furthermore, mRNA expression of the transcription factor FOXO3A was significantly down-regulated by insulin, IL-1&amp;#946; and TNF-&amp;#945;. However, neither FOXO3A nor the desaturases were cooperatively regulated by these stimulating factors. TRAIL, TWIST and BTG1 on the other hand, were significantly up-regulated in a synergistic manner when cells were treated with a combination of insulin, IL-1&amp;#946; and TNF-&amp;#945;. The observed regulation of gene expressions in THP-1 monocytes treated with insulin and cytokines suggests that insulin may affect the regulation of inflammatory related genes in circulating human monocytes. As insulin is secreted in the bloodstream followed by elevated levels of glucose after a meal, these results may reflect possible diet-induced changes in gene expression.

Lite er kjent i forhold til proteiners interaksjon med alginat, noe som er viktig å ha kjennskap til ved bruk av biopolymeren som immobiliseringsmateriale for celler og proteiner. Denne oppgaven er derfor en studie av alginat – protein interaksjoner. En interaksjon med albumin, fibrinogen og insulin ble undersøkt, da proteinene har relevans i forhold til release systemer av proteiner, og en immunrespons mot alginatkuler ved celletransplantasjon. Lysozym ble brukt som positiv kontroll, ettersom proteinet er kjent å interagere med alginat.Ettersom alginat er negative ladet er det naturlig å tro at elektrostatiske krefter har betydning for polymerens vekselvirkning med andre molekyler. For å undersøke elektrostatiske krefters innflytelse på alginat – protein interaksjoner, ble proteinenes elektrostatiske overflatepotensial modellert ved hjelp av Adopter Poisson-Boltzmann Solver (APBS). Isoterm titreringskalorimetri (ITC) ble videre brukt for å måle vekselvirkningsvarmen ved interaksjon mellom proteiner og alginat i løsning. Proteinbinding til alginatkuler ble kvantifisert ved konsentrasjonsmålinger på nanodrop og mikroplateleser, og visualisert i mikroskop. Det ble tatt utgangspunkt i fysiologiske betingelser (pH = 7 og ionestyrke = 160mM), slik at forholdene samsvarte med det alginatkuler opplever ved transplantasjon.Ved ITC interagerte proteinene med alginat etter synkende styrke: lysozym (positivt ladet) >> albumin (negativt ladet) > fibrinogen (negativt ladet). Albumin og fibrinogen viste å interagere svakt med alginat, til tross for deres negative ladning. Dette kan forklares ved at proteinene har en stor overflate, med enkelte positive regioner selv ved pH > pI. Den høye ionestyrken brukt i studiet bidrar til å redusere de tiltrekkende kreftene mellom molekylene. Ingen målbar interaksjon ble observert mellom alginat og insulin (negativt ladet), noe som kan forklares ved at proteinet har en liten overflate med få positive regioner. Proteinbinding til alginatkuler viste samme trend som ITC, noe som indikerer at alginatets elektrostatiske egenskaper i gel og løsning er den samme. Binding av lysozym ga kulekollaps ved høye proteinkonsentrasjoner på grunn av kondensering til gelnettverket i alginat. Den svake bindingen av albumin og fibrinogen påvirket ikke kulenes stabilitet.Studiet indikerer at elektrostatiske tiltrekkende krefter har betydning ved alginat – protein interaksjoner, og at proteinenes overflateladning har betydning for deres interaksjon med alginat. APBS viste å gi et bra bilde på proteinenes mulige interaksjon med alginat, ettersom det var bra korrelasjon mellom antatt og observert interaksjon. Dette kan forklares ved at APBS implementerer eksperimentelle forhold (pH, ionestyrke etc.) som har betydning for proteinenes elektrostatiske overflatepotensial. De eksperimentelle metodene brukt i studiet ga i flere tilfeller bare et kvalitativt mål på proteinenes interaksjon med alginat. Dette skyldes mest sannsynlig den svake (nanodrop og plateleser) og uspesifikke (ITC) interaksjonen som ble observerte.I videre arbeid vil det være interessant å se nærmere på adsorpsjon og absorpsjon av proteiner til alginatkuler eksponert for helblod, et modellsystem som samsvarer mer med det alginatkuler opplever ved transplantasjon. Hvordan proteinsammensetningen på kuleoverflaten endres over tid er også av interesse, ettersom proteinbinding ikke er en statisk prosess.

The most common cancer among women in Europe and the United States is breast cancer. However, treatment remains a major challenge. If prediction of prognosis and treatment response become more reliable, treatment can be improved. An approach to overcome this challenge is to establish a technique for identification of subtypes. Metabolite profiling of breast cancer has shown potential to identify subtype. The aim was divided into three parts, including metabolite extraction and profiling and isotope labelling of metabolites.The first aim was to optimise a method for extraction of polar and non-polar metabolites for mass spectrometry-based analysis. To achieve the aim, beads-based homogenisation of xenograft tissue in 60% methanol solution and in chloroform was performed in a series of experiments. Requirements were fulfilled, including complete extraction, simple performance and high reproducibility. Method was therefore stated successfully optimised.The second aim was to obtain and compare metabolite profiles of luminal-like and basal-like subtypes of breast cancers. To achieve the aim, polar metabolites were extracted from xenograft tissue using the optimised method prior to gas chromatography mass spectrometry (GC-MS) analysis. Profiles comprising more than 30 metabolites and their concentration were obtained. For comparison of subtypes, data analysis including log2 ratios, principal component analysis (PCA) and Student&apos;s t-test were used. All data analyses indicated differences in metabolite concentrations between subtypes. 15 metabolites were found by Student&apos;s t-test to significantly differ in concentration between subtypes, including lactate, glycine, citrate, lysine and aspartate. Therefore, metabolite profiling is a potential tool for identification of subtype. Furthermore, metabolites shown to significantly differ may provide insight into metabolic changes in breast cancers that remain poorly understood. The third aim was to investigate metabolic pathways in luminal-like subtype, possessing reduced tumour growth rates due to treatment. To achieve the aim, 13C-labelled glucose was injected into xenograft models prior to tumour excision, extraction, GC-MS analysis and calculation of summed fractional labelling (SFL). 8.3% lactate, 2.2% citrate and 1.6% fumarate were found labelled using SFL. 13C labelling was therefore shown retained throughout glycolysis, to enter the tricarboxylic acid cycle (TCA) cycle and to give rise to TCA intermediates.

Doxorubicin is a topoisomerase-targeting anthracycline that is one of the most effective anticancer drugs currently known. However, its clinical use is restricted by cardiotoxicity and the development of drug resistance. The main goal of this thesis has been to increase the knowledge of doxorubicin mechanism in addition to evaluate if predictive biomarkers for doxorubicin response could be identified. A total of 128 tumor samples collected from breast cancer patients before and after neoadjuvant treatment with doxorubicin were studied. mRNA expression level in tumor tissue was assessed using whole-genome mRNA microarray analysis (Agilent Human GE 4x44K microarray).More than 5000 genes were found to be up- and down regulated following doxorubicin treatment. The molecular and cellular functions as well as canonical pathways found to be enriched in the list of genes up regulated after doxorubicin exposure were involved in among other cardiovascular system development and function, cellular movement and immune responses. p53 was found to be the transcription factor regulating the highest number of target molecules within the list of up regulated genes. RNA processing, splicing and translation were shown to be overrepresented in the list of down regulated genes. The association between doxorubicin response and changes in gene expression revealed several genes such as CTGF, ITGB4 and IGF1 to be up regulated in the samples collected from patients characterized with a partial response to doxorubicin compared to those with minimal change and/or stable disease following treatment. In addition, the gene expression profiles between samples having wild type compared to mutated TP53 were studied, and a lower induction of expression were found for several genes such as FGF9 and COL11A2 in the samples having a mutated p53. This study showed that the gene expression profile in breast cancer tumors is altered as a response to doxorubicin exposure. Identifying genes significantly altered after therapy and associate their change with response to treatment may help identify the subgroup of patients benefitting from doxorubicin treatment. Patients with little or no effect of treatment could receive alternative therapy and be spared unnecessary treatment and risk of side effects.

Breast cancer is the most common cancer type amongst women, and closer to 3000 women in Norway will be diagnosed with this disease in 2012. Although major improvements have been achieved in the treatment, and thus the outcome, of breast cancer patients in the past years, little has been accomplished for those with an advanced disease. B7-H3 is an immunoregulatory protein, and its overexpression has been associated with advanced disease and poor prognosis in breast cancer. A previous study has shown that B7-H3 silencing increased Paclitaxel sensitivity in B7-H3 expressing breast cancer cell lines. Resistance to treatment is a general challenge in systemic management of advanced breast cancer, and increased knowledge about the molecules and pathways involved in this process is important in order to improve the outcome for these patients.To further study the function of B7-H3 and its putative involvement in lack of treatment response in breast cancer, we compared the efficacy of 22 different anti-cancer drugs in two B7-H3 expressing triple negative metastatic breast cancer cell lines, MDA-MB-435 and MDA-MB-231, and their B7-H3 silenced counterparts. In particular two drugs targeting the PI3K/Akt pathway, API-2 and Everolimus, showed a significantly better efficacy in the B7-H3 silenced cells. To elucidate the cellular mechanisms involved in the observed sensitization in the B7-H3 knockdown cells, we performed Western blot analysis on several proteins in the PI3K/Akt/mTOR pathway. The cells that did not express B7-H3 had lower levels of both phospho-Akt and the downstream signaling molecule phospho-p70S6K following drug exposure, indicating B7-H3 associated the regulation of proteins in this pathway. This, together with the previously observed relationships between B7-H3 expression in metastasis and chemoresistance, suggest that this protein might be a therapeutic marker to increase the effect of current anti-cancer treatment, although the specific roles of B7-H3 in this context need to be investigated further.

Recombinant protein production in Escherichia coli provides a cheap and efficient way of producing medically and industrially relevant proteins. Sequence features of individual genes and especially their 5&#146; terminal coding sequences act on the efficiency of gene expression by complex regulatory mechanisms which are still not fully understood. This study aimed to investigate the features of the 5&#146; coding region of recombinant mRNAs, and to optimize them for increased expression in E. coli. A previous study had found that a synonymous change of the bla reporter gene 2nd codon leads to an increased expression, and accordingly a synonymous library in the 5&#146; bla coding sequence was created by a directed evolution approach building on this feature. Variants conferring up to three-fold increases in active enzyme amounts were identified, and the increased expression was shown to stem from increased transcriptional efficiency. The effect of changing the 2nd codon synonymously was further investigated by synonymous substitutions of the 2nd codons of the bla and two other reporter genes, phoA and celB. These experiments showed that the effect of 2nd codon changes on the gene expression is determined by the sequence context, as changes in expression levels appeared to be gene specific. All the coding sequences of the study were also analysed in silico, and an application for calculating the tRNA adaptation index was programmed in Python and made freely available online.As the synonymous codon changes did not lead to a great improvement in protein amounts and any sequence features affecting the expression were hard to pinpoint, an alternative strategy involving 5&#146; terminal gene fusions was investigated. Combinatorial mutagenesis coupled to an effective screening technique was applied to further optimize a 5&#146; terminal fusion partner, previously shown to improve expression of several eukaryotic genes. The application of the best identified fusion partner candidate yielded a 3.8-fold improvement in IFN-&amp;#945;2b protein amounts over the original fusion, and showed twice as high protein amounts than a pelB-IFN-&amp;#945;2b fusion previously proven to give industrial expression amounts. The developed peptide fusion is thus an eligible candidate for further development for use in heterologous protein production.