digitalisering@umu.se

Genetic polymorphism is by definition the occurrence in the same population of two or more alleles at one locus,each with a frequency high enough not to be maintained by recurrent mutation only (1). Among the human plasmaproteins two major categories of polymorphism have been described, allotypic and electrophoretic heterogeneity. Allotypy is defined by Oudin as individual antigenic differences among proteins within a species (2). The first discovered polymorphism of this category was the Gm system of immunoglobulin G by Grubb (3). The first described electrophoretic heterogeneity in plasma proteins was theHp (haptoglobin) system discovered by Smithies (4). Sincethen genetic variants of several other human plasma proteins have been found. This dissertation is concerned with thegenetic and immunological aspects of the polymorphism of the third component of human complement, C3. / digitalisering@umu.se

Cerebral stroke has become one of the leading causes of death and disability worldwide. During an ischemic stroke, oxygen and nutrient deprivation occurs, which combined lead to cell starvation, anoxia, and eventually cell death. However, when blood flow is restored, reperfusion damage occurs resulting in increased cell death through several mechanisms. One of the main reasons behind ischemia/reperfusion damage is oxidative stress due to elevated production of reactive oxygen species (ROS) during reperfusion. There are several proteins and processes that are thought to be involved in elevated oxidative stress and the formation of ROS during reperfusion, among which the NADPH oxidase (Nox) family is suggested to be the main contributor of ROS.To examine this hypothesis, in the present work, we inhibited activity of the Nox2 and Nox4 enzymes during ischemia/reperfusion with the Glucox Biotech AB (Sweden) inhibitors M4, M107, and M114 to evaluate whether reducing Nox activity could reduce the ischemia/reperfusion-induced cell death, hence be used as a potential stroke treatment, the cell viability was measured with MTS after ischemia/reperfusion induction and treatment with the Nox substances. We also examined the gene expression levels of the Nox enzymes Nox2 and Nox4 with qPCR after induced ischemia/reperfusion in the neuroblastoma cell line NB69.Our results showed a decrease in Nox4 gene expression after 1h ischemia/8h reperfusion and an increased expression after 1h ischemia/24h reperfusion in NB69 cells. Treatment with M114 resulted in increased cell viability after 2h ischemia/72h reperfusion. However, the toxic effect of ischemia/reperfusion-induced response was found to be inadequate, as indicated by extensive proliferation and lack of cell death. This unfavorable outcome is suggested to be excess of oxygen in medium, metabolization of L-glutamine, and effects of growth factors in the serum used in cell culture medium during the ischemic phase. Therefore, the cell culture protocol was modified to the use of PBS instead of glucose-free medium under serum-free condition during the ischemia. The altered ischemic conditions resulted in continuous reduction in cell viability at increasing ischemic time points with total cell death at 2h ischemia, suggesting applicable conditions for ischemia/reperfusion studies. Even though a conclusion could not be made about the inhibitors M4, M107, and M114 as the cell viability assay was performed under insufficient conditions; the Nox inhibitors shows high potential as future ischemic stroke treatments, which may help save lives and improve life quality for affected patients.

Lignocellulosic biomass has potential to chip in the chemical and biofuels supplies in future societies,even though lignocellulose is a recalcitrant structure that has to be treated in several steps. After theirproper life cycle, wood-derived materials such as particleboards have few outcomes today apart fromenergy recovery for heat production. Then, they may be used as lignocellulosic biomass sources in theproduction of molecules of interest. Fermentation from wood-derived monosaccharides imposespreliminary sugar retrieval, for instance through pre-treatment and enzymatic hydrolysis. This studyfocuses on the potential of particleboards waste for chemical and biofuel production by comparingsaccharification through simulated steam explosion pre-treatment and enzymatic hydrolysis betweennative and particleboard-derived wood, with an insight in subsequent fermentation by Saccharomycescerevisiae. Urea-Formaldehyde bound particleboard was investigated, as well as some aspects ofMelamine-Urea-Formaldehyde bound particleboard.Pre-treatment resulted in apparition of lignocellulosic degraded compounds in a much larger extent innative wood than in particleboard, which seemed to be only superficially impacted. Formation ofdegraded compounds from sugars – furfural and 5-hydroxymethylfurfural – was enhanced when pretreatmentwas prolonged. Removal of a substantial fraction of the adhesive contained in theparticleboards was observed, leading to comparable concentrations in free urea, its degradedproducts, and formaldehyde between native wood and particleboards during enzymatic hydrolysis.Enzymatic hydrolysis with cellulases and hemicellulases highlighted a critical role of pre-treatment toenhance final yields, both in native wood and in Urea-Formaldehyde particleboard. Adding 20 minutessteam-explosion type pre-treatment at 160 °C resulted in glucose yields increase from 18.5 % to 32.8% for native wood and from 15.6 % to 37.4 % for particleboard. Prolonging pre-treatment residencetime to 35 minutes resulted in much better glucose extraction for native wood but only slight progressfor the particleboard, as glucose yields reached 64.5 % and 41.1 % respectively. Maximalconcentrations achieved were 277 and 184 mg/gbiomass respectively.Fermentation brought to light high inhibition from both native wood and particleboard sources ofmedia, which were attributed to components or degraded products of lignocellulose that were notanalysed in this project. Ethanol was formed during fermentation, with reduced productivity butincreased yields as compared with the control sample. Inhibition was so strong that no difference couldbe given between native and particleboard wood. In this situation, no inhibition potential of resin orits degradation products could be proved. / Lignocellulosic biomassa har potential att bidra till kemikalier och biobränsletillförsel i framtidasamhällen, trots att lignocellulosa är en rekalcitrant struktur som måste behandlas i flera steg. Idagträmaterial som spånskivor bara används för energiåtervinning och värmeproduktion efter deraslivscykel. De kan därför användas som råvara för framställning av värdefulla molekyler.Fermenteringsprocesser behöver frisättningen av trä monosackarider genom förbehandlingsprocesseroch enzymatisk hydrolys. Studien fokuserar på potentialen för avfall från spånskivor för kemisk ochbiobränsleproduktion. Vi har jämfört sackarifiering mellan nativt trä och spånskivor genom simuleradångaxplosion och enzymatisk hydrolys, med en inblick i efterföljande fermentering av Saccharomycescerevisiae. Spånskivor bunden av urea-formaldehyd undersöktes, liksom vissa aspekter av spånskivorbundna med melamin-urea-formaldehyd.Förbehandlingen producerade högre koncentration av lignocellulosa nedbrytningsprodukter frånnativt trä jämfört med spånskivor. Bildningen av nedbrytningsprodukter från sockerarter - furfural och5-hydroxymethylfurfural - ökade med längre förbehandlingar. En väsentlig fraktion av limmet borttogsfrån spånskivorna, vilket ledde till jämförbara koncentrationer i fri urea, dess nedbrytningsprodukteroch formaldehyd mellan naturligt trä och spånskivor under enzymatisk hydrolys.Enzymatisk hydrolys med cellulaser och hemicellulaser avslöjade den kritiska rollen av förbehandlingför att förbättra utbytet, både i naturligt trä och i urea-formaldehyd spånskiva. Längre (20 minuter)ångexplosion vid 160° C resulterade i högre glukosutbytet (från 18,5% till 32,8% för naturligt trä ochfrån 15,6% till 37,4% för spånskivor). Förlängning av uppehållstiden före behandlingen till 35 minuterresulterade i mycket bättre glukosekstraktion för nativt trä (64,5%) men endast liten framsteg förspånskivan (41,1%). Detta resulterade i maximalt utbyte av 277 mg Glc/g biomassa och 184 mg Glc/ gbiomassa för nativt trä och spånskivor, respektive.Fermentering visade hög hämning från lignocellulosa nedbrytningsprodukter som inte analyserades iprojektet för både nativt trä och spånskällor för media. Etanol bildades under fermentering medreducerad produktivitet men ökade utbyten jämfört med kontrollprovet. Hämningen var så stark attingen skillnad kunde ges mellan naturligt trä och spånskivor. I denna situation kunde ingenhämningspotential för lim eller dess nedbrytningsprodukter bevisas.

Lignocellulosic biomass has potential to chip in the chemical and biofuels supplies in future societies,even though lignocellulose is a recalcitrant structure that has to be treated in several steps. After theirproper life cycle, wood-derived materials such as particleboards have few outcomes today apart fromenergy recovery for heat production. Then, they may be used as lignocellulosic biomass sources in theproduction of molecules of interest. Fermentation from wood-derived monosaccharides imposespreliminary sugar retrieval, for instance through pre-treatment and enzymatic hydrolysis. This studyfocuses on the potential of particleboards waste for chemical and biofuel production by comparingsaccharification through simulated steam explosion pre-treatment and enzymatic hydrolysis betweennative and particleboard-derived wood, with an insight in subsequent fermentation by Saccharomycescerevisiae. Urea-Formaldehyde bound particleboard was investigated, as well as some aspects ofMelamine-Urea-Formaldehyde bound particleboard. Pre-treatment resulted in apparition of lignocellulosic degraded compounds in a much larger extent innative wood than in particleboard, which seemed to be only superficially impacted. Formation ofdegraded compounds from sugars – furfural and 5-hydroxymethylfurfural – was enhanced when pretreatmentwas prolonged. Removal of a substantial fraction of the adhesive contained in theparticleboards was observed, leading to comparable concentrations in free urea, its degradedproducts, and formaldehyde between native wood and particleboards during enzymatic hydrolysis.Enzymatic hydrolysis with cellulases and hemicellulases highlighted a critical role of pre-treatment toenhance final yields, both in native wood and in Urea-Formaldehyde particleboard. Adding 20 minutessteam-explosion type pre-treatment at 160 °C resulted in glucose yields increase from 18.5 % to 32.8% for native wood and from 15.6 % to 37.4 % for particleboard. Prolonging pre-treatment residencetime to 35 minutes resulted in much better glucose extraction for native wood but only slight progressfor the particleboard, as glucose yields reached 64.5 % and 41.1 % respectively. Maximalconcentrations achieved were 277 and 184 mg/gbiomass respectively. Fermentation brought to light high inhibition from both native wood and particleboard sources ofmedia, which were attributed to components or degraded products of lignocellulose that were notanalysed in this project. Ethanol was formed during fermentation, with reduced productivity butincreased yields as compared with the control sample. Inhibition was so strong that no difference couldbe given between native and particleboard wood. In this situation, no inhibition potential of resin orits degradation products could be proved. / Lignocellulosic biomassa har potential att bidra till kemikalier och biobränsletillförsel i framtidasamhällen, trots att lignocellulosa är en rekalcitrant struktur som måste behandlas i flera steg. Idagträmaterial som spånskivor bara används för energiåtervinning och värmeproduktion efter deraslivscykel. De kan därför användas som råvara för framställning av värdefulla molekyler.Fermenteringsprocesser behöver frisättningen av trä monosackarider genom förbehandlingsprocesseroch enzymatisk hydrolys. Studien fokuserar på potentialen för avfall från spånskivor för kemisk ochbiobränsleproduktion. Vi har jämfört sackarifiering mellan nativt trä och spånskivor genom simuleradångaxplosion och enzymatisk hydrolys, med en inblick i efterföljande fermentering av Saccharomycescerevisiae. Spånskivor bunden av urea-formaldehyd undersöktes, liksom vissa aspekter av spånskivorbundna med melamin-urea-formaldehyd. Förbehandlingen producerade högre koncentration av lignocellulosa nedbrytningsprodukter frånnativt trä jämfört med spånskivor. Bildningen av nedbrytningsprodukter från sockerarter - furfural och5-hydroxymethylfurfural - ökade med längre förbehandlingar. En väsentlig fraktion av limmet borttogsfrån spånskivorna, vilket ledde till jämförbara koncentrationer i fri urea, dess nedbrytningsprodukteroch formaldehyd mellan naturligt trä och spånskivor under enzymatisk hydrolys. Enzymatisk hydrolys med cellulaser och hemicellulaser avslöjade den kritiska rollen av förbehandlingför att förbättra utbytet, både i naturligt trä och i urea-formaldehyd spånskiva. Längre (20 minuter)ångexplosion vid 160° C resulterade i högre glukosutbytet (från 18,5% till 32,8% för naturligt trä ochfrån 15,6% till 37,4% för spånskivor). Förlängning av uppehållstiden före behandlingen till 35 minuterresulterade i mycket bättre glukosekstraktion för nativt trä (64,5%) men endast liten framsteg förspånskivan (41,1%). Detta resulterade i maximalt utbyte av 277 mg Glc/g biomassa och 184 mg Glc/ gbiomassa för nativt trä och spånskivor, respektive. Fermentering visade hög hämning från lignocellulosa nedbrytningsprodukter som inte analyserades iprojektet för både nativt trä och spånskällor för media. Etanol bildades under fermentering medreducerad produktivitet men ökade utbyten jämfört med kontrollprovet. Hämningen var så stark attingen skillnad kunde ges mellan naturligt trä och spånskivor. I denna situation kunde ingenhämningspotential för lim eller dess nedbrytningsprodukter bevisas.

Sepsis is aggressive and severe inflammatory body response to an infection and is considered to be one of the most common death causes in patients. The current diagnosis of sepsis is not fast enough to help those who get sepsis, due to its fast progression. The current golden standard for sepsis diagnosis is blood culturing. However, the biggest downside of it is the long time. Research is now focused on finding a faster way to diagnose sepsis on early stages. The most promising one tends to be the usage of biomarkers. Today, there are 260 defined sepsis biomarkers, however, only few of them are clinically used. Among them, C-reactive protein, and procalcitonin. Another potential biomarker could be miRNAs. The research about that today is at early stage. To use miRNAs as biomarkers, they need to be quantified. One way to quantify miRNAs is the two-tailed RT-qPCR method together with absolute quantification. This study focused on evaluating the best extraction method of small RNA for later quantification of specific miRNA. The blood plasma from healthy donors was divided into spiked and non-spiked samples, where the synthetic miRSeps-3 served as a spike-in positive controls. All samples were extracted using two methods, manual and robotic with Qiacube (Qiagen). Absolute quantification was applied to quantify miRNA in all samples. The successful results indicated that the two-tailed RT-qPCR was sensitive enough. More optimization is required for the methods, however, the whole method has a good potential to become helpful for clinical usage in the future

digitalisering@umu

Detection of circulating tumor cells (CTC) in peripheral blood is indicative of early recognition of tumor progression and such an important biomarker for early diagnosis, staging, monitoring and prognosis of cancer. However, CTC are found in very low concentrations and reliable isolation of these rare cells is challenging. Microfluidics enables precise manipulation of fluids and cells and is ideal for cell sorting methods for clinical diagnostics. The thesis contributes towards the development of microfluidic based CTC isolation methods from peripheral blood. The methods are based on size and immunoaffinity. The first part of the thesis describes the phenomenon of inertial focusing for size based cell separation at high throughputs. In paper 1, we demonstrate continuous filtration of leukocytes from diluted blood, with an efficiency of 78% at a flow rate of 2.2ml/min. In the paper 2, separation of total and subpopulation of leukocytes with a purity of 86% for granulocytes and 91% for lymphocytes is demonstrated. Furthermore, cancer cells spiked into whole blood could be separated at a yield of 88%. Finally, in paper 3 and 4 we unravel parts of the unexplored elasto-inertial microfluidics and was utilized to precisely focus the cells, as part of an integrated optofluidic micro flow cytometer device, capable to simultaneously measure fluorescence and scattering of cells and particles at a rate of 2500 particles/sec (paper 4). Second part of the thesis focuses on acoustophoresis. In (paper 5), a multifunctional acoustic microdevice was developed for isolation of cancer cells from red blood cells with a separation efficiency of 92.4% and trapping efficiency of 93%. In (paper 6), microbubbles activated acoustic cell sorter was developed for affinity based cell separation. As a proof of principle, cancer cells in a suspension were separated at an efficiency of 75%. In the third part, using cellulose nano fibrils (paper 7), we demonstrate efficiently capture and release of cancer cells at a release efficiency of 95%. Finally, a novel, single step self-assembly of spider silk proteins is introduced inside microfluidic channels for effective capture of cancer cells with 85% capture efficiency and subsequent release of captured cells with 95% release efficiency (paper 8). The novel recombinant silk modified microfluidic device was validated using pancreatic cancer patients. In summary, we have developed different microfluidic based isolation technologies for the capture and characterization of CTC. / <p>QC 20170321</p>

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

Sulforafan är en isotiacyanat som beskrivs ha effektiva cancerpreventativa egenskaper. Kemikalien görs tillgänglig för människan genom konsumtion av korsblommiga grönsaker så som broccoli och grönkål. Cancer är vanligt, och i Sverige räknar man med att var tredje människa kommer att drabbas under sin livstid. I följande litteraturstudie var syftet att sammanställa information om på vilket sätt sulforafan påverkar koloncancerceller, samt söka evidens för att konsumtion av sulforafanrika grönsaker bidrar till minskad risk att drabbas av koloncancer. Sulforafan har cancerpreventativa egenskaper i cellkultur så som inhibering av histondeacetylas-aktivitet, inducering av cellcykelarrest och apoptos och minskad proliferation hos cancercellerna. Det minskar även uttryck av gener som är inblandade i angiogenes.Det finns inte nog med evidens om broccolikonsumtion, på grund av sitt höga innehåll av sulforafan, skulle vara cancerpreventativt hos människan. / Sulforaphane is an isothiocyanate that is described as having chemopreventative effects. The phytochemical is made available to humans by dietary consumption of cruciferous vegetables such as broccoli and kale. Cancer is a common disease, and in Sweden it is estimated that one in three will be diagnosed with cancer during their lifetime. This review study aims to summarize the effect of sulforaphane on human colon cancer cells, and seek evidence that consumption of cruciferous vegetables reduces the risk of developing colon cancer. Sulforaphane is considered chemopreventative in vitro through inhibition of histone deacetylas activity, induction of cell cycle arrest and apoptosis and through reduction of cell proliferation. It has also been shown to reduces expression of genes involved in angiogenesis.There is not enough evidence to confirm that dietary broccoli consumption, through its high content of sulforaphane, would be chemopreventative.