Hundens domesticering är en av de äldsta och tros ha startats 35000 f.n. Under domesticeringen från varg till hund, har hunden utvecklat både morfologiska och beteendemässiga skillnader så som bredare snot, högre skallar och minskat risktagande. Många av dessa skillnader tros bero på skillnader i aktiva regulatoriska regioner mellan arternas genom. Syftet med detta examensarbete är att kartlägga genomiska interaktioner för hjärnvävnader hos både hund och varg för att identifiera regulatoriska skillnader mellan arterna. Förhoppningsvis kan detta leda till nya insikter i genomiska skillnader som utvecklats under hundens domesticering. För att jämföra arternas regulatoriska regioner användes metoden Capture Hi-C (HiCap) på vävnadsprover av både hypotalamus och prefrontala cortex för varg och hund. HiCap är en metod utvecklad från chromosome conformation capture-metoden 3C. I HiCap så fixeras interagerande delar av DNA:t så att promotor-enhancerinteraktioner förblir. Dessa interagerande regioner ligeras sedan samman och biblioteksbereds för sekvensering. Genom sekvensering fastställs vilka promotorer som aktivt regleras av enhancers i cellkärnan. Hi-C bibliotek förberedes för alla vävnader för båda arterna. I vissa av biblioteken upptäcktes längre DNA-fragment som kan renas bort. På grund av avsaknad av probes för sequence capture så kunde laborationen ej fullföljas och därmed inga särskilda resultat erhållas. Om laborationen fullföljs kan resultaten förhoppningsvis ge nya epigenetiska insikter i hundens domesticering. / The domestication of dog to wolf started around 35000 BP and is believed to be the oldest domestication event among both plants and animals. During this event, dogs have developed differences in morphological and behavioural traits to their ancestors, such as wider snouts, higher skulls, and lower tendencies to taking risks. It is now suggested that many of these differences can be explained by differences in active regulatory regions. The main objective of this thesis is to map chromatin interactions in the genome of wolf and dog brain tissues to annotate regulatory variants between the canine species. This will hopefully provide novel information regarding genomic changes mediating traits gained through domestication. We will perform Capture Hi-C (HiCap) on tissue samples of hypothalamus and prefrontal cortex of wolf and dog. HiCap is a method derived from the chromosome capture method 3C. In HiCap, the interacting regions of the DNA are crosslinked, ensuring that promoter-enhancer interactions will not be lost. These interacting regions are then ligated together, followed by sequencing library preparation. Subsequently, sequencing of these libraries will provide information of which promoters are actively regulated by enhancers in the nucleus. We successfully prepared Hi-C libraries for all tissues and animals. However, there were longer fragments in some libraries which can be removed. Due to lack of necessary probes for sequence capture, the laboratory work was cut short and no major results were obtained. By continuing the laboratory work, hopefully these libraries will result in novel insights in the domestication of the dog.

Bovint β-laktoglobulinprotein (bLG) är ett litet globulärt protein med 162 aminosyrarester, som vanligtvis finns i mjölkvassle. Under sura förhällanden dissocierar dessa dimera proteiner och bildar amyloidliknande fibriller. Studien av β- laktoglobulinfibriller kan vara ett värdefullt verktyg för att förstå strukturen och dynamiken hos patogena amyloidproteiner och relaterade sjukdomar (t.ex. Alzheimers). Det är dessutom viktigt att förstå den korrekta formationen och elucideringen av dessa protein-nanofibriller (PNF) och deras sammansättningutgör också en grund för vidare design av nya biobaserade material. Således är framställningen av en signifikant homogen morfologi av nano-fibriller från bLG för proteinstrukturstudier huvudsyftet med detta projekt. Studien omfattar också användning av rekombinant β-laktoglobulin renat från Escherichia coli Origami (DE3) - celler. Omfattningen av bildandet av dessa PNF kan påverkas genom att variera experimentets olika förhållanden. Huvudsyftet med denna avhandling är att modifiera reaktionsparametrarna såsom inkubationstid, temperatur, koncentrationer, såningsanalyser och även hitta nya för att maximera homogeniteten hos den beredda PNF. En detaljerad analys av alla effekter av olika förhållanden på reaktionsprocessen och vilken provtyp eller beredningsprocess som leder till ökad mängd fibriller är huvudresultatet av denna avhandling. Detta kan i sin tur fungera som en bas för framtida modeller eller proteiner som kan användas för att få en bättre förståelse för amyloidrelaterade patologier eller andra associerade applikationer, till exempel i livsmedelsindustrin eller design av nya material. I slutet av studien visade det sig att den högsta mängden fibriller bildades för de prover som inkuberades vid 70 ℃, förvarades i 48 timmar, vid 300 rpm, med 10 % fröprov som sonikerades två gånger med ett intervall på 60 minuter . / Bovine β-lactoglobulin protein (bLG) is a 162 residue small globular protein, usually found in the whey component of milk. These dimeric proteins under acidic conditions and high temperatures dissociates and form amyloid-like fibrils. The study of bLG fibrils can be a valuable tool for understanding the structure and dynamics of pathogenic amyloid proteins and related diseases (e.g., Alzheimer’s). Also, proper formation and elucidation of these protein nano-fibrils (PNF’s) and their assembly provides a foundation for further design of new bio-based materials. Thus, producing a significant homogenous morphology of the nano-fibrils from bLG for protein structure study is the main objective of this project. The study involves also the use of recombinant β- lactoglobulin purified from Escherichia coli Origami (DE3) cells. The extent of formation of these PNF’s can be influenced by varying the different conditions of the experiment. The main aim of this thesis is to modify the reaction parameters such as the incubation time, temperature, concentrations, seeding assays and also find new ones so as to maximize the homogeneity of the prepared PNF. A detailed analysis of all the effects of different conditions on the reaction process and which sample type or preparation process leads to increase in the amount of fibrils is the main outcome of this thesis. This can in turn serve as a base for future models or proteins that can be used to gain a better understanding of amyloid-related pathologies or any other associated applications such as in food industries or design of new materials. In the end of the study, it was found that highest amount of fibrils were formed for those samples incubated at 70 ℃, kept for 48 hours, at 300 rpm, with the 10 % seed sample that was sonicated twice at an interval of 60 minutes.

Since the first human spaceflight in 1961, hundreds of humans have been in space. Microgravity and high radiation are the main spaceflight hazards. The space environment is known to impact several aspects of human health, such as bone density and cognitive performance. However, the effects of long­duration spaceflights on a cellular and molecular level, utilizing biosamples and multiomic approaches, is poorly studied. In this project, the method Spatial Transcriptomics has been utilized to compare brain tissue from the hippocampus region of mice that have been in space with a control group of mice that have stayed on Earth. Spatial Transcriptomics allow for the quantification of gene expression, while maintaining the spatial information of the transcriptome. The results of this study suggest that spaceflights cause mitochondrial stress.   This thesis work is part of a more extensive study in collaboration with NASA, and more studies will be conducted to investigate the effects of spaceflights further. If these findings are confirmed, medicines used on Earth to treat patients with mitochondrial dysfunction could increase the well­being of astronauts in space. / Sedan den första människan skickades till rymden år 1961, har hundratals astronauter lämnat jordens atmosfär.   De mest signifikanta hälsoriskerna i rymden är mikrogravitation och hög strålning och rymdmiljön har stor påverkan på oss. Exempelvis upplever astronauter ofta minskad benmassa och nedsatt kognitiv funktion. Men kunskapen kring hur människor påverkas av långtidresor i rymden är begränsad. Särskilt få experiment har genomförts på stora dataset från biologiska prover, på en molekylär och cellulär nivå. I detta projekt har genuttryck hos möss som varit i rymden jämförts med en kontrollgrupp av möss som stannat på jorden. Metoden Spatial Transcriptomics (ST) har använts för att undersöka vävnadssnitt från hippocampus i mushjärna. Med ST är det möjligt att undersöka RNA­molekyler och kartlägga deras position i vävnaden. Resultatet från denna studie indikerar att miljön i rymden leder till dysfunktion i mitokondrierna. Detta arbete är en del av en större studie i samarbete med NASA och fler experiment kommer genomföras för att undersöka hur vi påverkas av miljön i rymden. Om fler studier stödjer detta resultat, kan mediciner som använts på jorden för att behandla patienter med dysfunktion i mitokondrierna, användas i förebyggande syfte för astronauter.

Colorectal cancer (CRC) is one of the most common and deadly cancers in the western world. The incidence of CRC shows the tendency to rise with the increase of obesity, which is caused by current increase in fat intake, suggesting the correlation between CRC and high-fat diet (HFD). HFD-induced obesity causes gut inflammation which is also noticed in inflammatory bowel diseases (IBD) and CRC and can be seen as an important factor in CRC development. Moreover, it has been demonstrated, that while both sexes are at risk of developing CRC, men have higher incidence compared to women, showing the protective effect of estrogen. In addition, since gut microbiome is first to respond to colon inflammation, we hypothesized, that intestinal estrogen signaling could contribute to reduced initiation and progression of colon cancer by modifying the microbiota composition. For that, two experiments with two different mouse models were conducted. First part of the study concentrated on the effect of (HFD, 60%) and different estrogenic ligands (17-β estradiol, and DPN) on microbiota. Bioinformatics analysis on whole genome sequencing (WGS) data and qPCR validation were used as the methods. Here we found that estrogenic ligands achieved restoration of close-to-normal microflora after significant change initiated by HFD. We also found that microbiome in males showed stronger reaction to HFD than female microbiome, implying protective actions in females. Furthermore, the effect of ligands also proved to be stronger in males. Second part of the study concentrated on the effect of estrogen receptor β (ERβ) on microbiota for which ERβ knockout mice were used in addition to cancerogenic AOM/DSS treatment. Bioinformatics analysis on WGS data was used as the method. We found that female mice were more affected by AOM/DSS treatment compared to males, especially the mice with knockout gene. The genotype alone, however, resulted in very few differences. In summary, this project shows the effect of HFD, estrogen and ERβ expression on gut microbiota diversity. It shows that microbiome of male mice is more susceptible to dietary changes and estrogen supplementation. Likewise, it demonstrates, that the microbiome of females reacts strongly to combination of carcinogenic treatment and lack of iERβ.

Current T-cell tracking techniques in vivo are limited. The ability to successfully target a gene in vivo in T-cells and track movement throughout its life cycle provides an exciting opportunity to elucidate the functions of genes. The aim of this study was to test an optogentically inducible Cre recombinase as well as a self-cleaving gRNA which can find and associate with Cas9 in vivo. Mouse T-cells which consecutively produce Cas9 (Cas 9, Jackson laboratory) were transduced and transplanted in immunodeficient mice (TCRb-/-, Jackson laboratory). The optogenetic component of the system is activated upon blue light stimulation and is introduced to the T-cell through a mouse stem cell virus (MSCV). The TCRb-/- mice underwent surgery which exposed their lymph nodes to blue light pulses from a fibre optic wire, this process is referred to as blue light surgery. BLU-VIPR T-cells which express self-cleaving gRNAs reduced the relative abundance of the target protein (Thy1.2), after blue light surgery in vivo. Furthermore, the optogenetic system showed minimal leakiness when used for gene targeting using gRNAs. This suggests that the gRNAs had associated with Cas9 and were able to successfully target the Thy1.2 gene. Results from the optogentically induced Cre recombinase showed that Cre was expressed in significant amounts without blue light stimulation, suggesting some background leakiness in the BLU-VIPR system.

The demand for the improvement of currently available tests for qualitative non-invasive diagnostic of COVID-19, i.e., the development of new methods for fast, low-cost and accurate tests for the conformation of SARS-CoV-2, is increasing rapidly. Among many different approaches, electrochemical biosensors, which have the capability of miniaturization and could be available globally in most remote areas, may also help in avoiding the transmission of COVID-19 disease. When properly designed, electrochemical tests might have higher sensitivity, specificity, and accuracy, which is very important for COVID-19 diagnostics. In this work a saliva based electrochemical biosensor developed for COVID-19 detection was tested using real human samples. First, 41 saliva samples from volunteers were collected during January-February 2022, when the rate of SARS-CoV-2 infection was the highest in Skåne region, Sweden. Second, cyclic voltammograms of SARS-CoV-2 biomodified electrodes were recorded in buffers with and without SARS-CoV-2 positive control, as well as in saliva samples. Third, the samples were analyzed using commercially available COVID-19 salivary tests, viz., rapid antigen test and RT-qPCR (quantitative reverse transcription polymerase chain reaction). It was shown that 8 samples were collected from COVID-19 positive volunteers. Based on the analysis of all experimental results it was concluded that compared to rapid antigen and RT-qPCR tests, the sensitivity and reproducibility of the biosensor is not enough for real practical applications. Thus, some suggestions for further improvement of basic parameters of the developed biodevice were made. / <p>x</p> / x

Interstitial Fluid is a complex sample, highly abundant in the human body that can give information regardingtissue secretion, intracellular signaling and tissue health status. The composition of the interstitial fluid can giveinformation regarding the processes occurring in muscles and alterations due to pathological changes occurringduring disease progression. Currently this sample has not yet been characterized within rare diseases like musculardystrophies. Facioscapulohumeral Muscular Dytrophy is an inherited progressive myopathy, characterized by thedegeneration and progressive muscular fiber necrosis of muscles from the face, upper arms and lower limbs. It canbe diagnosed; but in an advanced stage where weakness in the muscles have already occur. Meanwhile there is nocurrent understanding of the mechanisms happening in the muscle. In this project an immunoassay protocol wasdeveloped using suspension bead array technology to create an optimal method to analyze the protein content ofthese samples. The technological platform allows antibody-based capturing and detection of protein targets frombiotinylated biological samples. By modifying an existing protocol for analysis of serum and plasma samplesabundance of 63 protein targets was measured in muscle interstitial fluid from healthy individuals and patientsaffected by facioscapulohumeral dystrophy (FSHD), The optimized steps were the sample pre-treatment, the assaybuffer dilution ratio and the incubation time for capturing the protein targets. The findings of this project indicatethat using 1 μl of muscle interstitial fluid sample with minimized dilution factor and 60-fold molar excess biotinrelative to sample protein concentration enables detection of Interstitial fluid protein components. The proteinsdetected are ret finger protein-like 4B (RFPL4B) and albumin in from affected muscle and histone cluster(HIST1H3A) and albumin in non affected muscle.

No description available.

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.