The epidermis is composed of multiple cell strata where viable keratinocytes, in the basal layer (stratum basale (SB)), go through a range of steps with the final stage of being dead corneocytes in the outer most layer (stratum corneum (SC)). The differentiation, which can be thought of as programmed cell death, include several key processes that are essential for an intact skin barrier. The route from SB to SC is accompanied by changes, such as osmotic pressure and pH, that are believed to trigger some of these processes. In this project, HaCaT cells were incubated with, commonly used, skin care substances (urea, glycerol, transcutol and salicylic acid) to assess their impact on cell viability, by MTT-assay, and gene expression, by qPCR. Further, the relationship between osmotic pressure, viability and gene expression was studied. The excipients showed a dose-dependent decrease of keratinocyte viability which also was explained by elevated osmotic pressure when concentration was increased. Exceptions were however observed for transcutol, which showed protective features against osmotic stress. Upregulation of the genes were mainly observed when cells were treated with high concentrations. Involucrin was affected by the substances to a greater extent when compared to other markers. The upregulation of involucrin was however seen to be driven by the osmotic pressure rather than biological effects of the molecules. The project conclude that the viability and gene expression of the keratinocytes are highly related to the osmotic pressure and probably influences the differentiation to a greater extent than the molecules themselves.

Natural products and their derivatives have and are still used by humans for various health ailments due to their rich sources of drug discovery. New biologically active compounds from natural products play a key role in drug development. Marine sponges and their associated microbes contain a lot of bioactive compounds that are potential for drug development. These compounds produce chemical compounds with useful pharmaceutical properties such as antitumor, anti-infective, anti-inflammatory, and antibacterial properties. The main focus of this project was on the antibacterial activity of six different sponge specimens. The aim was to screen the antibacterial activity of the sponge specimen’s extracts. In order to do so, a Minimum Inhibitory Concentration assay was performed to screen the sponge's antibacterial activity against E. coli and S. aureus. Analytical HPLC was used for separation and Solid Phase Extraction (SPE) was used for determining the effect of salts towards the inhibition of anti-bacterial activity for two selected extracts. Ethanolic extract of Stylissa massa showed antibacterial activity against S. aureus. SPE would be a rapid purification step to remove the salts present in sponges at a high concentration but it has not shown a significant effect on the inhibition of antibacterial activity. However, further separation and purification need to be done to be able to completely screen for all the six different sponge specimens.

<p>Cerebral radiation necrosis as a consequence of radiation therapy is often observed in patients several months to years after treatment. Complications include painful headaches, seizures, and in the worst-case death. Radiation necrosis is an irreversible condition with the options available to manage it all having noticeable downsides. As such, there is a critical need for better ways of either preventing the onset of necrosis and/or managing its symptoms. As radiation necrosis cannot be induced in humans for ethical reasons, a mouse model that mirrors the features of radiation necrosis observed in patients would allow for new techniques to be tested before being used in human clinical trials. This thesis will explain how our lab designed a murine model of cerebral radiation necrosis that uses a 320 keV cabinet irradiator to produce radiation necrosis and MRI and histology to evaluate the development of radiation necrosis at multiple time points.</p><p><br></p> <p> </p> <p>Our model required the development of a mouse positioning apparatus that could be used in the cabinet irradiator used as well as the machining of lead shields so that focal semi-hemispheric irradiations could be conducted with other critical structures spared. The MRI scans used as well as the algorithm used to draw radiation necrosis lesions were based off what has been used in previous Gamma Knife models of radiation necrosis. Our initial work showed that since the cabinet irradiator has a relatively flat dose distribution unlike the Gamma Knife, the radiation lesion volumes produced in the former either plateaued or decreased, unlike in the case of the latter where lesion volumes tended to decrease over time. Further work analyzed the effects of fractionation and found minimal sparing using four different fractionation schemes. The effects of strain and sex on the development of radiation necrosis were also analyzed, with strain being found to be a statistically significant parameter while sex was not. Future research should focus on testing the effects of new drugs and techniques for better dealing with radiation necrosis.<b></b></p>

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The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.

The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using the two technologies combined in pump probe experiments. Three main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding on the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; and the development of theory, with sample applications, for infrared X-ray pump probe spectroscopy - the main theme of the thesis. Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found that the X-ray photoelectron spectrum of the water dimer differs strongly from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. Our simulations of infrared X-ray pump-probe spectra were carried out using wave packet propagation techniques. The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-ray pump probe spectroscopy - are examined in detail in two sample applications - on the NO molecule and on the dynamics of proton transfer in core ionized water dimer. It is found that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential. This results in a phase dependence of the X-ray pump probe spectra. A proper choice of the delay time of the X-ray pulse allows to directly observe the X-ray transition in the proton transfered well of the core excited potential. / QC 20101125

Cardiac hypertrophy is when heart muscles thicken as an adaptive response to several stimuli. Prolonged pathological cardiac hypertrophy can lead to heart failure and severe cardiovascular diseases. Scientists have faced challenges in studying cardiac hypertrophy due to the lack of human cardiomyocytes available. Recently, hypertrophic model using human induced pluripotent stem cell-derived cardiomyocytes was introduced. In this study, expression profiles of in vitroendothelin-1 induced cardiac hypertrophy model were investigated at different time points. The study aimed to examine molecular pathways associated with cardiac hypertrophy, identify biomarker candidates for cardiac hypertrophy, and investigate if there were known pharmaceuticals that putatively are targeting the suggested candidate biomarkers. Using the Ingenuity pathway analysis (IPA) software, GRM1, NPPA, and STC1 gene were identified as biomarker candidates for cardiac hypertrophy model across all time points. More biomarker candidates unique to the cardiac hypertrophy-stages were also identified using IPA. In vivomicroarray data of hypertrophied heart profiles were also used to compare to the in vitro data and preliminarily validate the gene candidates identified by IPA. Four genes were identified by IPA and were presented in the in vivo data. IPA also revealed the in activation of specific pathways of the early-stage cardiac hypertrophy model. The result suggested that the molecular mechanisms of the in vitro cardiac hypertrophy model did not fully represent the actual hypertrophic condition of the heart. More research and validation are required to understand the underlying mechanism fully and potentially, in the future, utilize the identified genes as cardiac hypertrophy biomarkers.

Följande studie behandlar utvecklandet av automatiserade cellsegmenteringsmodeller med avsikt att identifiera gränser mellan celler i hjärtvävnad. Syftet är att möjliggöra analys av data genererad från multiplexed error-robust in situ hybridization (MERFISH). MERFISH är en spatial transcriptomics-teknik som till skillnad från exempelvis single-cell RNA sequencing (ScRNA-seq) och single molecule fluorescence in situ hybridization (smFISH), möjliggör profilering av hundratals RNA-sekvenser hos enskilda celler utan att förlora dess rumsliga kontext. I Kosuri laboratoriet på Salk Institute of Biological Studies i San Diego tillämpas MERFISH på mushjärtan. Syftet är att få en djupare insikt i hur celler är organiserade i friska hjärtan, och hur denna struktur ändras i och med åldring och sjukdom. Att extrahera meningsfull information från MERFISH medför dock en betydande utmaning - en exakt cellsegmentering. Studien bidrar följaktligen till utvecklandet av segmenteringsmodeller för att kringgå de utmaningar som står i vägen för all efterföljande analys. Då klassiska segmenteringsalgoritmer är otillräckliga för att segmentera den komplexa vävnad som hjärtat utgörs av, tillämpades några av dagens mest avancerade och framstående maskininlärningsalgoritmer inom fältet, kallade Cellpose och Omnipose. Givet den täta och heterogena hjärtvävnaden, som härstammar från en bred distribution av celltyper och geometrier, utvecklades två separata modeller; en för att täcka både mindre celler och kardiomyocyter skurna på tvärsnittet; och en för att enbart segmentera kardiomyocyter skurna i longitudinell riktning. Den förstnämnda modellen utvecklades och tränades i Cellpose, och uppnådde en träffsäkerhet på 91.2%. Modellen för longitudinella kardiomyocyter utvecklades istället både i Cellpose och Omnipose för att utvärdera vilket nätverk som är bäst lämpat för ändamålet. Ingen av nätverken lyckades uppnå en tillräckligt hög träffsäkerhet för att vara applicerbar, och är därmed i behov av fortsatt träning. Modellen genererad i Omnipose bedöms dock vara mest lovande, givet dess mer heltäckande segmentering. Ytterligare utvecklingsområden för framtiden innefattar segmentering av celler i fibros-täta regioner, samt att utveckla en 3D-segmentering av hela hjärtat för att uppnå en mer komplett MERFISH-analys. Sammanfattningsvis har de genererade segmenteringsmodellerna banat väg för möjliggörandet av en rigorös MERFISH-analys av hjärtat. Genom att avslöja några av de strukturella och funktionella orsakerna till hjärtsvikt på en cellulär nivå, kan vi således på sikt bidra till utvecklingen av mer effektiva terapeutiska strategier. / The following study delves into the development of automated cell segmentation models, with the intention of identifying boundaries between cells in the cardiac tissue for analysing spatial transcriptomics data. Addressing the limitations of alternative techniques like single-cell RNA sequencing (ScRNA-seq) and single molecule fluorescence in situ hybridization (smFISH), the study underscores the innovative use of multiplexed error-robust fluorescence in situ hybridization (MERFISH) deployed by the Kosuri Lab at Salk Institute for Biological Studies. This advanced imaging-based technique allows for a single-cell transcriptome profiling of hundreds of different transcripts while retaining the spatial context of the tissue. The technique can accordingly reveal how the organization of cells within a healthy heart is altered during disease. However, the extraction of meaningful data from MERFISH poses a significant challenge - accurate cell segmentation. This thesis therefore presents the development of a robust model for cell boundary identification within cardiac tissue, leveraging some of the advanced supervised machine learning algorithms in the field, named Cellpose and Omnipose. Due to the dense and highly heterogeneous tissue- stemming from a wide distribution of cell types and shapes- two separate models had to be developed; one that covers the smaller cells and the cross-sectioned cardiomyocytes, and correspondingly one to cover the longitudinal cardiomyocytes. The cross-section model was successfully developed to achieve an accuracy of 91.2%, whereas the longitudinal model still needs further improvements before being implemented. The thesis acknowledges potential areas for improvement, emphasizing the need to further improve the segmentation of longitudinal cardiomyocytes, tackle the challenges with segmenting cells within fibrotic regions of the diseased heart, as well as achieving a precise 3D cell segmentation. Nonetheless, the generated models have paved the way towards enabling efficient downstream MERFISH analysis to ultimately understand the structural and functional dynamics of heart failure at a cellular level, aiding the development of more effective therapeutic strategies.

To improve the efficacy of the dendritic cell vaccine Alv B DC from Alv B, the PD-L1 expression in cancer cells was attempted to be reduced through a transfection with a custom designed siRNA. Before transfecting the dendritic cells, the siRNA functionality had to be tested through flow cytometry, that resulted in negative results and therefore led to a RT-qPCR protocol that indicated that the siRNA was functional. Protocols for the two methods were developed and a cell line expressing PD-L1 was set up as a tool for testing. The final goal of testing the effects in Alv B DC was never performed due compromising time but the positive result from the PCR provides a promising start to further testing.

Giardia intestinalis is a unicellular protozoan parasite responsible for 280 million gastrointestinal infections every year. When colonizing its host, Giardia interacts closely with the small intestine epithelium by attaching to enterocytes and releasing multiple proteins to the extracellular environment. Some of the released proteins have been shown to aid the parasite’s survival in the intestine by disrupting various host defense mechanisms. Here, we attempt to characterize the specific localization of five proteins after their secretion by Giardia. In parallel we aim to produce and identify parasite’s molecules potentially working as triggers of the immune response built during infection. To study the localization of specific secreted proteins during in vitro interactions with differentiated Caco-2 cells, we started by creating transgenic parasites expressing the ADI, EF1α and G3PD proteins with a downstream detectable tag. To identify candidate proteins from Giardia, thought by our lab to be involved in immune system activation, we established a mammalian expression system for the production of recombinant versions of the selected candidate giardial PAMPs. We achieved the expression of the VSP1267 protein, natively present on the parasite’s surface. However, we found that this protein was not secreted after expression, thus complicating its purification and later use in TLR-activation experiments. In the future, we aim to localize the tagged proteins, expressed by the produced transgenic trophozoites, and optimize the mammalian expression system in order to identify candidate immune triggers during giardiasis.