Ultraviolett (UV) strålning genererar fotodimerer i celler som leder till komplikationer med replikering och transkribering. Dessutom bidrar det också med DNA lesioner och brott. Förståelsen för hur de molekylära mekanismerna fungerar är av högsta vikt för att förhindra uppkomsten och bota hudsjukdomar som orsakats av UV-strålning. Den här uppsatsen fokuserar på hudens komposition och hur den förändras med åldern, av både inre och yttre faktorer samt cellernas egna reparationsmekanismer. Huden består av cellulära och extracellulära matriskomponenter, båda är fint fördelade och organiserade för att bidra med skydd, elasticitet och spänst. Åldrande leder till att hudens förmågor försämras, rynkor uppstår och det sker en förlust av spänst. Detta sker naturligt som konsekvens av inre åldrande, men detta påskyndas av yttre faktorer som miljöföroreningar och UV-strålning. Olika mekanismer i celler reparerar dessa skador, det här arbetet tar upp nukleotidreparation (NER) och hur det reparerar via att klyva bort de mutagena delarna av genomet för att förhindra uppkomst av tumörer. Trots hudens olika reparationssystem förblir vissa celler skadade, personer med ljusa pigment är mer känsliga för mutationer än personer med mörka pigment. För att förhindra och förebygga uppkomst av farliga hudförändringar rekommenderas idag inte bara solskyddsfaktorer, utan även antioxidanter som skyddar mot oxidativ stress som även den orsakas av UV-strålning.

Den snabba utvecklingen av det nya genredigerande verktyget CRISPR-Cas9 har öppnat dörren för behandlingen av svåra, virusorsakade sjukdomar. Herpes simplexvirus typ 1 (HSV-1), en av de mest vanligt förekommande virus i världen, kan etablera sig latent i känselnervceller. Stressfaktorer kan orsaka herpeskeratit, som är en sekundär infektion av ögat vilket leder till inflammation och potentiell ärrbildning. Vid upprepade återfall kan herpeskeratit resultera i synnedsättningar och i allvarligare fall blindhet, och därför anses det som en stor samhällsbörda.  Denna uppsats undersöker förutsättningarna att CRISPR-Cas9 i framtiden ska användas för att effektivisera behandlingen av herpeskeratit. Detta görs först genom att beskriva hur HSV-1 infekterar människokroppen, etablerar latens och skadar hornhinnan. Sedan introduceras utvecklingen av CRISPR-Cas9 som ett molekylärt verktyg där dess fördelar och nackdelar beskrivs. Slutligen sammanfattas de tre olika tillvägagångssätten av forskare som undersöker möjligheten att skapa en CRISPR-Cas9-baserad behandling.  Den första metoden baseras på användningen av CRISPR-Cas9 för att dämpa virusreplikation vilket resulterar i att konsekvenserna blir lindrigare vid eventuella återfall. Detta alternativ har visat sig vara framgångsrikt in vitro och det kringgår en del utmaningar gällande leveranssystemet, men är begränsad i sina användningsområden. Den andra metoden baseras på att redigera känselnervceller så att de konstant utsöndrar CRISPR-Cas9 samt relevanta guide- RNA för dämpningen av virionbildandet likväl som förebyggandet av en primärinfektion. I praktiken blir detta svårt att tillämpa som behandlingsmetod på grund av ett antal tekniska svårigheter. Det tredje alternativet är att klyva det latenta HSV-1 genomet. Detta skulle omöjliggöra en sekundärinfektion. In vitro-experiment har visat att det är möjligt, men svårigheter med leveranssystem och effektiviteten har gjort det problematiskt att dra några relevanta slutsatser om CRISPR-Cas9 är rätt väg eller inte. Andra genredigerande enzymklasser, exempelvis meganukleaser, har haft större framgång i musmodeller. Forskningen gällande CRISPR-Cas9 är vid ett tidigt stadium, men har väckt stort intresse inom forskningsvärlden. Med fortsatt optimeringen av CRISPR-Cas9, och dess framgång i andra virussammanhang är det troligt att CRISPR-Cas9 eventuellt kommer vara en del av lösningen för att bota herpeskeratit.

CCM is a human disease in which vascular malformations develop in the central nervous system, predominantly in the brain. The malformations which can measure up to several centimeters in size are dilated and leaky vessels, with unstable cellular junctions and thin vessel walls. Depending on the size and the location of the malformation, the lesion might impose a direct threat to the health of the patient. The symptoms vary from more mild health issues such as headaches, to more severe symptoms such as loss of vision or hearing, or even epileptic seizures, paralysis, and hemorrhagic strokes. CCM is caused by a mutation in either of the CCM genes: CCM1, CCM2, or CCM3. The molecular mechanisms underlying the development of the vascular malformations are still poorly understood, but it has recently been shown that the GTPase CDC42 interacts with the CCM protein complex. By inducing endothelial cell-specific deletion of Cdc42, similar malformations to that of CCM arise in the mouse brain. In this project, our aim was to investigate the molecular changes underlying the vascular malformations caused by endothelial specific Cdc42 deletion in mice. To do so, we isolated endothelial cells from Cdc42iΔEC and Control mouse brain at postnatal day seven and performed single-cell RNA sequencing of these cells. When analyzing the data, we noticed that a subset of KO cells clustered differently and showcased a unique molecular profile. These cells expressed pan-endothelial markers but did not classify as arteries, capillaries, or veins as they did not express markers for those endothelial sub-types. Instead, these cells expressed markers for fenestrated choroid plexus endothelium, such as Igfbp3, and Gpihbp1, yet the absence of Aqp1 expression distinguishes them from choroid plexus endothelial cells. To validate the results from the RNA sequencing experiments we performed immunohistochemistry on brain sections of Cdc42iΔEC and Control mice.   In summary, we identified a unique expression pattern in a subset of Cdc42 depleted endothelial cells. While further investigations are needed, this is an important starting point for the identification of potential biomarkers, drug targets and the development of urgently needed treatment strategies.

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No description available.

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Platelet-derived growth factor receptors (α and β) are included in the family of receptor tyrosine kinases (RTKs). The dimeric PDGF ligands bind to the PDGF receptors and this results in receptor dimerization, autophosphorylation, and thus activation. The autophosphorylated receptor dimer activates numerous signaling pathways. The responses of the cell to the activation of these pathways include proliferation, migration and survival. After ligand stimulated activation, PDGF receptors are internalized mainly via clathrin-coated pits and subsequently degraded in the lysosomes. However, the proteasomes have also been reported to play a role in PDGFRβ degradation. The main aim of this project was to study the mechanism of ligand-stimulated cleavage of PDGFRβ and to explore its possible consequences. By using antibodies that recognize the extracellular and intracellular parts of the receptor, respectively, we showed that ligand stimulation of PDGFRβ leads to the formation of an extracellular (~130 kDa) and an intracellular (~70 kDa) fragment. These fragments were shown to be specific fragments of the receptor located at the plasma membrane and not newly synthesized receptor. The presence of calcium ions intracellularly was found to be essential for cleavage. Interestingly, we observed that with the use of the proteasomal inhibitor called bortezomib, receptor cleavage was inhibited. On the other hand, lysosomal inhibition did not inhibit PDGFRβ fragmentation. Subsequently, we showed that cleavage occurs after the receptor has been endocytosed. Then, we examined whether blocking PDGFRβ cleavage had any effect on the downstream signaling of the receptor. We revealed an increase in receptor, PLCγ and STAT3 phosphorylation and a decrease in Erk phosphorylation upon bortezomib treatment. Moreover, we observed that PDGFRβ undergoes another type of cleavage upon ligand stimulation in fibroblasts, known as ectodomain shedding. Finally, we showed that receptor internalization is decreased upon bortezomib treatment, suggesting that bortezomib blocks receptor cleavage by inhibiting its internalization. The cleavage site, responsible enzyme, as well as potential functional consequences of ligand-stimulated PDGFRβ cleavage, remain to be determined.

Ribosome biogenesis is the maturation and accurate assembly of ribosomal RNA and proteins to form functional ribosomes competent to enter the translation cycle, facilitated by the action of ribosome biogenesis factors. RbfA and KsgA are two factors involved in late stages of the 30S subunit biogenesis, yet before translation initiation. Despite the extensive research on both factors, their precise mechanisms remain unclear in translation initiation. Initiation factors constitute a key component of the translation initiation system, especially IF3 that proofreads the codon-anticodon interaction of the mRNA and initiator tRNA to maintain the accuracy of the initiation step. However, defective ribosomes with knockout RbfA (ΔrbfA) or KsgA (ΔksgA) appear to weaken IF3 binding affinities and subsequent proofreading. Previous results suggest that when defective ribosomes enter in vivo translation with cognate (AUG) or even near-cognate (AGG) start codons could bypass the fidelity checkpoints created by initiation factors, leading to the inaccurate initiation of translation. This study aimed to characterize the assembly defective ΔrbfA and ΔksgA ribosomes in in vitro initiation assays on cognate and near-cognate codons and with the interplay of initiation factors for the rate and accuracy in translation initiation. The two assays, BOP-Met-tRNAfMet binding and subunit association, reveal that ΔrbfA ribosomes are defective in both rate and accuracy in translation initiation, which largely contribute to its growth defect at 37°C. In contrast, ΔksgA ribosomes are not defective in the initiation step of translation when comparing to the wildtype ribosome. Therefore, RbfA seems to have the primary role in ribosome biogenesis compared to KsgA. Further, the results show that the initiation factors, in particular IF3, adds to the fidelity of initiation by dissociating the initiator tRNA on near/non-cognate codons, with no major difference between Wt and mutant ribosomes in selection against near/non-cognate codons. Therefore, we do not see any loss of fidelity for the ΔrbfA and ΔksgA ribosomes in in vitro initiation based assays, contrary to previous in vivo based results.

Loss of appetite is together with fever and malaise hallmarks of infection. Loosing appetite during an acute infection such as influenza does not result in any longlasting effects, but loosing appetite during chronic diseases such as cancer or AIDS constitutes a risk factor for mortality. Food intake regulation during inflammation is orchestrated by the brain in response to peripheral inflammatory signals. It is known that expression of the prostaglandin synthesizing enzyme cyclooxygenase 2 (COX-2) is crucial for the mechanisms underlying inflammation-induced anorexia, and that prostaglandin E2 (PGE2) is involved in anorexia induced by interleukin-1 beta (IL-1β). In this thesis I examined the prostaglandin-pathways proposed to be involved in anorexia. We show that acute anorexia is dependent on COX-2 expression, while cancer-induced anorexia is mediated by cyclooxygenase 1 (COX-1), at least in the initial stages, suggesting that the signaling pathways for chronic- and acute anorexia are distinct. We were able to demonstrate that the pathway underlying acute anorexia is distinct from that of fever, and that taste aversion is prostaglandin independent. We could also show that both acute and chronic anorexia-cachexia is dependent on expression of myeloid differentiation primary response gene (MyD88) in hematopoietic/myeloid cells. In summary, the findings presented in this thesis suggest that anorexia is a result of many different signaling pathways, as opposed to what is the case for several other inflammatory symptoms such as fever and malaise, where the pathways have been shown to be very exclusive. This provides new insight into the diversity of the pathways underlying inflammatory symptoms, which is fundamental for the ability to present potential, symptom-specific drug targets.

<p>Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients.</p><p>In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains.</p><p>The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified.</p><p>In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.</p>