The commonly used materials in endodontic treatment lack the ability to prevent microleakage, which can lead to failure of the treatment. This study aimed to evaluate the effectiveness of adding Coltosol plug in preventing coronal microleakage in endodontically treated teeth using the in vitro dye penetration method. Ten single canal plastic teeth were prepared and obturated using the SG-endodontic technique. The teeth were randomly divided into two groups, with and without Coltosol plug, and dye penetration was measured at 6 and up to 72 hours. No difference in leakage was observed after 6 and 72 hours. Specimens with a plug of Coltosol showed similar dye penetration compared to the ones with only Gutta-percha and sealer.

Antibiotic resistance in multidrug resistant bacteria cause high mortality rates worldwide, where there has been over 1,000,000 deaths reported as of the year 2019. Antibiotics were thought to be the cure for fighting infectious diseases and preventing further spreading of infection. This became a major problem due to bacteria evolving and developing mechanisms for resistance. The purpose of this review was to see if there are differences in the resistance mechanism of gram negative and gram positive bacteria, focusing mainly on the six most common multidrug resistant pathogenic bacteria; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae. The results show that there is a difference in the resistance mechanism between gram positive and gram negative multidrug resistant bacteria. The difference in resistant mechanisms is due to the cell wall compositions of gram negative and gram positive bacteria. The main difference as to why the gram negative bacteria have more resistance is due to the outer membrane. Antibiotics have a hard time to diffuse through and into the cell, that is they can easily decrease their outer membrane permeability. Gram positive bacteria lack an outer membrane which makes them become more susceptible to antibiotics. The most common antibiotic resistance mechanisms in gram negative bacteria are outer membrane mechanisms such as lipid A and lipopolysaccharide modification as well as mutations in porin channels. On the other hand, the most common resistance mechanisms for gram positive bacteria are point mutations especially in penicillin binding proteins as well mutations in the rpoB gene. One important gram positive bacteria is Methicillin resistant Staphylococcus aureus, which developed a new mechanism against antibiotics, a missense mutation and mutation on the promoter region in penicillin binding protein 4. Recently new research has come forward showing that N-chlorotaurine (NCT) inhibits resistance in both gram positive and gram negative multidrug resistant bacteria. The research on NCT is still fairly new and only time will tell if this method of inhibiting resistance will be used in the future. This review highlights the importance and concern of multidrug resistance bacteria, especially due to bacteria being able to rapidly evolve when antibiotics are used incorrectly. It is important to understand the differences in resistance between gram negative and gram positive bacteria and how resistance spreads. This knowledge can be used to develop antibiotics that treat infections. It is however still a challenge to overcome resistance in multidrug resistant bacteria due to evolutionary adaptation especially through horizontal gene transfer, where resistant bacteria can adapt to changing conditions. / Antibiotikaresistens hos multiresistenta gramnegativa och grampositiva bakterier orsakar hög dödlighet över hela världen, där det har rapporterats över 1,000,000 dödsfall för år 2019. Antibiotika ansågs vara botemedlet för att bekämpa infektionssjukdomar och förhindra ytterligare spridning av infektioner. Detta blev ett stort problem på grund av att bakterier utvecklades mekanismer för resistens, vilket gör att de kan överleva när de behandlas med antibiotika. Syftet med denna studien är att se om det finns skillnader i resistensmekanismener för gramnegativa och grampositiva bakterier, med fokus på de sex vanligaste multiresistenta bakterierna; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae och Streptococcus pneumoniae. Resultaten visar att det finns en skillnad i resistensmekanismen mellan grampositiva och gramnegativa multiresistenta bakterier. Skillnaden i resistenta mekanismer beror på cellväggssammansättningen av gramnegativa och grampositiva bakterier. Den största skillnaden till varför de gramnegativa bakterierna har mer resistens beror på det yttre membranet. Antibiotika har svårt att penetrera genom och in i cellen genom att minska deras yttre membranpermeabilitet. Grampositiva bakterier saknar ett yttre membran som gör att de blir mer mottagliga för antibiotika. De vanligaste antibiotikaresistensmekanismerna hos gramnegativa bakterier är yttre membranmekanismer som lipid A och lipopolysackaridmodifiering samt mutationer i porinkanaler. De vanligaste resistensmekanismerna för grampositiva bakterier är punktmutationer, särskilt i penicillinbindande proteiner samt mutationer i rpoB genen. En viktig grampositiv bakterie är Meticillin-resistent Staphylococcus aureus, som utvecklade en ny mekanism mot antibiotika, en missense-mutation och mutation på promotorregionen i penicillinbindande protein 4. Nyligen har ny forskning kommit fram som visar att N-klorotaurin (NCT) hämmar resistens i både grampositiva och gramnegativa multiresistenta bakterier. Forskningen om NCT är fortfarande ny och bara tiden kommer att utvisa om denna metod för att hämma resistens kommer att användas i framtiden. Den här studien belyser vikten och oron för multidresistena bakterier, särskilt på grund av att bakterier snabbt kan utveckla antibiotikaresistens när antibiotika används på fel sätt. Det är viktigt att förstå skillnaderna i resistens mellan gramnegativa och grampositiva bakterier och hur resistens sprids inom resistenta bakterier. Denna kunskap kan användas för att utveckla antibiotika som behandlar infektioner orsakade av både gramnegativa och grampositiva bakterier. Det är fortfarande en utmaning att övervinna resistens hos multiresistenta bakterier på grund av evolutionär anpassning särskilt genom horisontell genöverföring, där resistenta bakterier kan anpassa sig till förändrande förhållanden.

The bacterium Streptococcus pneumoniae (pneumococcus), is considered to be a leading cause of morbidity and mortality globally, particularly in infants and the elderly. It is one of the most frequent causes of respiratory tract infections, which sporadically have the potential to develop into serious invasive symptoms including sepsis and meningitis. The development of effective vaccination against this pathogen is essential for reducing the morbidity and mortality it causes since the currently available vaccines can protect against only a limited number of the 100 pneumococci serotypes which target the polysaccharidic capsule of the bacterium. The potential use of conserved protein antigens could provide a defense to a wider range of serotypes and clonal types. The immunogenic properties of the proteins MalX and PrsA as well as their role in vital biological functions of S. pneumoniae have made them stand out as potential targets. MalX is a crucial membrane protein involved in the metabolism of maltose, whereas PrsA is a chaperone-like protein that is connected to the cell envelope. To understand these proteins' potential as vaccine candidates, it is essential to understand their immunogenic characteristics and physiological roles. In this project, we tried to characterize the two antigens to determine the functional significance of different regions and domains in antigen recognition and their expression dynamics in bacterial host. A better understanding of the antigenic properties of the PrsA and MalX proteins will drive the construction of improved versions of antigens for vaccine prototypes. Some approaches were used to clarify the structural characteristics and antigenic determinants associated with these proteins including, protein expression, purification, and structural characterization. Additionally, their expression in E. coli was examined using immunological assays including ELISA and Western blot. The identification of antigenic regions of these proteins also provides insight into how to develop epitope-based vaccinations that specifically target S. pneumoniae. This project discusses the possibility of using membrane vesicles (MVs) as a platform for vaccination. Membrane vesicles made from bacterial cells have innate immunogenic qualities that expose the immune system to a wide variety of antigens. Incorporating MalX and PrsA into such vesicles can improve the vaccine candidate's overall immunogenicity and effectiveness and trigger a stronger immune response against S. pneumoniae.

The H9N2 avian influenza virus (AIV) has emerged, spread and established itself in poultry globally, in just under 30 years. During this time, multiple reassortants of H9N2 with increased zoonotic potential have been isolated in poultry and humans, causing a major threat to the economy and global health. Curiously, H9N2 appears to be compatible with multiple Hemagglutinin (HA) and Neuraminidase subtypes, in nature. Here, the aim was to investigate the HA reassortment dynamics of the poultry adapted H9N2 AIV, in a laboratory setting. Firstly, HA subtypes from wild bird isolates were cloned, before being co-transfected with the backbone of a chicken H9N2 AIV. The rescued H9N2 reassortants were titred on cells before the replication kinetics of a subset of the HA reassortants was assessed. The cDNA sequence of seven HA subtypes induced extensive recombination in E. coli, but ultimately ten out of eleven available HA subtypes were successfully cloned. Further, the chicken H9N2 AIV was compatible with all ten HA subtypes, producing infectious viral particles after co-transfection. However, all HA reassortants displayed decreased replicative fitness in MDCK-2 cells, compared to the wild-type virus. Interestingly, HA subtypes with similar genotypes cluster into distinct HA clades and groups, but these HA clades did not correlate with the replicative fitness of the reassortants. This study suggests that poultry adapted H9N2 AIV is compatible with many HA subtypes, highlighting the importance of reducing its spread in poultry, to reduce reassortment opportunities.

Recent studies proposed that mammalian cell lines with slower growth rates were infected more efficiently by Trypanosoma cruzi, and that cell-to-cell transmission could be occurring during infection. This open the question of whether host cell-growth rate is a cellular characteristic influencing Trypanosoma cruzi invasion and infection. To prove if this was the case, the cell growth-rate was inhibited by starvation, reducing the foetal bovine serum concentration in the medium, and using a cell-cycle arresting drug, Baicalein. Then the percentage of infected cells of the control and the growth-modified group was measured via epifluorescence microscopy. The results did not show a strong evidence of negative correlation between the cell-growth rate and infection efficacy. To assess if cell-to-cell infection was occurring, the percentage of infected cells in contact with other infected cells was measured. This value was compared to the stochastic probability of this event happening. The results showed that the random probability of an infected cell being next to another infected cell was much lower than the percentage obtained from the empirical data. This suggests that cell-clusters found in infections are not a random event and that the dominant mechanism of infection is a short-range one: either by cell-to-cell infection, or by the proliferation of already infected cells. To support cell-to-cell transmission, parasites potentially passing from one cell to another were inspected through confocal microscopy and actin-rich regions were found at the parasite location. To determine whether actin played a role in this event, cells with a mutation in the actin gene were infected and compared to the mock group. The results showed that the percentage of infected cells in contact with other infected cells was lower for the mutant cells, suggesting that actin could play a key role in this event.

Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), has been considered as an economically important disease around the world because of the extreme reduction in sugar beet yield and sugar content in affected plants. The spread of rhizomania all over the world, including the emergence of resistance- breaking virus isolates, have been become a major concern for the plant pathologists and plant breeders aiming at improving sugar beet resistance to   BNYVV as well as better understanding sugar beet-virus interactions during disease development. The main focus of this project is to elucidate the role of P25-interacting partner, the VIP1 transcription factor, in activation of transcription.  The isolation of the gene-of-interest (VIP1) was performed by RT-PCR on total RNA preparations extracted from root tissue of sugar beet (Beta vulgaris ssp. vulgaris).  The isolated gene of interest was cloned using gateway system into a binary expression vector and the obtained construct was then transformed into Agrobacterium tumefaciens for analysing transient expression in the experimental host (Nicotinana benthamiana).  Dual-luciferase promoter activity assay was performed on isolated leaf discs co-expressing P25 and VIP1 and compared to appropriate controls.  Six promoter constructs were tested. However, we observed an increase in luciferase activity (1.8-4.2-fold) upon co-expression of P25 and VIP1 only for two constructs tested, although the increase was not supported by Student’s t-test at 0.05 significance level. Nevertheless, the luciferase activity assay data for these two constructs were consistent with RNA-seq and RT-qPCR data obtained previously showing upregulation of the expression of these two specific sugar beet genes during BNYVV infection in sugar beets.     Thus, the results support our hypothesis that the interaction of the virus virulence factor P25 with VIP1 transcription factor is needed to activate transcription of certain genes in the nucleus for the virus benefit.

Argonaute proteins play substantial roles in post-transcriptional regulation of gene expression within RNA interference (RNAi) pathways, making them crucial subjects for research, aimed at understanding their interactions with small non-coding RNAs (ncRNAs) and other RNAi components. This study focuses on investigating these properties of Argonaute proteins, particularly Argonaute protein A (AgnA), in the social amoeba Dictyostelium discoideum that is renowned for its broad genetic toolbox and unique life cycle. While previous studies have examined the disruption of three Argonaute genes (agnB, agnC, agnE) and their effect on mRNA levels and small ncRNA expression, this study extends to agnA gene, which remains less studied. Key questions surrounding the influence of AgnA on the cellular processes such as the cell growth rate, development, gene expression, as well as potential targets and small ncRNA binding, remain unanswered. A well-established approach that could provide the necessary answers is the disruption of the gene through traditional homologous recombination, by insertion of a drug-resistance cassette flanked by homology arms complementary to the target locus. However, the emerging CRISPR/Cas9 gene editing tool on contrary offers straightforward protocols for disruption of gene expression through efficient induction of genomic knockouts, point mutations and deletions. In this study, both approaches were applied in parallel to knockout the agnA gene, enabling comparison of knockout efficiency and further study of the growth rate, development and gene expression in the knockout strains. Moreover, important information regarding the growth patterns of both wild-type and agnE knockout strains were also elucidated, complementing the previous growth rate analyses. The obtained data from this research could provide valuable insights for future studies ofthe RNAi machinery components and particularly the function of Argonaute proteins in D. discoideum.

The plastid is an important organelle that allows eukaryotes to photosynthesize. The endosymbiotic event that led to the development of a primary plastid occurred more than one billion years ago. Since then the organelle has undergone a significant genome reduction, losing a large portion of non-essential genes whose function is covered by the eukaryotic host. The majority of the proteins needed for essential plastid function are transcribed in the nucleus and translated in the cytosol. These proteins are then translocated across the inner and outer plastid membranes. The difference in where proteins are translated and transported can be used to study plastid evolution, this can be done by examining what proteins are present in the red algae plastid and comparing it to proteins found in other algae groups. In this project, the exact placement of the proteins, mainly plastid proteins is of interest. In order to localize and identify proteins in the algal cell, an efficient method of cell lysis, both total and incomplete, where the preservation of organelles is essential needs to be developed. This thesis examines a set of different methods of cell lysis, both complete and incomplete, coupled with organelle fractionation, to achieve the isolation of proteins belonging to the different cellular compartments. I show that complete cell lysis with bead milling using 0.1-0.5 mm silica beads is more efficient than a method using a Dounce homogenizer. For incomplete lysis, I show that nitrogen cavitation at 750 psi for 15 min and 1,000 psi for 30 min provides the same level of cellular lysis, indicating that the nitrogen gas equilibrates within 15 minutes. Organelle fractionation with OptiprepTM gradient showed that the majority of the sample did not travel through the gradient, staying in the first layer, which also prevented revelation of the protein pattern on an SDS-PAGE gel, indicating insufficient centrifugation. A great deal of optimization is still required to make these methods as efficient as possible. The step that requires the most optimization is sample preparation for nitrogen cavitation and the use of an ultracentrifuge.

Flera miljoner människor världen över är idag drabbade av depression vilket skapar ett stort lidande. Depression leder även många gånger till självmord. Känt är att tarmmikrobiotan är en viktig del i sjukdomsförloppet [MB1] och att kunna karakterisera tarmmikrobiota som är av psykobiotisk karaktär blir därför viktigt i ett behandlande syfte men också för att kunna ställa diagnos mer objektivt. Syftet med den här litteraturstudien är att genom artikelgranskning i PubMed undersöka vad senaste forskningen säger om tarmmikrobiotans roll i tryptofanmetabolismen med efterföljande effekt på depression. Skiljer sig tarmmikrobiotan och tryptofanmetabolismen mellan individer med och utan depression och kan tarmmikrobiotan och tryptofanmetaboliter användas som biomarkörer för att diagnostisera depression? Resultatet visar att tarmmikrobiotan och vissa tryptofanmetaboliter skiljer sig signifikant åt mellan friska individer och individer med depression. Studiens slutsats är att tarmmikrobiota och tryptofans metaboliter skulle kunna användas som biomarkörer för att kunna diagnostisera depression mer objektivt. Dock förekommer flera olika potentiella confounders som bör tas i beaktning. / Several million people worldwide are today affected by depression, which creates a lot of suffering. Depression also often leads to suicide. It is known that the gut microbiota is an important part of the course of the disease and being able to characterize gut microbiota that is of a psychobiotic nature therefore becomes important for a treatment purpose but also to be able to make a diagnosis more objectively. The aim of this literature study is to examine, through article review in PubMed, what the latest research says about the role of gut microbiota in tryptophan metabolism with subsequent effect on depression. Do gut microbiota and tryptophan metabolism differ between individuals with and without depression and can gut microbiota and tryptophan metabolites be used as biomarkers to diagnose depression? The results show that the gut microbiota and certain tryptophan metabolites differ significantly between healthy individuals and individuals with depression. The study's conclusion is that gut microbiota and tryptophan metabolites could be used as biomarkers to diagnose depression more objectively. However, there are several different potential confounders that should be taken into account.

Neisseria meningitidis, also known as the meningococcus, is a globally spread obligate human bacterium causing meningitis and/or septicaemia. It is responsible for epidemics in both developed and developing countries. Untreated invasive meningococcal disease is often fatal, and despite modern intensive care units, the mortality is still remarkably high (approximately 10%). The continuously increasing antibiotic resistance in many bacterial pathogens is a serious public health threat worldwide and there have been numerous reports of emerging resistance in meningococci during the past decades. In paper I, the gene linked to reduced susceptibility to penicillins, the penA gene, was examined. The totally reported variation in all published penA genes was described. The penA gene was highly variable (in total 130 variants were identified). By examination of clinical meningococcal isolates, the association between penA gene sequences and penicillin susceptibility could be determined. Isolates with reduced susceptibility displayed mosaic structures in the penA gene. Two closely positioned nucleotide polymorphisms were identified in all isolates with reduced penicillin susceptibility and mosaic structured penA genes. These alterations were absent in all susceptible isolates and were successfully used to detect reduced penicillin susceptibility by real-time PCR and pyrosequencing in paper II. In papers III and IV, antibiotic susceptibility and characteristics of Swedish and African meningitis belt meningococcal isolates were comprehensively described. Although both populations were mainly susceptible to the antibiotics used for treatment and prophylaxis, the proportion of meningococci with reduced penicillin susceptibility was slightly higher in Sweden. A large proportion of the African isolates was resistant to tetracycline and erythromycin. In paper V, the gene linked to rifampicin resistance, the rpoB gene, was examined in meningococci from 12 mainly European countries. Alterations of three amino acids in the RpoB protein were found to always and directly lead to rifampicin resistance. A new breakpoint for rifampicin resistance in meningococci was suggested. The biological cost of the RpoB alterations was investigated in mice. The pathogenicity/virulence was significantly lower in rifampicin resistant mutants as compared with susceptible wild-type bacteria.