Giardia intestinalis is a unicellular protozoan parasite, causing giardiasis – a gastro-intestinal disease of variable outcome and severity, in a broad range of mammalian species. The parasite has a comparatively simple life cycle with two stages, proliferative trophozoites and infectious cysts, as well as a reduced set of eukaryotic-specific organelles. Its metabolic pathways are simplified if compared to non-parasitic organisms. Giardia compensates for this apparent simplicity with unique inventions and complex regulation of metabolic processes. Transition from a fragile trophozoite to a protected, compact cyst is called encystation. This process starts upon changes in the growing conditions: cholesterol deprivation and elevated pH, and leads to changes in membrane lipids, elevated cAMP, and induction of encystation-specific gene expression, starting with activation of Myb-like protein expression. Within hours postencystation induction, cyst wall components (GalNAc and CWPs) are produced and transported to the cell membrane, flagella and adhesive disc are disassembled and stored in cytoplasm, followed by DNA replication and diplomixis. One of the encystation-specific upregulated genes in Giardia (assemblage A, isolate WB) is GL50803_1470 (termed ORF1470). Its predicted protein product has an Alba_2 domain, binding nucleic acids, presumably DNA. To study its function in G. intestinalis, we created knockout mutants, using CRISPR/Cas9 technique, Cas9- HA cell line and pGdelp-BbsI-B00826, with integrated pac- and gRNA cassettes. Transfected Giardia Cas9-HA cells were PCR verified for the complete knockout of the gene, encysted, and effect of ORF1470 was studied using cyst counting, morphometric analysis, cell imagining and Western blot for detection of CWPs. We have found minor phenotypical differences between the parental strain Cas9-HA and wild-type WB and ORF1470 deficient cells. Future plans are further experiments with obtained ΔORF1470 strains including further KO verification, visualization of ORF1470 product during encystation and determination of its binding site in the genome using ChIP-seq technology.

En av smittvägarna för patogena bakterier inom vården är via kontakt med ytor kontaminerade av bakterier. Miljöodlingar är ett sätt att hitta smittrisker i miljön. Det finns dock inga tydliga riktlinjer för hur dessa ska utföras inom vården. Syftet med studien var att vid miljöodling testa om provtagning med E-swab och FLOQSwab fungerar lika effektivt som en tryckplatta vid detektion av vankomycinresistenta enterokocker (VRE) på fyra typer av ytor som är vanligt förekommande inom sjukhusmiljöer. Olika bakteriekoncentrationer av VRE användes för att kontaminera fyra olika typer av ytor. De tre olika miljöodlingsmetodernas effektivitet bestämdes genom att jämföra antalet detekterade kolonier. Resultatet visade att tryckplattan var mer effektiv än provtagningspinnarna på att detektera VRE. Vid jämförelse mellan E-swab och FLOQSwab visade de vara likvärdiga, då det inte förekom någon skillnad i antalet detekterade kolonier. Desto lägre bakteriekoncentration som användes, desto färre blev de positiva odlingarna. Från den lägsta koncentrationen var det endast tryckplattan som genererade enstaka positiva odlingar. Slutsatsen var att tryckplattan var den mest effektiva metoden när det kommer till att detektera VRE i miljön då de genererade flest positiva odlingar och detekterade fler kolonier vid detektionsgränsen än provtagningspinnarna. / One source of infection for pathogenic bacteria in healthcare is due to contact with surfaces contaminated by bacteria. Environmental sampling is a good way to find risks of infection in the environment. However, there are no clear guidelines on how to perform them in healthcare. The aim of this study was to test if sampling with E-swab and FLOQSwab are as effective ascontact plates for detection of vancomycin-resistant enterococci (VRE) on common surfacesin hospital environments. Different bacterial concentrations of VRE were used to contaminate four types of surfaces. The efficiency of the three types of environmental sampling methodswere determined by comparing the quantity of detected colonies. The result showed that the contact plate was more efficient than the swabs at VRE detection. When comparing the swabs with each other they were shown to be equivalent, they detected the same quantity of colonies.Lower bacterial concentration resulted in fewer positive cultures, and the lowest concentration only the agar plate generated a few positive cultures. In conclusion, the contact plate was the most efficient method for detecting VRE in the environment as it generated the most positive cultures and detected more colonies around the detection limit than the swabs.

Klebsiella pneumoniae is a Gram-negative bacterium commonly giving rise to nosocomial infections especially in immunocompromised individuals. The high occurrence of antibiotic resistant K. pneumoniae strains in combination with the bacterium’s ability to form biofilms that are naturally more tolerant to antibiotic treatment represents a burden for the healthcare system due to potential therapeutic failure. In this study, we were investigating the effect of gentamicin, cefotaxime and ciprofloxacin on biofilm formation and eradication of a multi-resistant ESBL-producing K. pneumoniae strain associated with an outbreak at the Uppsala University Hospital in Uppsala, Sweden, from 2005 to 2007. Multidrug resistance was encoded on the pUUH239.2 plasmid. We also studied how resistant bacteria are selected for in mixed biofilm populations consisting of both resistant and susceptible bacteria under antibiotic treatment. Biofilms were grown in vitro by using an in-the-lab developed peg model. Biofilms were either allowed to form in the presence of the antibiotics or pre- formed and then exposed to antibiotic treatment at different time points. Our results suggest that gentamicin, cefotaxime and ciprofloxacin inhibit biofilm formation at concentrations below the minimum inhibitory concentration (MIC) and that biofilms become more tolerant to antibiotic treatment with increasing maturation. We further observed that increasing antibiotic concentrations select for the presence of the plasmid in less mature biofilms. Overall, these findings highlight the need of early antibiotic treatment during infection and give insight into the dynamics of resistant and susceptible bacteria in mixed biofilm populations in the presence of antibiotics.

<p>Malaria is the most common parasitic disease of humans worldwide. A factor that aggravates the many attempts to control the epidemiologic malaria situation is the spreading of resistance against anti-malarial drugs. In this project the point mutation at position 86 of the <em>Plasmodium. </em><em>falciparum</em><em> </em>multidrug resistance gene (<em>pfmdr1</em>), which is thought to contribute to Chloroquine resistance, was analysed in 188 samples from a low transmission area in eastern Sudan, where malaria endemicity is seasonal. The patient group studied had asymptomatic and sub patent parasitemia that persisted during the transmission-free dry season, after being treated with Chloroquine. To differentiate between wild type and mutant genotypes, nested PCR and restriction fragment length polymorphism with the enzyme Apo1 was used. Out of 188 samples 79 (42%) were successfully analysed. Of those, 72% had parasites with mutant genotypes or where mixed infection. No conclusions on the relevance of the <em>pfmd</em><em>r</em><em>1</em> gene in the studied samples are made due to the many remaining gaps. However, eventual sources of error and previous findings in the study area are discussed.</p>

<p>The third domain of life, Archaea is one of the three main evolutionary lineages together with the Bacteria and the Eukarya domains. The archaea are, despite their prokaryotic cell organisation, more closely related to eukaryotes than to bacteria in terms of the informational pathways (DNA replication, transcription and translation). Organisms from the archaeal hyperthermophilic genus <i>Sulfolobus</i> thrives in a hot (80°C), acidic (pH 2-4) and sulphur-rich environment.</p><p>In my thesis, I have used a variety of different approaches to study the <i>Sulfolobus</i> cell cycle. After dilution of a stationary phase cell culture with fresh medium, synchronous cell cycle progression was obtained. From the synchronised cell culture experiment we could conclude that the major cell cycle events (nucleoid segregation, cell division and chromosome replication) were tightly coupled to each other and to cellular mass increase. </p><p>Inhibitors of the elongation stage of chromosome replication, and of cell division, as well as drugs arresting the cell cycle in the post-replicative phase, were found in an in vivo screening of a range of antibiotics. The cell cycle was found to be regulated such that the previous cell cycle step had to be successfully accomplished before the next could initiate, except for DNA replication which could occur without an intervening cell division event.</p><p>The replication pattern of <i>Sulfolobus solfataricus</i> was analysed using a marker frequency assay. From the results, we were able to determine that a single origin is utilized in vivo, that the replication directionality is bidirectional, and also an approximate location of the replication origin within the genome.</p><p>Intracellular virus production in vivo of SIRV2 (<i>Sulfolobus islandicus</i> rod-shaped virus2) in <i>Sulfolobus islandicus</i> was also analysed. The effects on the host cell were determined, including loss of cell viability, inhibited initiation of replication at virus infection and DNA degradation and loss of cell integrity at the time of virus release. Also, for the first time intracellular virus DNA was visualized with flow cytometry.</p>

<p>Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. </p><p>In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using <i>in vivo</i> reporter gene fusion expression and different <i>in vitro </i>methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.</p><p>Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.</p><p>Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.</p>

<p><i>Paenibacillus polymyxa</i> belongs to the group of plant growth promoting rhizobacteria (PGPR). Activities associated with <i>P. polymyxa</i>-treatment of plants in earlier experiments include, e.g., nitrogen fixation, soil phosphorus solubilization, production of antibiotics, auxin, chitinase, and hydrolytic enzymes, as well as promotion of increased soil porosity. My thesis work showed that, in stationary phase, <i>P. polymyxa</i> released the plant hormone cytokinin isopentenyladenine, in concentrations of about 1.5 nM.</p><p>In a gnotobiotic system with <i>Arabidopsis thaliana</i> as a model plant, it was shown that <i>P. polymyxa</i>-inoculation protects plants; challenge by either the pathogen <i>Erwinia carotovora</i> (biotic stress) or induction of drought (abiotic stress) showed that pre-inoculated plants were significantly more resistant than control plants. By RNA-differential display on RNA from <i>P. polymyxa</i>-treated or control plants, changes in gene expression were tested. One mRNA, encoding ERD15 (drought stress-responsive gene) showed a strong inoculation-dependent increase in abundance. In addition, several biotic stress-related genes were also activated by <i>P. polymyxa</i>. </p><p>Antagonism towards the fungal pathogens <i>Phytophthora palmivora</i> and <i>Pythium aphanidermatum</i> was studied. <i>P. polymyxa</i> counteracted the colonization of zoospores of both oomycetes on <i>A. thaliana</i> roots, and survival rates of plants treated with <i>P. polymyxa</i> were much higher when challenged by <i>P. aphanidermatum</i>. </p><p>Using a green fluorescent protein-tagged isolate of <i>P. polymyxa</i>, colonization of <i>A. thaliana</i> roots was investigated. Two main conclusions can be drawn. Firstly, the bacterium enters the root tissue (but not leaves) and is abundantly present in intercellular spaces. Secondly, the root becomes severely damaged, indicating that – under some conditions – <i>P. polymyxa</i> is a "deleterious bacterium", and in others it promotes growth. Based on work presented in my thesis, I argue that a balance between the activities of a PGPR, the genetic background and physiological state of a plant, and the environmental conditions employed in test systems, ultimately determines the resulting effect. </p>

<p>Cell division in the Gram-positive bacterium <i>Streptomyces coelicolor</i> starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of <i>S. coelicolor</i> exploit two types of cell division with strikingly different outcomes depending on the developmental stage. </p><p>The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery.</p><p>By isolation and characterization of a non-sporulating <i>ftsZ</i> mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The <i>ftsZ17</i>(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation.</p><p>An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. </p><p>The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of <i>ftsZ</i> expression and the turnover of the FtsZ protein. </p><p><i>S. coelicolor</i> is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like <i>Escherichia coli</i>. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves. </p>

Scaling down operations and functions into the fascinating micro world not only improve performance, lower costs, and enable easier integration, but also opens the door to new functionalities. This truly multidisciplinary thesis presents novel solutions to current and relevant challenges in the areas of 1) on-chip liquid manipulation which has applications in micro total analysis systems, medical diagnostics, and drug discovery and 2) pressure sensing which has an established market in the automotive and industrial processes industry. Especially in the area of liquid manipulation, the aim was to take advantage of forces and properties dominating on the micro scale whenever possible, rather than compensating for these effects, and to create solutions with universal appeal and application areas. In the area of liquid manipulation, this thesis discusses a novel method of passively synchronizing liquid movement on-chip based on liquid surface tension and device geometry. This technique has potential applications in timing independent processes, liquid-liquid interactions, and digitizing liquid movement. A fast and passive discrete sample micromixer is also presented based on the same principles. A unique way of direct access, bubble tolerant sample interfacing with flow-through microfluidics using a closed-open-closed channel is also introduced. This method can be used to regulate flow on-chip without the need for any moving parts or electrical contact. Moreover, work is presented on two types of out-of-plane electrospray ionization mass spectrometry (ESI-MS) emitter tips which mimic ideal mass spectrometry tips. Fabrication of these tips is uncomplicated and results in robust structures with good performance. In the field of pressure sensing, this thesis investigates a form based resonating principle. The Q factor of the sensor is improved by low pressure encapsulation and structure design. A novel technique for excitation and detection of resonant microsensors using 'burst' technology is also demonstrated. This method involves temporally separating excitation and detection, thereby eliminating crosstalk and the need for electrical feedthroughs. It also allows high voltages to be used with sensitive circuitry and a single electrode to be used for both excitation and detection.

The third domain of life, Archaea is one of the three main evolutionary lineages together with the Bacteria and the Eukarya domains. The archaea are, despite their prokaryotic cell organisation, more closely related to eukaryotes than to bacteria in terms of the informational pathways (DNA replication, transcription and translation). Organisms from the archaeal hyperthermophilic genus Sulfolobus thrives in a hot (80°C), acidic (pH 2-4) and sulphur-rich environment. In my thesis, I have used a variety of different approaches to study the Sulfolobus cell cycle. After dilution of a stationary phase cell culture with fresh medium, synchronous cell cycle progression was obtained. From the synchronised cell culture experiment we could conclude that the major cell cycle events (nucleoid segregation, cell division and chromosome replication) were tightly coupled to each other and to cellular mass increase. Inhibitors of the elongation stage of chromosome replication, and of cell division, as well as drugs arresting the cell cycle in the post-replicative phase, were found in an in vivo screening of a range of antibiotics. The cell cycle was found to be regulated such that the previous cell cycle step had to be successfully accomplished before the next could initiate, except for DNA replication which could occur without an intervening cell division event. The replication pattern of Sulfolobus solfataricus was analysed using a marker frequency assay. From the results, we were able to determine that a single origin is utilized in vivo, that the replication directionality is bidirectional, and also an approximate location of the replication origin within the genome. Intracellular virus production in vivo of SIRV2 (Sulfolobus islandicus rod-shaped virus2) in Sulfolobus islandicus was also analysed. The effects on the host cell were determined, including loss of cell viability, inhibited initiation of replication at virus infection and DNA degradation and loss of cell integrity at the time of virus release. Also, for the first time intracellular virus DNA was visualized with flow cytometry.