Background: Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens AdvanceTM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS). Methods: Peripheral blood samples from 522 patients with a clinical diagnosis of "suspected malaria" were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. Results: Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM. Conclusions: The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians' clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

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Bacterial infections are a leading cause of death worldwide with over 7 million bacterial infection-related deaths reported in 2019 alone. Metal oxide- and polyelectrolyte-based antimicrobials are widely studied and used, but their mechanisms of action and interactions with bacterial cells and other substances found in nature are incomplete. The overall goal of this work is to gain insight on the mechanisms of metal oxide and polycationic disinfectants. The first part of this work presents the development and study of antimicrobial facemasks that kill 99.9% of pathogenic bacteria responsible for hospital acquired infections, Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae within 30 minutes. These facemasks were made by adhering cuprous oxide (Cu2O) microparticles to polypropylene textiles. Furthermore, we investigated whether surface contact was necessary to kill bacteria in drying droplets on the facemask surface and found that surface contact produced better killing than exposure to Cu+ ions that dissolve into the droplet. The second part of this work describes the mechanism of action of polycationic antimicrobials. The goals of this section were twofold. Our first aim was to develop a method of studying both polycation adsorption on bacterial cells and antimicrobial activity with single-cell and time-resolved measurements. Our second aim was to determine whether the surface density of antimicrobial required for kill is a better metric of effectiveness than solution concentration required for kill. We adsorbed bacteria to the surface of glass coverslips inside a flow cell. We then flowed through the cell a mixture of fluorescently tagged polycation, polydiallyldimethyl ammonium chloride (PDADMAC) tagged with Cy3, and a fluorescent dye, Sytox Blue, that indicated membrane permeability. This allowed us to image the density of PDADMAC on the cell surface, as well as the time taken for individual cells to permeabilize using fluorescence microscopy. We found a time lag of 5–10 minutes between adsorption and death and found that the time-to-die of an individual cell was well correlated with the rate of adsorption. We also found that the time-to-die and equilibrium adsorption differed among species but followed a trend of more adsorption onto bacterial species with a more negative zeta potential. Most importantly, we found that there was a wide range of cell responses, highlighting the usefulness of single-cell measurements in addition to ensemble-average measurements. Polycationic disinfectants need to be deployed in a wide range of environments, ranging from almost pure water to hypertonic salt. Owing to their cationic charge, one would expect the salt content of the medium to affect the antimicrobial action. So, we investigated the effect of ionic strength on polycation antimicrobial activity. We used our previous method to measure the time-course of adsorption and kill of our labelled cationic polymer in NaCl solution. We found that addition of NaCl decreased the density of polymer adsorption and diminished efficacy of PDADMAC. At salt concentrations at or above 0.15 M, which is similar to normal saline, PDADMAC was no longer bactericidal but instead bacteriostatic (stops growth). Fluorescence depolarization measurements showed that PDADMAC rigidified model bacterial membranes, but salt reduced this rigidity. We also found that the bacteriostatic effect is reversible, and cell growth resumed once PDADMAC was removed. The third section of this work focused on the effect of capillary geometry on the height of capillary rise. Angled capillaries are common in natural and engineered systems such as porous media. However, this system has not been studied by experiment or modelling. The goal of this section is to examine the equilibrium height of a meniscus in a trapezoidal capillary as an example of an angled capillary. To do this, we constructed glass capillaries from hydrophilic borosilicate glass microscope slides and used pure water or ethanol-water solutions as the liquid. This system was modelled by numerical solution to the Laplace equation to obtain the shape of the vapor-liquid interface. Both experiment and theory showed that there is less capillary rise for greater wall angles of the trapezoid, and the rise is more sensitive to the wall angle (α) than the contact angle (θ) at angles close to vertical. / Doctor of Philosophy / Bacterial infections are a leading cause of death, with over 7 million deaths from bacterial infections reported in 2019. Many disinfectants use metal oxide particles or polymers as the active ingredient. While these are very effective at cleaning surfaces, the exact mechanism of how they work is still unknown. The overall goal of our research is to understand how metal oxide and polymer-based antimicrobials work so we can design better disinfectants and deploy them in environments where they are effective. The first part of my work focused on making a facemask that could both filter microbes out of the air like a regular facemask but then kill microbes that have landed on the mask. To make the facemasks, we adhered antimicrobial solid (cuprous oxide) particles to material (polypropylene) that is used to make facemasks. We found that our treated facemasks killed 99.9% of adsorbed bacteria within 30 minutes, and we found that bacteria die faster when they were forced to contact the surface. The second section of this work focused on understanding how polymer-based disinfectants work. Polycations are polymer molecules that have positive charges on one or more of the repeat units. Polycations are widely studied disinfectants, but their mechanism is not completely understood. The goals of our research were to 1) find a method of determining the amount of polymer on a bacterium and whether or not the bacterium was alive on a single-cell level, and 2) determine which was more important in determining bacterial kill: the amount of antimicrobial on the cell surface or the amount in solution. We found that cells died about 5 – 10 minutes after the polymer adhered to the cell. We also found that the time-to-die and maximum amount of polymer that can adhere to the cell differed among species but followed a trend of more adherence on more negatively charged species. Most importantly, we found that individual bacteria of the same species had a very large range of response, which highlights the usefulness of measuring the response of individual cells. We continued our polymer research to study how salt affects polymer antimicrobial efficacy. Antimicrobials need to be deployed in a wide range of environments ranging from pure water to saltwater. As they are charged, they will interact with other charged species in the sample, such as salts. Once the salt concentration rose to ~1% - which is the same concentration as found in blood serum – the polycation no longer killed bacterial cells, but instead, halted their growth. Additionally, polycations make bacterial cell membranes more rigid, and this effect disappears at high salt concentrations. Last, we found that the halted cell growth is reversible, and cells will regrow once the polymer is removed. The third section of this work focused on capillary rise in angled pores. Capillary rise is a well-studied effect where surface tension pulls liquid confined in a thin capillary against gravity. Capillaries with angled walls occur in nature and in engineered systems. However, there is no work that describes how the capillary rise is affected by an increase in capillary width with height. Our goal was to use experiment and theory to examine the liquid rise in trapezoidal capillaries. The test liquids were pure water or water-ethanol mixtures. Both theory and experiment showed that capillary rise decreased as the angle of the capillary increased, and that the rise was more sensitive to the wall angle (α) than the contact angle (θ).

L'expression génétique est un processus cellulaire fondamental réglé de manière ne. Les promoteurs inducibles perme ent de perturber l'expression génétique en changeant l'expression d'une protéine par rapport à son niveau physiologique de référence. Ce e propriété en fait un outil incontournable pour décrypter le fonctionnement des processus biologiques via la comparaison du comportement de la cellule sous divers niveaux d'induction. Toutefois, une limite actuelle à l'utilisation des promoteurs inducibles provient de la difficulté à appliquer des perturbations précises et dynamiques. Les deux obstacles principaux étant: (i) la variabilité intercellulaire ainsi qu'à la nature aléatoire de l'expression génétique qui limite la précision de la perturbation appliquée. (ii) la difficulté à prèdire quantitativement le comporteement des systèmes biologiques sur les longues periodes requises pour des objectifs d'expression variables dans le temps. Or des perturbations précises et changeant dans le temps perme ent d'obtenir de riches informations sur la dynamique d'un système biologique. Est présenté ici une plate-forme de contrôle temps réel en boucle fermée qui permet le contrôle quantitatif sur une longue durée de l'expression génétique chez la levure. Ce e plateforme utilise la microscopie par uorescence pour suivre l'expression génétique, un système micro uidique pour interagir avec l'environnement cellulaire ainsi qu'un logiciel perme ant l'analyse d'image en temps réel et le calcul de la stratégie de contrôle à appliquer. Ce système permet le contrôle de l'expression d'un gène chez la levure, tant au niveau d'un population cellulaire qu'au niveau de la cellule seule et ceci pour un objectif d'expression constant ou dépendant du temps. Le système de réponse au chocs hyper-osmotiques de la levure S. cerevisiae (HOG pathway) a été utilisé pourin uencer l'expression génétique. Toutefois, la possibilité d'utiliser un autre système d'induction sans profondes modi cations de la plate-forme est démontrée. De surcroît au développement de ce e plate-forme est également ici démontré la possibilité de contrôler le système HOG. A n de comprendre la dynamique cellulaire et de pouvoir la quanti er, il est nécessaire de pouvoir appliquer des perturbations précises. La plate-forme de contrôle de l'expression génétique présentée ici permet de perturber avec précision le niveau d'expression d'une protéine et représente donc une contribution majeure dans ce e direction.

The understanding of how white blood cells react when coming into contact with various surfaces is of major importance for a wide range of biomaterials and biosensor applications. In this study it is investigated if it is possible to determine how neutrophils react to a certain type of sensor chip called cell clinic being developed. This study investigates the cell surface coverage on the sensor chip and how it correlates to the signal response of the sensor at hand. Neutrophils, as other white blood cells, are cells that quickly adhere to surfaces and during the adhesion process they activate at different levels depending on i.e. type of surface or surface functionalization, this activation can be visualized by the change in morphology. While measuring the change of capacitance with the cell clinic sensor during cell adhesion, the cell surface coverage is of main importance. The main focus of this diploma work has been to develop an image analysis script capable of conducting automated analysis on a large body of images estimating the surface coverage. Input data for this modeling is taken from fluorescent microscopy images. The experiments conducted during this project have indicated that white blood cells adhered to the sensor surface shows signs of being activated also without external activation. This clearly shows that knowledge of how neutrophils react to surface modifications is of great importance as well as the awareness that any surface may trigger a response from the immune system i.e. neutrophil activation, so also in the cell clinic. It is a fact that it might be difficult to evaluate the effect of a foreign substance on the neutrophils while a significant amount is activated from being in contact with the surface. Regarding different surfaces the white blood cells does not display any preference of adhering to any specific surface. The surfaces used in this project was silicon oxide wafers, silicon oxide wafers with a nitride surface functionalization and the intended sensor chip; however the addition of PMA clearly shows an effect on how many cells that adheres to the surface as well as the average area of each cell.

Natural killer (NK) cells represent indispensable part of the innate immunity as they are capable of promptly identifying virally infected or tumor cells and participating in the regulation of adaptive immune responses. These functions are ensured by the interplay between NK receptors, creating a complex regulatory system. Solving the receptors' structure may contribute to an overall understanding of NK cell biology. Presented thesis describes an elucidation of the structure of the inhibitory C-type lectin-like receptor (CTLR) Nkrp1b with an emphasis toward structural features (stalk, loop and oligomerization state) which might affect conformation or interactions of this receptor. The interaction of Nkrp1b with its ligand, Clr-b protein, is immunologically significant as it regulates NK cells' activity independently and monitors changes that are not visible to cytotoxic T lymphocytes. To study individual structural aspects of Nkrp1b, two protein variants were recombinantly prepared in bacterial expression system: entire ectodomain and ligand-binding domain lacking the stalk. Using a range of mass spectrometric techniques in combination with homology modeling and molecular dynamics, we proposed the Nkrp1b structure including its monomeric and dimeric arrangements. In addition, the oligomerization...

Extracellular vesicles are important players in cell-to-cell communication and have the potential to be used as biomarkers for decease. The mechanical properties of extracellular vesicles is an active field of research, with new methods and models being developed. In this thesis, two samples of extracellular vesicles containing different levels of membrane protein expressions are investigated using atomic force microscopy. Three models for determining stiffness are applied to the samples: the Hertz model, an adhesion angle dependent model, and the modified Canham-Helfrich model. The Hertz model indicated a higher Young’s Modulus for vesicles without membrane proteins, but with large errors. The adhesion angle dependent model did not provide high enough sensitivity to determine any difference in stiffness between the two samples. The modified Canham-Helfrich model indicated a higher bending modulus for the vesicles with membrane proteins. The results highlight the importance of taking several measurements on each vesicle, in contrast to how the method is currently applied in research. / Extracellulära vesiklar är essentiella komponenter inom cell-till-cell-kommunikation, och har potentialen att kunna användas som sjukdomsmarkörer. Extracellulära vesiklars mekaniska egenskaper är ett aktivt forskningsfält där nya experimentella metoder och teoretiska modeller är under utveckling. I den här arbetet används atomkraftsmikroskopi för att undersöka de mekaniska egenskaperna av två prover av extracellulära vesiklar med olika mängd membranproteiner. Tre modeller för att utvärdera deras styvhet tillämpas: Hertz-modellen, en adhesionsvinkelberoende modell, och den modifierade Canham-Helfrichmodellen. Hertz-modellen indikerade högre elasticitetsmodul för provet med lägre antal membranprotein, men med stora fel i mätningarna. Den adhesionsvinkelbaserade modellen var inte känslig nog att påvisa några skillnader i styvhet mellan proverna. Den modifierade Canham-Helfrich-modellen indikerade att vesiklarna med membranprotein har högre böjmodul än veiklarna utan membranprotein. Resultaten understryker att det är viktigt att göra flertalet mätningar på varje vesikel, i kontrast mot hur modellen tillämpas i dagsläget.

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The prokaryotic adaptive immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a defense mechanism that helps to protect the prokaryotic cell from invading mobile genetic elements. This project was performed at Uppsala University and served to answer whether the expression of Cascade, which is part of the CRISPR defense system, will have a negative effect on the cell that expresses it and to also determine whether the CRISPR defense system is effective enough to stop the spread of a conjugative plasmid. A microfluidic system was used in order to perform the experiments and images were taken with the help of fluorescent microscopy. Three different donor strains from E.coli were used. These strains had their own version of the RP4 conjugative plasmid which had the ability to infect recipient E.coli cells with said plasmid. The recipient cells had the ability to express the CRISPR system in order to defend themselves from the plasmid and CRISPR was also inducible with the help of IPTG. The different versions of the RP4 conjugative plasmid had different amounts of spacer targets that Cascade, the recognition complex in the CRISPR system, could recognize. When the recipient cells were induced and had a known target sequence of the plasmid they were able to defend themselves and keep the number of transconjugant cells low. When the recipient cells did not know the target the amount of transconjugant cells were higher. It was also noted that when the cells were induced inside the microfluidic PDMS chip they had a slower generation time. It was also noted that recipient cells had begun to die towards the end of the microfluidic experiments when the cells were induced. This raised the question as to whether the CRISPR defense system was targeting itself as well as the RP4 conjugative plasmid.