Monoploid genotypes (2n = x = 12), derived by anther culture of a diplandrous genotype of Solanum phureja, a South-American diploid potato species, were examined for their utility in germplasm development. Nine monoploid genotypes and the diploid anther-donor plant were grown in photoperiod chambers at The Southeastern Plant Environment Laboratories (SEPEL) at North Carolina State University to examine the effect of photoperiod on tuber yield and to determine the variability for critical photoperiod for tuberization. Significant differences were found among the monoploid genotypes for total tuber weight and tuber number. Longer photoperiod treatments both decreased and delayed tuberization. Axillary tuber formation from single-node cuttings was used to estimate the onset of tuber induction and demonstrated variability among monoploid genotypes for critical photoperiod for tuberization. Leaf-disc culture of 24 monoploid genotypes yielded calli which regenerated plants from three genotypes. SDS-polyacrylamide gel electrophoresis of leaf extracts demonstrated variability among diploid and tetraploid calliclones of one monoploid genotype for total protein banding pattern. Absence of stainable pollen and lack of seed set after crosses to diploid species and tetraploid cultivars illustrated infertility among doubled (2n = 2x = 24) and twice doubled (2n = 4x = 48) monoploid-derived lines. Flow-cytometric analysis of pollen obtained from the diploid anther-donor genotype grown under three photoperiods at SEPEL yielded two populations of pollen based on propidium iodide staining of DNA. These populations corresponded to pollen separation based on size parameters alone, introducing the potential for flow sorting of pollen to increase seed set in 4x-2x crosses to tetraploid cultivars. Protoplast isolation from in vitro material and extraction of leaf nuclei both in vitro and in vivo were performed on the anther-donor plant, one of its anther-derived monoploids, and a diploid and tetraploid plant derived from callus culture of the monoploid genotype. Flow-cytometric analysis of propidium-iodide Stained cells and nuclei showed a greater ploidy stability for plant material grown in vitro and a limit to endopolyploidization imposed by initial ploidy level. Flow-cytometric analysis of protoplast-derived nuclei from nine monoploid genotypes derived from anther culture of a single diploid genotype exhibited Significant differences for 4C DNA content, but not for 1C DNA content, indicating that ploidy stability, rather than monoploid status per se, is influenced by genotype. / Ph. D.

Over the last 50 years, flow cytometry has evolved from a modest cell counter into an invaluable analytical tool that measures an ever-expanding variety of phenotypes. Flow cytometers interrogate passing samples with laser light and measure the elastically scattered photons to ascertain information about sample size, granularity, and basic morphology. Obtaining molecular information, however, requires the addition of exogenous fluorescent labels. These labels, although a power tool, have numerous challenges and limitations such as large emission spectra and cellular toxicity. To move beyond fluorescent labels in microscopy, a variety of techniques that probe the intrinsic Raman vibrations within a sample have been developed, such as coherent anti-Stokes Raman scattering (CARS) and Raman microspectroscopy. In this dissertation, I present the first development of a label-free flow cytometer that measures the elastically scattered photons and probes the intrinsic Raman vibrations of passing samples using multiplex coherent anti-Stokes Raman scattering (MCARS). MCARS, a coherent Raman technique that probes a large region of the Raman spectrum simultaneously, provides rich molecularly-sensitive information. Furthermore, I present its application to sorting polymer microparticles and its use in two example biological applications: monitoring lipid bodies within cultures of Saccharomyces cerevisiae, a model yeast with numerous human homologs, and monitoring the affect of nitrogen starvation on Phaeodactylum tricornutum, a diatom, which is being genetically engineered to efficiently produce biofuels.

Considerable variation exists both within and between species in nuclear DNA content. Despite there being no obvious functional role for much of this DNA, many studies have reported phenotypic correlations with genome size at various taxonomic levels. This suggests that DNA plays a functional role beyond the traditionally understood mechanisms. One such example of a phenotypic correlation with DNA content is present in the genus Silene, where a negative correlation between DNA content and flower size exists within and between species. This relationship is consistent with the direction of sexual dimorphism in DNA content (caused by heteromorphic sex-chromosomes) and flower size in the most studied species in the genus: S. latifolia. This thesis takes a comparative approach between two closely related species in the genus (S. latifolia and S. marizii), which differ markedly in their nuclear DNA content, in order to investigate the nature and phenotypic impacts of variation in DNA content. A phenotypic survey from a number of S. marizii populations reveals that the pattern of DNA content variation in this species is very different to that in S. latifolia. In particular, phenotypic correlations with DNA content appear be much weaker, whilst sexual dimorphism in DNA content, when present, appears to occur in either direction. A survey of interspecific hybrids suggests that this may be due to an enlarged S. marizii X-chromosome and that DNA content in hybrids may be biased with regard to their parents. Repetitive elements may be significant constituents of plant genomes. A study of Ty1-copia class retrotransposons in the two species reveals that they are present as a large and highly heterogeneous population. Phylogenetic analysis of these elements suggests a substantial degree of genetic isolation between the two species. Finally, an assessment of the flow-cytometric method, used to estimate DNA content, reveals substantial error associated with the method, but only limited evidence for stoichiometric effects.

Biotechnological processes are distinguished from classical chemistry by employing bio-molecules or whole cells as the catalytic element, providing unique reaction mechanisms with unsurpassed specificity. Whole cells are the most versatile \'factories\' for natural or non-natural products, however, the conversion of e.g. hydrophobic substrates can quickly become cytotoxic. One host organism with the potential to handle such conditions is the gram-negative bacterium Pseudomonas putida, which distinguishes itself by solvent tolerance, metabolic flexibility, and genetic amenability. However, whole cell bioconversions are highly complex processes. A typical bottleneck compared to classical chemistry is lower yield and reproducibility owing to cell-to-cell variability. The intention of this work was therefore to characterize a model producer strain of P. putida KT2440 on the single cell level to identify non-productive or impaired subpopulations. Flow cytometry was used in this work to discriminate subpopulations regarding DNA content or productivity, and further mass spectrometry or digital PCR was employed to reveal differences in protein composition or plasmid copy number. Remarkably, productivity of the population was generally bimodally distributed comprising low and highly producing cells. When these two subpopulations were analyzed by mass spectrometry, only few metabolic changes but fundamental differences in stress related proteins were found. As the source for heterogeneity remained elusive, it was hypothesized that cell cycle state may be related to production capacity of the cells. However, subpopulations of one, two, or higher fold DNA content were virtually identical providing no clear hints for regulatory differences. On the quest for heterogeneity the loss of genetic information came into focus. A new work flow using digital PCR was created to determine the absolute number of DNA copies per cell and, finally, lack of expression could be attributed to loss of plasmid in non-producing cells. The average plasmid copy number was shown to be much lower than expected (1 instead of 10-20). In conclusion, this work established techniques for the quantification of proteins and DNA in sorted subpopulations, and by these means provided a highly detailed picture of heterogeneity in a microbial population.

Light allows to non-invasively study the complex and dynamic biological phenomenon undergoing within cells and tissues in their native state. The development of super-resolution microscopes in recent years has helped to overcome the fundamental limitation imposed by Abbe’s diffraction limit, thereby revolutionizing the field of molecular and cellular biology. With the advancement of various super-resolution techniques (like STED, PALM, and 4Pi) it is now possible to visualize the nanometeric cellular structures and their dynamics in real time. The limitations of existing fluorescence microscopy techniques are: poor axial resolution when compared to their lateral counterpart, and their inability to produce high resolution images of dynamic samples. This thesis covers two broadly connected areas of fluorescence imaging techniques while addressing these limitations. First, the PSF engineering and spatial filtering technique for axial super-resolution microscopy and second, the integration of light sheet illumination PSF with microfluidic cytometry for imaging cells on-the-go. The first chapter gives an explicit description on the fundamentals of fluorescence imaging. This introductory chapter includes a variety of optical microscopes, PSF engineering, the resolution limit imposed by the wave nature of light, the photochemistry of the fluorescent dyes, and their proper selection for fluorescence experiments. In addition to the state-of-art imaging techniques, namely Laser Scanning Confocal Microscopy and Light Sheet Microscopy, this chapter also gives a brief explanation on the evolution of imaging cytometry techniques. Their high speed analytic capability (i.e sorting and counting) makes this technique an important tool in health care diagnosis and other various biomedical applications. The chapter ends with a discussion on the operating principle of the flow cytometers and their limitations. The second chapter in this thesis describes the spatial filtering technique for engineering the PSF to eliminate the side-lobes in the system PSF of the 4Pi Confocal Microscopes. Employing an amplitude mask with binary light transmission windows (also called binary filters), the incident light is structured to minimize the secondary lobes. These lobes are responsible for exciting the off-focal planes in the specimen, hence provide incorrect map of the fluorophore distribution in the object. The elimination of the side-lobes is essential for the artifact-free axial super-resolution microscopy. This second chapter describes the spatial filtering technique in details (its mathematical formulation, application in fluorescence microscopy for generation of desired PSF including Bessellike beam). Specifically, spatial filtering technique is employed in 4Pi type-C Confocal Microscope. The spatial mask used results in the reduction of the side-lobes in 1PE case while they are nearly eliminated in 2PE variant of the proposed technique. The side-lobes are reduced by 46% and 76% for 1PE and 2PE when compared to the existing 4Pi type-C Confocal Microscope system. Moreover, OTF of the proposed system confirms the presence of higher frequencies in the Fourier domain indicating high resolution imaging capability. Apart from the resolution in lateral and axial dimension, achieving high resolution while imaging dynamic samples is another challenge that is limiting the field of fluorescence microscopy to flourish. The third and fourth chapters are entirely dedicated towards the work that was carried out to develop imaging techniques on a microfluidic platform for imaging dynamic samples. The fusion of microscopy and flow cytometry has given rise to the celebrated field of imaging flow cytometry. In recent years, the focus has shifted towards miniaturized cytometry devices. Apart from the reduced cost of the sample reagents and the assays, portability and easy handling make the microfluidic devices more relevant to developing countries. The commercially available cytometers are bulky and quite costly. In addition to these practical concerns, they are complex in operation and limited in performance. Most of the existing cytometers use different inlets for sheath and sample flow to achieve the hydrodynamic focusing of the sample assays in a narrow and confined region. The laser beam in the illumination arm interrogates with the flowing samples at this region and the response is captured by the detection optics. The same principle is extensively used in most of the microfluidic based flow cytometers reported till date. Apart from the hydrodynamic force other effects like electro-osmotic, acoustic, and dielectrophoresis have also been exploited to achieve flow focusing in the microfluidic channel. Despite omitting the necessity of external syringe pump as required in pressure driven based cytometers, they all rely upon point-source based excitation scheme and thereby can not interrogate the cells flowing through the entire microfluidic channel. The third chapter describes the integration of light sheet illumination PSF with microfluidic flow cytometry for simultaneous counting and imaging cells on-the-go. The chapter starts with the description on photolithography procedure for preparing SU8 master and PDMS casting procedure adopted to prepare dedicated microfluidic chips for the developed imaging system. The research work reported here demonstrates the proof-ofprinciple of light sheet based imaging flow cytometer. A light sheet fills the entire microfluidic channel and thus omits the necessity of flow focusing and point-scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the acquired images. Compared to the existing state-of-the-art techniques, the proposed technique shows marked improvement. Using fluorescently coated Saccharomyces cerevisiae cells, cell counting with throughput as high as 2090 cells/min was recorded. Overall the proposed system is cost-effective and simple in channel geometry. Apart from achieving efficient counting in operational regime of low flow rate, high contrast images of the dynamic samples are also acquired using the proposed cytometry technique. Further, visualization of intra-cellular organelles is achieved during flow in light sheet based high-throughput cytometry system. The fourth chapter demonstrates the proof of concept of light-sheet-based microfluidic cytometer in conjugation with 2π/3 detection system for high-throughput interrogation and high resolution imaging. This system interrogates the flow channel using a sheet of light rather than the existing point-scanning based techniques. This ensures single-shot scanning of specimens flowing through the microfluidic flow channel at variable flow rates. In addition to high throughput counting at low flow rate, visualization of the intra-cellular organelle (mitochondrial network in human cancerous cells) during flow is achieved with sub-cellular resolution. Using mitochondrial network tagged HeLa cells, a maximum count of 2400 cells/min at the optimized flow rate of 700 nl/min was recorded. The 2π/3 detection system ensures efficient photon collection and minimal background caused by scattered illumination light. The other advantage of this kind of detection system which includes 8f detection optics, is the capability to produce variable magnification using the same high NA objective. This thesis opens up in vivo imaging of sub-cellular structures and simultaneous cell counting in a miniaturized flow cytometry system. The developed imaging cytometry technique may find immediate applications in the diverse field of healthcare diagnostics, lab-on-chip technology, and fluorescence microscopy. The concluding chapter summarizes the results with a brief discussion on the future aspects of this field (e.g., live-cell imaging of infectious RBC in microfluidic device and 3D optical sectioning of flowing cells). The field of imaging flow cytometry has immense applications in the overlapping areas of physics and biology. The hydrodynamic forces which are used to achieve flow focusing of the sample assays can have an adverse effect in the cell morphology, thereby altering the cellular functions. Light sheet based cytometry system lifts off the requirement of flow focusing and ensures a single shot scanning of entire samples flowing through the microfluidic channel. The similar concept can be used to study the developmental biology of an entire organism, such as C. elegans. This enables the direct observation of developmental and physiological changes in the entire body. Such an organism can be kept alive for a longer duration in microfluidic chambers, and the neural development and mating behaviors can be extensively studied.

Biotechnological processes are distinguished from classical chemistry by employing bio-molecules or whole cells as the catalytic element, providing unique reaction mechanisms with unsurpassed specificity. Whole cells are the most versatile \''factories\'' for natural or non-natural products, however, the conversion of e.g. hydrophobic substrates can quickly become cytotoxic. One host organism with the potential to handle such conditions is the gram-negative bacterium Pseudomonas putida, which distinguishes itself by solvent tolerance, metabolic flexibility, and genetic amenability. However, whole cell bioconversions are highly complex processes. A typical bottleneck compared to classical chemistry is lower yield and reproducibility owing to cell-to-cell variability. The intention of this work was therefore to characterize a model producer strain of P. putida KT2440 on the single cell level to identify non-productive or impaired subpopulations. Flow cytometry was used in this work to discriminate subpopulations regarding DNA content or productivity, and further mass spectrometry or digital PCR was employed to reveal differences in protein composition or plasmid copy number. Remarkably, productivity of the population was generally bimodally distributed comprising low and highly producing cells. When these two subpopulations were analyzed by mass spectrometry, only few metabolic changes but fundamental differences in stress related proteins were found. As the source for heterogeneity remained elusive, it was hypothesized that cell cycle state may be related to production capacity of the cells. However, subpopulations of one, two, or higher fold DNA content were virtually identical providing no clear hints for regulatory differences. On the quest for heterogeneity the loss of genetic information came into focus. A new work flow using digital PCR was created to determine the absolute number of DNA copies per cell and, finally, lack of expression could be attributed to loss of plasmid in non-producing cells. The average plasmid copy number was shown to be much lower than expected (1 instead of 10-20). In conclusion, this work established techniques for the quantification of proteins and DNA in sorted subpopulations, and by these means provided a highly detailed picture of heterogeneity in a microbial population.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Circulating tumor cells (CTCs) have become a key component in the identification and treatment of cancer. Once dislodged from the main tumor, CTCs travel through the bloodstream and cause metastasis. Early detection and identification of these cells can help in the evaluation and prognosis of various types of cancer, as well as assisting in patient treatments by determining the spread of the disease. Here, a high-throughput microfluidic analysis technique is described that can efficiently detect and identify cells, with the specific identification of CTCs as a future application through fluorescent labeling in mind. As proof of principle, the device has been shown to detect and characterize individual human Jurkat (T-lymphocyte) cells at a rate of 100 cells/minute. The device employs micro-scale flow focusing to isolate individual cells. The cells are detected using both light scattering and laser-induced fluorescence to evaluate cell size and surface functionality.

Experimental studies into the use of cold atmospheric plasmas for inactivating foodborne pathogens are presented in this thesis. Eliminating the possibility that treatment delivered by a plasma to a population or assemblage of micro-organisms is unevenly distributed is an essential pre-requisite to attempting to interpret inactivation kinetics with a view to elucidating mechanisms of inactivation. A filtration method of depositing cells evenly on the surface of a membrane without cell stacking was developed and used throughout the work described here. Two atmospheric plasma systems were evaluated and each brought about microbial inactivation in a distinct way. A pulsed radio frequency plasma jet operated at 3.47 MHz caused gross morphological changes to L. innocua whereas a low frequency air mesh plasma system operated at a frequency of 24 kHz led to the inactivation of these bacteria without inducing observable structural changes. Changing the operating parameters of the plasma jet system had a significant effect on the composition of the reactive plasma species generated as revealed by changes to the mode of inactivation of bacteria. In addition to inactivating bacteria, the pulsed plasma jet was shown to be highly effective in degrading and removing amyloid aggregates from the surface of mica coupons. Amyloids have widely been used as a non-infectious model for prions, and the results obtained here show potential for the application of gas plasma technology for removing prions from abiotic surfaces in medical and other applications. It has widely been assumed that bacterial envelopes are the principal sites at which reactive plasma species bring about damage to cells. However, changing the composition of the bacterial membranes of E. coli and Listeria innocua by cultivating them at widely different temperatures to induce changes proved not to result in enhanced inactivation. Flow cytometry was also used to provide additional insights into possible mechanisms of inactivation. The following fluorescent dyes were used either singly or in combination; SYTO 13, DiBAC4(3), cFDA and PI. The results obtained with the dyes DiBAC4(3) and PI showed that Gram positive bacteria became depolarised prior to the bacterial membrane becoming compromised, possibly suggesting that the inactivating plasma species are affecting membrane proteins responsible for maintaining the bacterial charge. Differences between the fluorescent dye staining of Gram negative and Gram positive species were obtained using SYTO13 and PI demonstrating that the different membrane structures affect their interaction with the plasma. In additional studies, the air mesh plasma was used to treat multi-drug resistant strains of Methicillin resistant Staphylococcus aureus (MRSA) in an attempt to reverse antibiotic resistance. MRSA PM 64 was shown to reverse its antibiotic resistance to Oxacillin, Kanamycin and Trimethoprim. Culturing the bacteria in a nutrient limited media led to increased resistance towards plasma treatment and maintenance of their high levels of antibiotic resistance.

The study of the formation of new cardiac muscle cells during postnatal development is a relatively new field. During fetal development, new cells are formed as the heart grows. However, the proliferative ability of postnatal cardiomyocytes is still debated. While several studies have been made on mammals, less is known about the chicken cardiac cells and their postnatal proliferation. As almost all previous studies have used microscopy-based cell counting methods, there has been some limitations on accuracy and amounts of cells that could be counted. The aim of this study is to develop a method for using flow cytometry to analyze proliferative ability of chicken cardiomyocytes and to investigate if any postnatal proliferation exists. For this study, 4 weeks old Red Junglefowl (Gallus gallus) chickens were used for isolating cardiomyocytes. In addition, 19 days old Red Junglefowl embryos were used to asses if a longer incubation time would yield a higher number of proliferative cells. Cells were stained using a commercial EdU imaging kit and analyzed using flow cytometry and imaging flow cytometry. The produced results could not be used for determining the proliferative ability of the cardiomyocytes, but provides crucial information for possible method improvements. In conclusion, this study has laid important groundwork for future studies on the proliferative ability of chicken cardiomyocytes.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction In the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus (HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiency syndrome (AIDS) related complications now prevails in the aging HIV infected population. Increased levels of inflammation and chronic immune activation are associated with HIV infection. In the era of ART people living with HIV are at an increased risk of cardiovascular disease (CVD). Platelets play a pivotal role in both inflammation and immune activation and upon activation platelets degranulate and secrete various inflammatory, coagulatory and adhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a key component of the coagulation pathway and serve as a link between inflammation and thrombosis. Activated platelets have been implicated in inflammatory and cardiovascular disease and have been identified as immune cells that play a crucial role in pathogen recognition and modulation of immune cells during infections. Several antiviral and antibacterial properties of platelet alpha granule contents have been established. Platelet aggregometry remains the most widely used technique to evaluate platelet function even though this technique is limited by many pre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement of platelet function in their physiological environment with minimal artefactual activation. Few studies have however reported on standardized methods to evaluate platelet function in the context of HIV. Platelet function remains unclear and data on HIV infected treatment naïve individuals remains scarce. The aim of this project was to examine the relationship between platelet function and immune activation in patients with HIV Materials and methods This study consisted of five sub-studies, firstly platelet indices and levels of platelet activation were determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfected healthy controls) using; flow cytometry and haemotology analyzers. The relationship between these indices and markers of platelet activation, disease progression and immune activation were assessed. Furthermore, levels of platelet activation and aggregation were evaluated in a cohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthy controls), using a novel whole blood flow cytometry based functional assay. These baseline levels were then correlated with markers of immune activation and disease progression in HIV. In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls was evaluated using flow cytometry. Platelet response was measured post stimulation with adenosine diphosphate (ADP) at concentrations known to induce reversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess platelet function in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP, arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry. Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flow cytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfected healthy controls. Associations between PLAs, immune activation and disease progression in HIV infected individuals were determined. The final study evaluated platelet aggregates, platelet derived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected (ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, platelet aggregates and markers of immune activation and disease progression were evaluated. Results HIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85 vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) and viral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated (r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) with platelet aggregation. HIV infected individuals showed increased levels of platelet activation (%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV, platelet function is retained and platelets showed increased response to submaximal concentrations of endogenous agonists. HIV infected individuals showed increased levels of circulating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36- 18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8 (r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levels of circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160); PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133); activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6], p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group 0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals. Conclusion This study supports the potential use of the MPV and PDW as readily available markers of platelet activation and immune activation in HIV. We also showed elevated levels of activated platelets in HIV infected individuals that were hyper responsive to endogenous agonists in a concentration dependent manner. Platelet flow cytometry is a rapid and valuable technique in the evaluation of platelet function in HIV. The measurement of platelet function using flow cytometry allows the evaluation of platelet signalling pathways that may be modified in HIV infected individuals. Lastly we describe an optimized whole blood flow cytometry based assay for the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) and levels of activated platelets and aggregates which mimics the in vivo physiological environment of MPs. To the best of our knowledge, this study is the first to report on a novel approach in evaluating platelet function in HIV using a series of optimised whole blood flow cytometry based platelet assays. In addition, minimal work has been performed previously on platelet function in the context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients as defined for this study. / AFRIKAANSE OPSOMMING: Inleiding In die era van antiretrovirale terapie (ART), het mense wat met die menslike immuniteitsgebreksvirus (MIV) leef, het nou 'n verlengde lewensduur. 'N opkomende neiging van nie-verworwe immuniteitsgebreksindroom (vigs) heers nou in die verouderende MIV-besmette bevolking. Verhoogde vlakke van inflammasie en chroniese immuun aktivering word geassosieer met MIV-infeksie en in die era van ART loop mense wat met MIV leef, 'n verhoogde risiko van kardiovaskulêre siekte (KVS). Plaatjies speel 'n belangrike rol in beide inflammasie en immuun aktivering en met aktivering degranulate en skei plaatjies verskeie inflammatoriese, coagulatory en adhesie molecule af. Geaktiveerde plaatjies druk oppervlak P-selectin (CD62P) is 'n belangrike komponent van die stollings weg en dien as 'n skakel tussen inflammasie en trombose. Geaktiveerde plaatjies is in beide inflammasie en kardiovaskulêre siekte betrokke en is geïdentifiseer as immuun selle wat 'n deurslaggewende rol speel in die patogeen erkenning en modulasie van immuun selle tydens infeksies. Verskeie antivirale en antibakteriese eienskappe van plaatjie alpha korrel inhoud is vasgestel. Plaatjie aggregometry bly die mees gebruikte tegniek om plaatjie funksie te evalueer, alhoewel hierdie tegniek is beperk deur baie pre-analitiese veranderlikes. Plaatjie vloeisitometrie aan die ander kant bied 'n vinnige meting van plaatjie funksie in hul fisiologiese omgewing met 'n minimale artefactual aktivering. Min studies het egter berig op gestandaardiseerde metodes om plaatjie funksie in die konteks van MIV te evalueer. Plaatjie funksie is steeds onduidelik en data oor MIV besmet behandeling naïef individue bly skaars. Die doel van hierdie projek was om die verhouding tussen die plaatjie funksie en immuun aktivering in pasiënte met MIV te ondersoek. Materiaal en metodes Hierdie studie het bestaan uit vyf sub-studies. In die eerste plekis plaatjie indekse en vlakke van plaatjie aktivering bepaal in 'n groep van 330 deelnemers (185 MIV-besmette ARV naïef en 145 onbesmette gesonde kontrole) met behulp van vloeisitometrie en hematologie ontleders. Die verhouding tussen hierdie indekse en merkers van plaatjie aktivering, die siekte se progressive en immuun aktivering is beoordeel. Verder is die vlakke van plaatjie aktivering en samevoeging in 'n groep van 82 deelnemers (41 MIV-besmette (ARV naïef) individue en 41 onbesmette gesonde kontrole) geëvalueer, met behulp van 'n nuwe vol bloed vloeisitometrie gebaseerde funksionele toets. Hierdie basislyn vlakke is dan gekorreleer met merkers van immuun aktivering en die progreessie van die siekte in MIV. In 'n daaropvolgende studie, is plaatjie funksie in 'n groep wat bestaan uit 58 MIV besmet te (ARV naïef) en 38 onbesmette beheer geëvalueer met behulp van vloeisitometrie. Plaatjie reaksie is na stimulasie gemeet met adenosine diphophate (ADP) by konsentrasies bekend omkeer (0.04mM) te oorreed en onomkeerbaar (0.2mm) plaatjie aggregasie. Ten einde plaatjie funksie in MIV te evalueer, is plaatjie reaksie in 'n groep wat bestaan uit 58 MIV-besmette (ARV naïef) en 38 onbesmette kontrole geëvalueer. Die plaatjies is geaktiveer deur gebruik te maak van wisselende konsentrasies van ADP, is aragidoonsuur (AA) en kollageen en plaatjie funksie gemeet met behulp van vloeisitometrie. Vlakke van sirkulerende plaatjie leukosiet gemiddeldes is ook gemeet met behulp van vloeisitometrie in 'n groep wat bestaan uit 35 MIV-positiewe (ARV naïef) individue en 32 onbesmette gesonde kontrole. Assosiasies tussen leukosiet gemiddeldes, immuun aktivering en die progressive van ie siekte in MIV-besmette individue is ook bepaal. Die finale studie het plaatjie-gemiddeldes, plaatjie afgelei mikrodeeltjies en mikrodeeltjies geëvalueer in 'n groep wat bestaan uit 46 MIV besmet (ARV-naïewe) en 40 onbesmette gesonde kontrole. Assosiasies tussen mikrodeeltjies, plaatjie afgelei, plaatjie gemiddeldes en merkers van immuun aktivering en die siekte se progressie is geëvalueer. Resultate MIV-besmette individue het gedaalde gemiddelde plaatjie volume vlakke getoon (HIV gemiddelde 7,91 ± 0,85 8,52 ± 1,12 teen, p <0,0001) wat direk gekorreleer het met CD4-tellings (r = -0,2898, p = 0,0075) en virale (r = 0,2680, p = 0,0177). Plaatjie verspreiding breedte vlakke het direk gekorreleer met (r = 0,3455, p = 0,0362) met 'n aktiewe koagulasie en omgekeerd gekorreleer (r = -0,3666, p = 0,0463) met plaatjie aggregasie. MIV-besmette individue het verhoogde vlakke van plaatjie aktivering getoon (% CD62P mediaan 11,33 [5,96-29,36] teen kontrole groep 2,48 [1,56-6,04], p = 0,0001). In MIV, was plaatjie funksie behou en plaatjies het 'n verhoogde reaksie op submaksimale konsentrasies van endogene agoniste getoon. MIVbesmette individue het verhoogde vlakke van sirkuleer plaatjie monosiet-gemiddeldes gedemonstreer (25.26 [16,16-32,28] teen kontrole groep 14,12 [8,36-18,83], p = 0,0001) wat direk gekorreleer het met merkers van immuun aktivering; % CD38 / 8 (r = 0,54624, p = 0,0155), virale lading (r = 0,633, p <0,009). Verder rapporteer ons op verhoogde vlakke van sirkulerende mikrodeeltjies (mediaan% LP 1.7 [0,95-2,83] teen kontrole groep 1,12 [0,63-1,57], p = 0,0160); PMPs (mediaan% PMPs 26,64 [11,33-36,62] teen kontrole groep 20,02 [18,08-26,08], p = 0,0133); geaktiveer PMPs (mediaan CD62P MFI 3,81 [3,46-4,54] teen kontrole groep 3,41 [3,16- 3,6], p = 0,0037) en plaatjie gemiddeldes (Mediaan% CD62P 14,10 [5,49-39,94] teen 0.17 [0,10- 10,99], p= 0.0097) in MIV besmet asimptomatiese individue. Gevolgtrekking Hierdie studie ondersteun die potensiële gebruik van die MPV en PDW as waardevolle geredelik waardevolle merkers van plaatjie aktivering en immuun aktivering in MIV. Ons het ook getoon verhoogde vlakke van geaktiveer de plaatjies in MIV-besmette individue getoon wat hyper reageer op endogene agoniste was in 'n konsentrasie-afhanklike wyse. Plaatjie vloeisitometrie is 'n vinnige en waardevolle tegniek in die evaluering van plaatjie funksie in MIV. Die meting van plaatjie funksie gebruik vloei cytometry maak die evaluering van plaatjie sein paaie wat in MIVgeïnfekteerde individue verander moontlik. Laastens het ons beskryf 'n hele bloed vloeisitometrie gebaseer de toets vir die evaluering van sirkulerende mikrodeeltjies, plaatjie afgelei mikrodeeltjies en vlakke van geaktiveer plaatjies en gemiddeldes wat lyk soos die in vivo fisiologiese omgewing van MP's. Na die beste van ons kennis, is hierdie studie die eerste om te rapporteer oor 'n nuwe benadering in die evaluering van plaatjie funksie in MIV met behulp van 'n reeks van new hele bloed vloeisitometrie gebaseer de plaatjie toetse. Daarbenewens is minimale werk voorheen uitgevoer op die plaatjie funksie in die konteks van MIV-infeksie; en veral in 'n groep van asimptomatiese, onbehandelde pasiënte soos vir hierdie studie. Hierdie projek het bewyse bygevoeg tot die teorie dat plaatjies, in MIV, kan 'n skakel wees tussen die aktiewe inflammatoriese reaksie en die toename in die aantal trombotische en kardiovaskulêre siekte waargeneem in pasiënte wat met hierdie siekte saamleef.