Mycobacteria, a genus within Actinobacteria Phylum, are well known for two pathogens that cause human diseases: leprosy and tuberculosis. Other than the obligate human mycobacteria, there is a group of bacteria that are present in the environment and occasionally cause diseases in immunocompromised persons: the non-tuberculosis mycobacteria (NTM). Mycobacterium kansasii, which was first discovered in the Kansas state, is the main etiologic agent responsible for lung infections caused by NTM and raises attention because of its co-infection with human immunodeficiency virus (HIV). Five subspecies of M. kansasii (Type I-V) were described and only M. kansasii Type I is pathogenic to humans. M. kansasii is a Gram-positive bacteria that has a unique cell wall and secretion system, which is essential for its pathogenicity. We undertook a comparative genomics and transcriptomic approach to identify components of M. kansasii Type I pathogenicity. Our previous study showed that espA (ESX-1 essential protein) operon, a major component of the secretion system, is exclusively present in M. kansasii Type I. The purpose of this study was to test the functional role of the espA operon in pathogenicity and identify other components that may also be involved in pathogenicity. This study provides a new molecular diagnostic method for M. kansasii Type I infection using PCR (Polymerase Chain Reaction) technique to target the espAoperon. With detailed manual curation of the comparative genomics datasets, we found several genes exclusively present in M. kansasii Type I including ppsA/ppsC and whiB6, that we believe are involved, or have an effect on ESX-mediated secretion system. We have also highlighted, in our study, the differences in genetic components coding for the cell membrane composition between the five subspecies of M. kansasii. These results shed light on genetic components that are responsible for pathogenicity determinants in Type I M. kansasii and may help to design better control measures and rapid diagnostic tools for monitoring these group of pathogens.

Die mechanischen Eigenschaften von Zellen charakterisieren und beeinflussen deren Zustand. Die vorliegende Arbeit zielt auf ein besseres Verständnis der biomechanischen Eigenschaften von Zellen ab. Der Fokus lag dabei auf der Biegesteifigkeit von Zellmembranen und der Deformierbarkeit der Zellen. Es werden drei Studien vorgestellt in der diese Materialparameter untersucht wurden. Die erste Studie befasst sich mit der Temperaturabhängigkeit der mechanischen Eigenschaften. Hierbei wurden acht verschieden Zelllienien bei jeweils fünf Temperaturen rheologisch vermessen. Zur Messung wurde der sog. \"optical stretcher\" verwendet der gleichzeitig die Zellen deformieren und aufheizen kann. Die Versuche zeigen, dass eine Zeit-Temperatur superposition dabei nicht für alle Zelltypen funktioniert. In der zweiten Studie wurden die Membransteifigkeit von Gewebeproben von Brust- und Gebärmutterhalskrebspatienten untersucht. Als Kontrollsystem wurde gutartiges Gewebe aus dem Umfeld des Tumors verwendet. Es konnte gezeigt werden, dass die Zellmembranen von Tumorzellen weicher waren als von gesundem Vergleichsgewebe. Die Änderung der Membrankomposition wurde dabei als mögliche Ursache massenspektroskopisch Untersucht und verschieden Ursachen der weichen Membrane diskutiert. Für die dritte Studie wurde der chemische Wirkstoff Soraphen A eingesetzt um die Membransteifigkeit von zwei Zelllienien zu erhöhen. Dies zeigte eine Verringerung von Zellbeweglichkeit und Invasivität.

Membrane traffic in polarised cells was investigated by growing Madin-Darby canine kidney (MOCK) cells on ·permeable polycarbonate filter supports which allowed access to both sides of the cell monolayer. Membrane glycoconjugates on the apical and basolateral cell surfaces were labelled enzymatically with ³H- and ¹⁴C-galactose, respectively, to provide covalent membrane markers. Experiments were done to quantitate membrane traffic during endocytosis at the respective plasma membrane domains and that due to transcytosis. Internalized label was quantitatively distinguished from label on the respective cell surface by its resistance to removal by glycosidases.

Bacterial membranes act as protective barriers and help to regulate molecular interactions between a cell and its surrounding environment. External chemical and physical influences have the potential to alter the properties of bacterial membranes and therefore impact the viability of the cell. This can stem from natural or seasonal changes to the local environment (e.g., temperature, pH, and salinity), or even deliberate application of an antimicrobial agent. Regardless, understanding exactly how such external stimuli influence bacterial membrane properties is of fundamental importance, both in terms of basic microbiology as well as for designing pharmaceutical interventions. Experimentally, this is a non-trivial task as this requires selective isolation of a signal arising from the membrane, which is typically buried in the overwhelming background response of the surrounding bulk environment. In particular, our lab has previously developed the surface-sensitive nonlinear optical method, second harmonic light scattering (SHS), as a means of interrogating molecular interactions at the membrane surfaces of living cells, even for multimembrane systems (e.g., Gram-negative bacteria). In this dissertation, time-resolved SHS was employed to study a variety of membrane properties across two separate projects, including 1) chemical and physical induced changes in membrane permeability and 2) temperature-induced membrane permeability changes. Specifically, in the first project (Chapter 4), the influence of the signaling molecule, indole, on the permeability of the bacterial cytoplasmic membrane was quantified. It was revealed that the interaction of indole with the tryptophan specific transporting protein, Mtr permease, resulted in enhanced passive diffusion across the membrane. For the second project (Chapter 5), we examined the influence of temperature on the rate of passive diffusion across a membrane, both in model systems (liposomes) and in living cells (E. coli). For both bacterial and liposome systems, increasing temperatures resulted in a modest increase in passive diffusion rates across the membrane. However, when the temperature range included a phase transition, passive diffusion increased by an order of magnitude. Therefore, by monitoring transport rate in relation to temperature, membrane phase transitions can be quantitatively determined based on the characteristic discontinuities in the measured trend. / Chemistry

Ivy geranium (Pelargonium peltatum) is a heat susceptible species with its heat tolerance varying among varieties. Reactive oxygen species (ROS) and in-vivo defense systems are related to plant heat damage and heat tolerance. Application of chelated-iron has also been reported to enhance ivy geranium heat tolerance; however, the correlation of ROS, relative enzyme stability, and iron content to differences in heat tolerance in ivy geraniums is unknown. Here we show that the H2O2 content and ROS scavenging enzyme stability in ivy geranium varies with varieties and active iron is not related to heat tolerance in ivy geranium. H2O2 content in mature leaves in both heat tolerant 'Beach' and sensitive 'Butterfly' increased under heat stress, but 'Butterfly' had a relatively greater increase of this toxic compound. Catalase (CAT) activities in young leaves in both varieties decreased. In young leaves of 'Butterfly', CAT activities decreased to a level significantly lower than that in old leaves while this did not occur in 'Beach'. Superoxide dismutase (SOD) activities in 'Butterfly' young leaves were also decreased. All these phenomenon coincided with the heat tolerance differences of the two varieties. Active iron content only changed with leaf age and did not vary between varieties or treatments. Our results demonstrated that ROS scavenging ability and relative enzyme stability may indicate heat tolerance in ivy geranium and that iron deficiency was not the cause of heat damage. Cell Membrane Themostability (CMT) and Triphenyl Tetrazolium Chloride (TTC) cell viability tests are alternative, laboratory-based screening methods for screening for heat-tolerance. Both CMT and TTC tests can represent the variance in heat tolerance observed in ivy geraniums. The results of both CMT and TTC tests correlated well with plant width and growth indexes although their correlations to plant chlorosis were low. Unlike TTC, CMT strongly correlated with plant width. CMT and TTC tests are complementary laboratory-based methods that can be applied to cultivar screening for heat tolerance in ivy geraniums.

Red blood cells (RBCs) are the predominant cell type in blood and have a two-layered outer shell which is composed of a cytoskeleton network tethered to a cytoplasmic membrane. In this thesis, I study the structure and mechanical properties of the RBC’s cytoplasmic membrane (RBCcm) on the nanoscale and utilize this knowledge to functionalize this biological structure on a molecular level. In a first case study, I measure the membrane’s bending rigidity from thermal fluctuations observed in X-ray diffuse scattering (XDS) and Neutron Spin Echo (NSE) experiments, as well as Molecular Dynamics (MD) simulations. I provide evidence of the RBCcm's highly deformable nature with a bending rigidity that is substantially softer as compared to synthetic membranes. The methods are applied to RBCs that were stored for up to 5 weeks. I demonstrate that storage of RBCs leads to an increased fraction of liquid ordered membrane domains and an increased bending rigidity. RBCs are ideal for pharmaceutical applications as they provide access to numerous targets in the body, however lack specificity. Functionalizing the cytoplasmic membrane is thus a prerequisite to use these cells in biotechnology. I develop protocols throughout two studies to tune the membrane's lipid and protein composition. I investigate the impact of synthetic lipid molecules on the membrane's structure and demonstrate that small molecules can be encapsulated into liposomes that are formed from these hybrid membranes. Further, I provide direct evidence that the SARS-CoV 2 spike protein can be anchored into the RBCcm through a detergent mediated insertion protocol. These virus-like particles are observed to trigger seroconversion in mouse models, which demonstrates the potential of functionalized RBC in biotechnology. / Thesis / Doctor of Philosophy (PhD)

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

Indiana University-Purdue University Indianapolis (IUPUI) / Proton exchange membrane fuel cells (PEMFC’s) offer an attractive alternative energy resource over traditional fossil fuels. The advantages such as high power density, relatively quick start-up, rapid response to varying loads and low operating temperatures make it a preferred technology option compared to other alternative energy sources. Nafion® by DuPont plays an integral role in the success of PEM fuel cells due to its high proton conductivity and high chemical and thermal stability. This research project aims to study the effect of mechanical and hygro-thermal stresses on the mechanical performance and proton conductivity of the membrane by subjecting it to realistic operating conditions such as those encountered in an automobile. In this thesis, the time-dependent behavior of the membrane has been modeled using a Prony series and the change in the conductivity due to mechanical loading was experimentally measured. The modeling of both electrochemical and mechanical properties can further be used in studying the degradation properties of the membrane and should guide the development of better membrane materials. Visco-elastic stress relaxation theory has been used in modeling the time-dependent behavior of the specimen. The EIS spectrum has been analyzed using a non-linear least squares method and an equivalent circuit method was also used to fit the spectra. This project was conducted in three phases. In the first phase a novel test facility was built to perform the experiments. A conductivity measurement test cell that measured the proton conductivity of a membrane was modeled and manufactured. The second phase included the design of different experiments that helped in modeling the interdependence of electrochemical and mechanical properties of the membrane. In this process, three series of experiments that tested the electrochemical and mechanical properties of the specimen were conducted. The membrane was held at constant strain and the through plane impedance was measured at different times during the test, specifically before and after stretching at ambient and varying environmental conditions. The membrane was also subjected to both mechanical and hygro-thermal loading conditions during the test. In the third phase, time-dependant mathematical model for the changes in the material properties were developed. The experimental apparatus thus tested the mechanical and electrochemical properties of the membrane simultaneously while the specimen was being subjected to constant mechanical and varying hygro-thermal conditions. Since the testing method is a novel procedure, the reliability and repeatability of the experimental facility has been verified before conducting the experiments. The experimental apparatus can further be used to test the membrane at varying strain rates and different hygro-thermal loading conditions in a consistent manner. The model developed can be used to analyze the degradation behavior of membrane and also to build better fabrication methods and membrane materials in future.

During apoptosis, changes occur in T-lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A2 (sPLA2). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was first applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: initial hydrolysis rate was elevated, total reaction product was increased four-fold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by substrate availability rather than enzyme adsorption. Fluorescence experiments identified three membrane alterations that might affect substrate access to the sPLA2 active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting increased lipid spacing. Second, laurdan detected increased solvation of the lower head group region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it was proposed that the apoptotic membranes become susceptible to sPLA2 through a reduction in lipid-neighbor interactions which facilitates migration of phospholipids into the enzyme active site. This proposal was then examined in T-lymphocytes treated with glucocorticoid, a more physiologically relevant apoptotic stimulant, using similar techniques. The following observations corresponded to induction of membrane susceptibility: increased merocyanine 540 intercalation; phosphatidylserine flip-flop, detected by annexin binding; and alterations in laurdan fluorescence properties. These observations implied a relationship among sPLA2 susceptibility, lipid spacing, and phosphatidylserine exposure. To clarify this relationship, additional assays were also performed using dibutyryl-cAMP to induce apoptosis, a drug reported to induce apoptosis in S49 cells without the typical translocation of phosphatidylserine. Our results indicated that in cells treated with dibutyryl-cAMP, the merocyanine 540 response and its correlation with sPLA2 susceptibility was similar to that observed with dex-treated samples. This suggests that the underlying mechanisms which promote sPLA2 hydrolysis lead to alterations that may be facilitated by but do not require phosphatidylserine exposure. Taken together, all of the results suggest that direct regulation of the biophysical microenvironment of the membrane is the mode of control of membrane susceptibility to the hydrolytic activity of sPLA2.

Secretory phospholipase A2 (sPLA2) behaves differently toward the membranes of healthy cells compared to those of damaged or dying cells. The enzyme catalyzes rapid and sustained hydrolysis of compromised cells consistent with a simple catalytic mechanism. In contrast, when healthy cells are incubated with sPLA2, they become resistant to hydrolytic attack as manifest by three unusual observations: First, hydrolysis is transient and represents only a small fraction of the total membrane phospholipid content. Second, subsequent addition of sPLA2 fails to generate additional product. Third, the apparent potency of the enzyme to cause the membrane to be refractory is much greater than the potency for catalyzing hydrolysis. The mechanism responsible for this resistance has not yet been identified. Using Monte Carlo and direct analytical methods, we have developed a model capable of explaining all three of these observations. The model requires two salient elements: only a small pool of phospholipids in the healthy cell membrane is available for catalysis by sPLA2, and hydrolyzed phospholipids are re-acylated and restored very slowly to the accessible pool. The requirement for initial hydrolysis (as opposed to the simple physical presence of the enzyme as previously thought) was confirmed experimentally. Additional evidence has shown that the membrane does not remain permanently in its resistant state. Over time, the membrane resets to its original state. The model also predicts that total substrate, reacylation rate, and the return rate of phospholipids to the membrane should all be constant as enzyme concentration is varied. This prediction was tested by quantitative analysis of hydrolysis time courses at varied enzyme concentrations. Experiments with fluorescent probes, merocyanine 540 and laurdan suggest, that resistance may also involve physical changes to the membrane beyond the kinetic mechanisms hypothesized in the model.