Eosinophils are end-point effectors of inflammation that contribute to the clinical severity of asthma. Eosinophil homing to the asthmatic lung is primarily guided by eotaxin-1, which is an eosinophil-selective chemokine. The mechanism by which eotaxin-1 augments intracellular calcium during cell migration is incompletely understood but is integral to the extravasation of eosinophils at sites of inflammation. We consequently report here that fluid shear stress, like eotaxin-1, unexpectedly activates human eosinophils in a calcium-dependent manner. We used confocal fluorescence microscopy to study calcium-handling in purified human eosinophils. Application of eotaxin-1 augmented the [Ca2+]i in a concentration-dependent manner. Pre-treatment of cells with ryanodine (10 μM) completely abolished the eotaxin-mediated calcium response, indicating that this phenomenon is dependent on Ca2+-release from the ER. Several SOCC blockers (2-APB, 100 μM; Gd3+, 10 μM; SKF-96365, 100 μM) attenuated SOCE, suggesting that these channels may directly contribute towards the eotaxin-1 calcium response in human eosinophils. In the presence of fluid-perfusion, eosinophils displayed a robust perfusion-induced calcium response (PICR) demonstrating that eosinophils are mechanically sensitive. The PICR rapidly induced adhesion and non-directional migration in eosinophils, suggesting that some hitherto unknown molecular mechanosensor permits these cells to detect and respond to changes in shear-stress. Pre-treatment of eosinophils with the non-selective tripeptide integrin receptor blocker, Arg-Gly-Asp (RGD), abrogated the PICR. The highly selective, dual α4β7/α4β1 integrin receptor blocker, CDP-323, was used to ascertain whether these highly expressed integrin subtypes mediate the PICR in eosinophils. Pre-treatment of cells with CDP-323 completely abolished the PICR, in addition to the eotaxin-mediated calcium response in a shear-dependent manner. Taken together, our results support a novel role for the α4β7/α4β1 integrin receptors as mechanosensors that directly modulate [Ca2+]i, adhesion and migration in human eosinophils. On-going experiments will seek to quantify the shear-response thresholds at which eosinophils activate and the time-course of the associated calcium response. This study suggests that the recruitment and activation of eosinophils are regulated by chemical and mechanical stimuli via overlapping, calcium-dependent signal transduction cascades. Given that the PICR is mediated by the eosinophil-specific α4β7/α4β1 integrin receptors, we conclude that integrin receptors are molecular mechanosensors that may facilitate eosinophil activation, adhesion and non-directional migration independently of, or in conjunction with, chemokine signaling. / Thesis / Master of Science in Medical Sciences (MSMS)

Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant. / Ph. D.

Doctor of Philosophy / Over 1.9 million new cases of cancer will pop up just this year alone. The prevalence of cancer, however, has not been met with the same magnitude of effective treatments, resulting in over 600,000 deaths in the United States. Before current treatments can be improved and new treatments can be developed, it is critical that we increase our understanding of what drives cancer to be so aggressive and maintain a fighting chance within the body despite our complex immune systems. The severity of cancer is not just a product of the cancer cell itself, but rather the components that make up the cell that define and drive metastatic behaviors and drug resistance. In order to improve diagnoses, prognoses, and treatment planning, the intracellular drivers of the disease must be better understood. Cells, electrical circuits in nature, reflect unique electrical properties dictated by their biophysical composition. These electrical properties can be revealed and exploited to characterize and treat contributors to disease progression. Using electric fields applied in several modalities, this work explores the electrical entities of malignant cell types towards improving in vitro treatment planning and developing a treatment modality cognizant of subcellular drivers. This dissertation details the use of dielectrophoresis and electroporation to detect and treat intracellular changes associated with poor prognosis.

It has been observed that the normal process of aging is associated with a decline in muscle strength and mass. It has also been observed that total body potassium and intracellular water (ICW) decrease with age, reflecting a loss of body cell mass (BCM), 60% of which is the skeletal muscle. It is generally accepted that traditional high-intensity strength training (ST) regimens can not only attenuate, but in some cases, reverse some of these aging-related changes. Periodization, a nontraditional approach to strength training, has been demonstrated to stimulate more rapid increases in muscle strength than traditional approaches in young adults; however, it has not been comprehensively evaluated in postmenopausal women. Investigators have consistently reported an increase in muscle strength in older adults undergoing both short- and long-term traditional ST programs. It is fairly well accepted that early increases in muscle strength are attributable to neurologic adaptations. There has been less consistency in the literature regarding the timing and nature of changes in muscle quality and mass with ST. Although several investigators have reported increased muscle protein synthesis rates as early as 2 weeks after ST initiation in older adults, the majority of published reports support the notion that significant NET gains in intracellular protein, and thus, gains in muscle mass/volume/hypertrophy do not occur before 9-10 weeks. Changes in intracellular water, which would be expected to occur with changes in intracellular protein, have not been studied during short-term ST interventions in older adults. Bioimpedance spectroscopy (BIS) has been validated as a field technique to accurately measure ICW (and BCM) changes in HIV infected individuals. The primary aim of the current study was to determine if muscle strength would increase in postmenopausal women undergoing a novel (periodized) ST intervention of 10 weeks duration. A secondary aim was to determine if BIS would detect a change in ICW in the study subjects from baseline to study conclusion. Study participants were eleven, healthy postmenopausal women between the ages of 60 and 74 (mean age: 65 ± 4.4 y) who had not engaged in ST in the six months preceding the study. ICW and muscle strength were assessed at baseline and at study conclusion. The ST program was conducted twice a week for 10 weeks at the Senior Center in Blacksburg, VA. Participants performed seven different exercises incorporating upper body and lower body muscle groups. The women performed one set of 8-12 repetitions at an intensity of 80% of one repetition maximum (1 RM) the first week, progressing to 2 sets of 8-12 repetitions at the same intensity during the second week. The remaining weeks consisted of three sets of 8-12 repetitions, performed at an intensity of 80%, 75%, and 70% of their current 1 RM, respectively. One RM was reassessed every other week. The major result from this study was that muscle strength of all trained muscle groups increased in postmenopausal women undergoing 10 weeks of pyramid ST (P<0.05). In addition, the pyramid ST protocol utilized in this study was well-tolerated and resulted in no injuries in any of the older women in the study, indicating that this approach may be used safely in this population. Mean ICW measured by the field method BIS did not change over the course of the study. This result was consistent with other published data reporting no changes in lean body mass or muscle volume/area by more sophisticated techniques. / Master of Science

Intracellular delivery of macromolecules is crucial for the success of many research and clinical applications. Several conventional intracellular delivery methods have been used for many years but are still inadequate for several applications because of the issues associated with toxicity, low-throughput, and/or difficulty to target certain cell types. In this study, we developed and evaluated new high-throughput intracellular delivery methods for the efficient delivery of macromolecules while maintaining high cell viability. First, we studied the feasibility of using an array of nanoneedles, with sharp tip diameters in the range of tens of nanometers, to physically make transient holes in cell membranes for intracellular delivery. Puncture loading and centrifuge loading methods were developed and assessed for the effect of various experimental parameters on cell viability and delivery efficiency of fluorescent molecules. In both methods, high-throughput intracellular delivery was feasible by creating transient holes in cell membranes with the sharp tips of the nanoneedles. The second physical intracellular delivery method we studied was a novel microfluidic device that created transient holes in the cell membrane by mechanical deformation and shear stress to the cell. We observed efficient delivery of fluorescent molecules and studied the effect of device design and flow pressure on the delivery efficiency compared to data in the literature. We accounted for cell loss and clogging in the microfluidic devices and determined the true loss of cell viability associated with this method. Lastly, we investigated the possibility of intracellular delivery using nanoparticles on a leukemia cell line. Among number of materials for nanoparticles tested, mesoporous silica/poly-L-lysine nanoparticles were selected for further intracellular delivery study based on cell viability and intracellular delivery capability. We demonstrated the co-delivery of protein and plasmid by encapsulating into and coating onto the surface of the nanoparticles, respectively, which would be advantageous for certain therapeutic strategies. In summary, this work introduced two new intracellular delivery methods involving nanoneedles and novel nanoparticles, and provided an early, independent assessment of microfluidic delivery, showing the strengths and weaknesses of each method. These methods can be further optimized for a number of laboratory and clinical applications with continued research.

Redox regulation and homeostasis are critically important in the regulation of cell function; however, there are significant challenges in quantitatively measuring and monitoring intracellular redox potentials. The work in this thesis details a novel approach to intracellular redox monitoring. The approach is based on the use of nanosensors, which comprise molecules capable of sensing the local redox potential, assembled on gold nanoshells. Since the Raman spectra of the sensor molecules change depending on their oxidation state, and since the nanoshells allow a large enhancement of the Raman scattering, intracellular potential can be calculated by simple optical measurements. A full description of the design, fabrication and characterisation (spectroscopic and electrochemical) of the nanosensors is provided within. The ability to deliver nanosensors into cells in a controllable fashion was confirmed using electron microscopy. Results from a range of assays are also presented which reveal that introduction of nanosensors does not result in any cytotoxicity. Sensor utility in monitoring redox potentials as cells responded to physiological and superphysiological oxidative and reductive stimuli was investigated. Importantly, the capability of the nanosensors in monitoring intracellular potentials in a reversible, non-invasive manner, and over a previously unattainable potential range, is demonstrated.

In the past fifteen years, ubiquitin has emerged as a central regulator of membrane protein trafficking. In this context, covalent attachment of this small protein to lysine residues of cargo proteins, a reversible modification termed ubiquitylation, provides a signal for their targeting to the vacuolar/lysosomal lumen where they are degraded, both in yeast and higher eukaryotes. Ubiquitylation is also used as a means of controlling the function of specific proteins in several trafficking machineries. The role of lipids - and in particular of membrane domains named lipid rafts - in controlling the intracellular trafficking of membrane proteins has also been the subject of intense investigation in recent years. One of the membrane proteins of the yeast Saccharomyces cerevisiae whose intracellular trafficking has been extensively studied is the general amino acid permease Gap1. Yet some aspects of the function of ubiquitin in the nitrogen-dependent control of this protein remain controversial. Moreover, the potential role of lipid rafts in regulating the functional properties and traffic of the Gap1 permease had not been investigated before this thesis work. The first part of our work readdresses the role of Gap1 ubiquitylation, and more precisely of the modification of the permease with polyubiquitin chains linked through the lysine 63 of ubiquitin, in controlling the fate of this protein in the secretory pathway. Our observations indicate that nitrogen-induced ubiquitylation of newly synthesised Gap1 occurs in the trans-Golgi complex. However, contrary to the generally accepted view, this modification is not necessary for the permease to exit this compartment en route to the endosome but only for its subsequent targeting to the vacuolar lumen via the multivesicular body (MVB) pathway. Our results also provide evidence that K63-linked polyubiquitylation is important mostly at the late endosomal level, for proper sorting of Gap1 into the MVB pathway, whether the permease comes from the cell surface by endocytosis or directly from the secretory pathway. In the second part of this work, we present a set of data providing novel insights into the controversial question of the exact nature of lipid rafts in yeast. We first showed that the Gap1 permease is associated with detergent-resistant membranes (DRMs) - the proposed biochemical equivalent of lipid rafts - when it is located at the cell surface. Our data further suggest that this may be true for most if not all yeast plasma membrane proteins. Moreover, we found that Gap1 production must be coupled to de novo synthesis of sphingolipids (SLs), major constituents of rafts, in order for the newly synthesised permease to be correctly folded, active, associated with DRMs, and stable at the cell surface. We propose a model where Gap1 would associate with newly synthesised SLs during its biogenesis and/or secretion, this association shaping the permease into its native conformation and ensuring its incorporation and stabilisation in specific lipid domains at the plasma membrane. Failure of Gap1 to acquire this lipidic microenvironment in turns leads to its ubiquitin-dependent degradation by a quality-control mechanism. This model might be valid for many other plasma membrane proteins and might account for their lateral distribution between distinct membrane domains.

Bacterial endosymbionts of eukaryotes are generally studied in terms of their benefit or detriment to their hosts. The constraints that the host's life history imposes on its endosymbionts are rarely considered, although bacterial genome content and size are influenced by both the biotic and abiotic factors in the environment. The host organism is the primary habitat of the endosymbiont. Thus, desecribing the environment a host provides its endosymbiont is essential for understanding the evolution of endosymbiotic bacteria. I propose a system to classify the endosymbiotic environment by three characteristics: 1) host life cycle 2) host metabolism, and 3) endosymbiont location relative to host tissues. Insect-bacterial mutualisms have been classified in terms of endosymbiont environment. The majority of insect-bacterial mutualisms currently studied involve monophagous, hemimetabolous hosts that provide a relatively constant endosymbiotic enviroment. A relatively constant environment may explain the extremely reduced genomes of their endosymbionts. In contrast, polyphagous, holometabolous hosts provide the most variable endosymbiotic environment. In this work, I examined the interactions between the polyphagous, holometabolous insect, Bactrocera oleae (Rossi), and the enteric gammaproteobacterium, Candidatus Erwinia dacicola, throughout host development. Candidatus Erwinia dacicola was found in the digestive system of all life stages of wild olive flies. PCR and microscopy demonstrated that Ca. Erwinia dacicola resided intracellularly in the gastric caeca of the larval midgut, but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. I document the widespread distribution and high frequency of Ca. Er. dacicola in ten populations of wild olive flies sampled in four countries (3 Old World and 1 New World). The relative abundance of the bacterium was highest in adults and less prevalent in the egg and pupal stages. Among adult flies, the bacterium was most common in ovipositing females. These results suggest that Ca. Er. dacicola is a persistent, autochthonous endosymbiont of the olive fly. Finally, mating initiation was examined to study the influence of Ca. Er. dacicola on mating between a laboratory and a wild population of olive flies from Israel. Behavioral differences between the two populations, not presence of the endosymbiont, explained mating initiation.

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Pathogenic Listeria are the causative agents of listeriosis, a severe food-borne infection. They are able to invade various non-phagocytic cell types including epithelial cells. The life cycle of these intracellular parasites involves penetrating into host cells, rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly spreading cell to cell. Each step of the listerial intracellular infection involves activation of virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated virulence gene activation occurs within host cells by mechanisms that remain unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen interaction. Methods for assessing PrfA-dependent gene expression were first developed and standardized, including a highly sensitive and accurate quantitative reverse-transcription real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with virulence using cell culture-based assays. These techniques were applied in an investigation into the structure-function of PrfA. We studied the role of a solvent-accessible pocket identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding (CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene activation. Site-directed PrfA mutants were constructed. Our data support the notion that PrfA activity is allosterically regulated and are consistent with a role for the pocket as putative binding site for the PrfA-activating allosteric effector. The characterization of spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*- suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated repression was investigated and found to explain the anomalous phenotype of strain NCTC 7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by cellobiose but not glucose. We also carried out experiments to address the intriguing activation of PrfAdependent virulence genes upon addition of an adsorbent to the culture medium, the socalled "charcoal effect". Using a chemically defined culture medium and resin, Amberlite™ XAD-4, we provided evidence that the virulence gene activation may involve the sequestration of a medium component rather than a bacteria-derived autorepressor, as initially thought. We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We showed that σB has no major effect on host cell invasion, and that L. monocytogenes invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in stress conditions. Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and developed normalized methods to accurately determine and quantify this cellular response in infected cell monolayers.