No description available.

Polymicrobial communities of bacterial and fungal species are present on the skin and mucosal surfaces of the body. Invasive infections caused by Candida species are commonly seen in immunocompromised individuals (HIV, transplants, cancer) and ranks as the third leading cause of infection in hospitalized patients. C. albicans is a polymorphic opportunistic fungus that infects critically ill patients and has the ability to change its morphology from yeast to hyphal form.The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.In this study, we examined two transporter mutants, cdr4 and ssu1, for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. To quantify biomass, wild type and mutant cells were grown overnight at 30˚C in YPD. The cells were washed, counted and diluted to a desired density of 106 cells/ml in medium 199, pH7.5. Cells were added to 96-well plates pre-incubated with 5% fetal bovine serum at densities of 105, 104, and 103 cells/well and allowed to adhere at 37˚C for one hour. The wells were then covered with fresh M199 media containing the QS molecule and monitored for 48 hours at 37˚C. After this time, the media and planktonic cells were removed. The biofilms were fixed with methanol, dried, then stained with 0.05% crystal violet. Bulk biomass was assessed by spectrophotometry. We did observe a difference in biofilm density when incubated in the presence of cyclic-diGMP. We noted that for the wild type and ssu1 strain their biofilms biomass increased by as much as 10% at 104 and 103 cell densities when compared with the control. While the cdr4 strain had a slight reduction in biofilm density when cyclic-diGMP was present. This result also indicates a potentially positive role in which cyclic-diGMP can help C. albicans develop denser biofilms, potentially in the presence of bacteria like P. aeruginsa, which secrete cyclic di-GMP, but kill hyphal forms of C. albicans. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1p, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.Given that mutations in CDR4 and SSU1 impact the QS response in C. albicans, we investigated whether these genes were regulated through two-component signaling by Chk1. To assess CDR4 and SSU1 expression, wild type and mutant strains were grown overnight in YPD media at 30˚C. Cells were then harvested and RNA was obtained by acid phenol extraction. Using RT-PCR, we determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. These results suggest the two genes are downstream targets in a pathway regulated by Chk1p. The finding that QS proceeds through a two-component pathway can be exploited in antifungal drug development. Given that two-component signaling is absent in mammalian cells, development of novel compounds that interfere with this pathway may be a useful alternative for treating patients with candidiasis.

No description available.

Inflammasome signaling during infections results in cell death and processing and secretion of cytokines from the IL-1 family, which facilitates control over an infection. Pseudomonas aeruginosa and Salmonella Typhimurium are opportunistic bacterial pathogens which may induce acute infections and activate various innate immune signaling pathways, including inflammasomes. However, under favourable conditions these pathogens may evade immune clearance resulting in the establishment of a chronic infection. In this study, I evaluated the modulation of host inflammasome signaling induced by P. aeruginosa and S. Typhimurium during chronic infections. I used a collection of P. aeruginosa clinical isolates obtained from the sputum of cystic fibrosis patients collected during stable and exacerbation periods of disease. I demonstrated that the majority of isolates displayed poor inflammasome signaling and only a small proportion of isolates retained their ability to induce inflammasome activation, which may be associated with pulmonary exacerbations in cystic fibrosis. Sequencing and bioinformatics revealed genetic variations within the type III and type VI secretion systems of P. aeruginosa. While an inactivation of the type III secretion system is expected to impair inflammasome signaling, my results indicate that the type VI secretion system inhibits inflammasome signaling in eukaryotic cells. Due to the lack of chronic animal models for P. aeruginosa, I utilized a murine model of chronic S. Typhimurium infection to assess the modulation of inflammasome signaling throughout the course of a chronic infection. I observed that S. Typhimurium isolated during the acute phase of infection displayed an increased potential to activate inflammasome signaling and this ability progressively declined during the chronic phase of infection. This reduction in inflammasome activation was associated with reduced expression of bacterial virulence factors, such as flagella and the type III secretion system, and was dependent on the NLRP3 inflammasome. Overall, these results reveal that the expression of virulence factors is modulated during chronic bacterial infections, which results in a reduction of inflammasome activation leading to co-survival of the pathogen and host.

No description available.

Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus of NHE1. To identify the p38 MAPK phosphorylation sites on NHE1, in vitro p38 MAP kinase assays coupled to deletion analysis of NHE1 and mass spectrometry, identified four possible p38 MAPK phosphorylation sites. To establish that NHE1 causes apoptotic alkalinization and determine whether the identified phosphorylation sites on NHE1 are functionally significant, we used PCR site directed mutagenesis to mutate T717, S722, S725, and S728 on the C-terminus of NHE1. Stable NHE1 deficient cell lines, expressing wild type (WT) NHE or the four mutated sites (F4MUTNHE), were assessed for apoptotic alkalinization using the pH-sensitive fluorescent protein, destabilized YFP. Our results show that NHE1 is required for apoptotic alkalinization, since expression of WT NHE restored alkalinization in an NHE deficient cell line, and that this process requires the phosphorylation of the p38 MAPK target sites, since mutation of all four sites prevented the apoptotic alkalinization response.

Ames dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells – somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice also have high circulating levels of anti-inflammatory and antidiabetic adiponectin. Plasma levels of this adipokine usually decrease with an increase in accumulation of visceral fat (VF). We thus believe that VF in df/df mice, developed in the absence of GH signaling, may be functionally different from the same fat depots in normal (N) mice and may be beneficial, rather than detrimental, to the overall health of the animal. We performed surgeries involving removal of VF depots (epididymal and perirenal fat) in both groups of mice and hypothesize that the beneficial effects of visceral fat removal (VFR) will be present exclusively in N mice as VF in df/df mice contributes to enhanced insulin sensitivity by producing decreased levels of pro-inflammatory adipokines like TNF and IL-6. We found that VFR improved insulin sensitivity only in N mice but not in the df/df mice. This intervention led to an upregulation of certain players of the insulin signaling pathway in the skeletal muscle of N mice only, with no alteration in df/df mice. The subcutaneous fat of df/df mice showed a downregulation of these insulin signaling genes upon VFR. Compared to N mice, epididymal fat of df/df mice (sham-operated) had increased gene expression of some of the players involved in insulin signaling and a decrease in transcript levels of TNFa. Ames dwarf mice had decreased levels of IL-6 protein in EF and in circulation. High circulating levels of adiponectin and iv decreased levels of IL-6 in circulation could contribute to the high insulin sensitivity observed in the Ames dwarf mice. Understanding the mechanisms responsible for VF having positive effects on insulin signaling in df/df mice would be important for future treatment of obese diabetic patients.

Problem Statement: The use of pulsed electric fields (PEFs) as a tumor treatment modality is receiving increased traction. A typical clinical procedure involves insertion of a pair of electrodes into the tumor and administration of PEFs (amplitude: ~1 kV/cm; pulse-width: 100 μs). This leaves a zone of complete cell death and a sub-lethal zone where a fraction of the cells survive. There is substantial evidence of an anti-tumor systemic immune profile in animal patients treated with PEFs. However, the mechanism behind such immune profile alterations remains unknown, and the effect of PEFs on cell signaling within sub-lethal zones remains largely unexplored. Moreover, different values of a PEF pulse parameter, for e.g. the pulse-widths of 100 μs and 100 ns, may have different effects on cell signaling. Thus, the challenge of answering the mechanistic questions is compounded by the large PEF parameter space consisting of different combinations of pulse-widths, amplitudes, and exposure times. Intellectual merit: This Ph.D. research provides proof that sub-lethal PEFs can enhance anti-tumor signaling in triple negative breast cancer cells by abrogating thymic stromal lymphopoietin signaling and enhancing stimulatory proteins such as the tumor necrosis factor. Furthermore, experimental evidence produced during this Ph.D. research demonstrates that PEFs may not directly impact the intracellular mitochondrial membrane at clinically relevant field amplitudes. As demonstrated in this work, PEFs may influence the mitochondria via an indirect route such as disruption of the actin cytoskeleton and/or alteration of ionic environment in the cytoplasm due to cell membrane permeabilization. Thus, a reductionist approach to understanding the influence of PEFs on cell signaling is proposed by limiting the study to membrane dynamics. To overcome the problem of investigating the entire PEF parameter space, this Ph.D. research proposes a first-principle thermodynamic approach of scaling the PEF parameter space such that an understanding developed in one regime of PEF pulse parameter values can be used to understand other regimes of the parameter space. Demonstration of the validity of this scaling model is provided by coupling Monte-Carlo methods for density-of-states with the steepest-entropy-ascent quantum thermodynamic framework for the non-equilibrium prediction of the lipid membrane dynamics. / Ph. D. / A complete cure for cancer is still far from being realized despite very promising developments on the front of molecular drug therapy. One promising conceptual approach would be to achieve the ability to re-tune the cancerous signals that drive disease progression. To overcome current challenges in tuning cancerous signaling a paradigm change in cancer treatment is necessary. For example, a treatment strategy to alter cell signaling which leverages both the physical and chemical properties that accompany malignancy may be required. Electric fields, be it in the form of low-amplitude steady state fields or high-amplitude pulsed electric fields (PEFs), can induce distinct physical and chemical effects on cells. Hence, the use of electric fields as a clinical tumor treatment modality is receiving increased traction. However, the effect of these electric fields on cell signaling and cell behavior remains largely unexplored. This Ph.D. work provides experimental evidence that PEFs can directly impact cancerous cell signaling towards a less inflammatory and possibly less cancerous state. Although a noteworthy finding, the data poses another challenging question, i.e., how does the electric field impact cell behavior? Answering this mechanistic question is essential for FDA approval and a broader clinical use of the electric field modalities. An impediment to answering this question is the vast parameter space of electric fields (e.g., amplitude, pulse width, and number of pulses), which makes performing experimental mechanistic studies untenable. It is argued via experimental evidence gathered during this work that applying scaling laws applicable to lipid membranes may provide a solution to reducing the candidate PEF parameters to a manageable number. A non-equilibrium thermodynamic model is proposed that allows studying the behavior of lipid species using scaled electric field parameters. Thus, the v understanding gained via the proposed model can direct the next level of extensive biological assays and animal studies and eventually lead to effective cancer treatments.

No / In the future mobile network, satellites will operate alongside cellular networks in order to provide seamless connectivity irrespective of the location of the user. Such a service scenario requires that the next generation of mobility management (MM) procedures are able to ensure terminal and user mobility on a global scale. This paper considers how the principles of Mobile-IP can be used to develop MM procedures for a heterogeneous access network, comprizing of satellite and cellular elements, connected to an IP core network.Initially, the system architecture is described. This is followed by a discussion of issues related to MM, where location, address and handover management are considered. A description of the signaling protocols for macro-mobility using Mobile-IP is then presented, emphasizing the need to minimize the change to the existing access network procedures. Finally, the performance of the protocols is analyzed in terms of the additional signaling time required for registration and handover.

When sending digital data from one device to another, both devices must agree on how to represent ones and zeros. This episode presents how signal levels affect the delivery of data and how line codes are used to represent the ones and the zeros.