Uncontrolled, systemic inflammation coupled to obesity is associated with increased morbidity and mortality of cardiovascular disease. As a consequence of adipose tissue expansion, hypoxia ensues resulting in inflammation, and the release of various factors to restore sufficient blood flow to the tissue. However, this is often inadequate and does not result in proper tissue oxygenation. Currently little is known about the potential for endogenously expressed immuno-modulatory cytokines to attenuate inflammation and also provide pro-angiogenic effects. Interleukin-19 is uniquely immuno-modulatory, pro-angiogenic and is expressed in adipose tissue. There is no known mechanism to explain the role of IL-19 in adipose tissue expansion. We hypothesize that IL-19 acts as a novel adipokine whose expression in inflamed adipose tissue promotes a compensatory, immuno-modulatory effect and is a counter-regulatory response to inflammatory stimuli. We report that IL-19 is expressed in adipose tissue at both the transcript and protein level and its expression is increased in inflamed visceral adipose tissue but not subcutaneous adipose tissue. Utilizing Il19-/- knockout mice, we found the loss of IL-19 leads to a metabolic phenotype characterized by reduced glucose and insulin tolerance, a reduction in protective gene expression with increased pro-inflammatory factors, increased adipose tissue hypoxia and fibrosis, decreased adipose tissue vessel density and increased adipocyte hypertrophy both in response to standard chow diet and chronic high fat diet. In cultured adipocytes the addition of IL-19 leads to increased metabolically protective factors while also increasing glucose uptake. Acute treatment with IL-19 reduced glucose and insulin intolerance in obese wild-type mice. These data suggest that IL-19 presents a novel therapeutic opportunity in that IL-19 can effectively allow adipose tissue expansion without concomitant inflammation and insulin insensitivity. Interleukin Enhancer-binding Factor 3 (ILF3), an RNA-binding protein, is best known for its role in innate immunity by participation in cellular anti-viral responses. A role for ILF3 in angiogenesis is unreported. Our working hypothesis is that ILF3 promotes angiogenesis through cytokine-inducible mRNA stabilization of pro-angiogenic transcripts. ILF3 expression in CD31+ capillaries of hypoxic cardiac tissue was detected by immunohistochemistry. Pro-angiogenic stimuli induce ILF3 mRNA and protein expression in cultured human coronary artery endothelial cells (hEC). Angiogenic indices including proliferation, migration and tube formation are all significantly reduced in hEC when ILF3 is knocked down using siRNA, but are significantly increased when ILF3 is overexpressed using adenovirus. Protein and mRNA abundance of several angiogenic factors including CXCL1, VEGF, and IL-8 are decreased when ILF3 is knocked down by siRNA. These factors are increased when ILF3 is overexpressed by adenovirus. ILF3 is phosphorylated and translocates from the nucleus to the cytoplasm in response to angiogenic stimuli. Pro-angiogenic transcripts containing adenine and uridine-rich (AU-rich) elements (AREs) were bound to ILF3 determined using RNA immunoprecipitation. ILF3 stabilizes pro-angiogenic transcripts including VEGF, CXCL1, and IL-8 in hEC. Together these data suggest that in endothelial cells, the RNA stability protein, ILF3, plays a novel and central role in angiogenesis. / Biomedical Sciences

The variation between males and females constitutes the largest phenotypic dimorphism in any given species and, in humans, contributes to differences seen in the risk, incidence and response to treatment for a number of diseases. A primary point of divergence is driven by variation in the sex hormone composition between males and females. However, we hypothesize that the sex chromosome composition alone drives dramatic events to which subsequent hormonal exposure either ameliorates or potentiates these differences. Methods to study early development are often limited to in vitro systems or in model organisms, such as in mouse. Human embryonic stem (ES) cell lines are available but are of limited access, are of high passages, and require more stringent growth conditions. Direct comparisons between male and female lines are extremely difficult, due to the abundance of multiple X chromosome statuses as well as non-random X chromosome inactivation. In the absence of more refined culture methods for human ES cells, we opted to use mouse ES cell lines derived in-house as this would enable us to determine allelic expression patterns as well as more easily maintain pluripotency and random X chromosome inactivation with established culture parameters. As such, to test our hypothesis, we derived an extensive panel of low passage mouse embryonic stem (ES) cell lines from reciprocal crosses between the C57BL/6 and CAST/EiJ mice. In total, I had access to over 20 unique mouse ES lines, including two of which show aneuploidy with loss of one of the sex chromosomes. The addition of these two lines to the experimental design grants us the ability to tease apart the individual contributions of the X and the Y chromosome in early development. Additionally, sex chromosome aneuploidies have yet to be evaluated in terms of their effects on the epigenome as well as their influence on directed differentiation in vitro. To set the foundation for our studies, we first performed a series of RNA-seq analyses in which we expanded the number and variation of sex-specific differences from previous reports using microarray. We then interrogated the contribution from each of the sex chromosome complements on gene ontology. Additionally, we identified and validated sex-specific alternative splicing events, for which there is very limited reporting. With an emphasis on genome-wide regulatory patterns, we then performed an unbiased weighted gene co-expression network analysis (WGCNA) for which we identified a key sex-specific expression module. The main driver of this module was the gene encoding Prdm14, a pivotal transcription factor involved in pluripotency. Luciferase assays with a known Prdm14-responsive enhancer showed higher expression when transfected into female than in male ES cells. Because Prdm14 is more abundant in female ES cells, this suggests that the dosage of this transcription factor is a key factor of its capacity to activate gene expression. This is the first ever documented sex-specific differential enhancer activity and further underscores the need to not only evaluate expression but functionality of the protein product within biological systems. Prdm14 has the dual capability of promoting and repressing transcription depending on its binding partners. Evaluation of histone modifications overlapping with known Prdm14 binding motifs in the promoters of the co-expressed genes revealed a unique signature between the male and female mouse ES lines. Based on our analyses, we hypothesize that the higher Prdm14 abundance in XX ES cells can activate gene expression even if genes harbor a repressive histone modification in their promoters. We propose that the lower abundance of Prdm14 in male ES cells can only activate genes that do not exhibit repressive histone modifications. With this information in hand, we then performed a directed differentiation assay to the cardiomyocyte lineage. From these experiments, we identified an XX-specific impairment to differentiate without chromosome loss. Additionally, the 39,X lines exhibited dysregulation of cardiac-related genes, potentially correlating with the defects seen in Turner syndrome patients. Overall these findings help to expand upon an underrepresented field in the basic sciences, namely the underlying contributions of the sex chromosome complement on gene expression and regulatory dimorphisms. / Biomedical Sciences

In recent years, the rapid decrease in size of personal communication devices has lead to the need for more compact antennas. At the same time, expansion of wireless systems has increased the applications for multi-functional antennas that operate over broad frequency bands or multiple independent bands. The civilian GPS system is quickly becoming the standard for personal and commercial navigation and position location. The difficulty with GPS is that there is no return link. That is, a GPS terminal determines its position, but that position is known only to the terminal user. A return link enables positional information derived from GPS to be communicated to a remote location. This is especially desirable for unmanned terminals. The next wide scale technology area for GPS is the integration of a GPS receiver with some type of wireless service to provide communication of the GPS - derived position as well as messaging. One of the most popular uses for this service is tracking of mobile cargo. This paper presents a design for a compact, low-profile antenna that operates at both the conventional cellular telephone band of 824 to 894 MHz and the civilian GPS L1 frequency of 1575 MHz. The combined antenna unit has a lateral diameter of less than 4 inches (10 cm) and its height is less than 2 inches (5 cm). The integrated unit is a hybrid design of two collocated antennas that operate at the two different bands. The planar inverted F antenna, PIFA, meets the specifications which are required in a reduced size environment. The PIFA is capable of achieving a bandwidth of 8% in the cellular band. The GPS portion of the hybrid unit consists of a dielectrically loaded patch located in a "piggyback" configuration on top of the top PIFA element. Computer simulation and design were performed using a combination of IE3D, a 2.5 dimensional commercial moment method code, and Fidelity, a commercial full 3D finite difference time domain code. Results will be presented from these calculations along with measurements on prototype antennas using both the Virginia Tech outdoor antenna range and the Virginia Tech near-field antenna range. / Master of Science

Control of neurosecretion of neurotransmitters appears to be regulated through second messengers that change the phosphorylation state of critical enzymes and proteins. The effect of protein phosphorylation promoting agents on secretion, desensitization and Na$\sp+$/Ca$\sp{2+}$ exchange activity were investigated and protein phosphatases were identified in bovine chromaffin cells. Cells exposed to 8-Br-cAMP, PDBu or okadaic acid alone show slightly decreased rates of desensitization. Okadaic acid plus 8-Br-cAMP potentiated secretion with repeated stimulations. The protein kinase inhibitor H7 increased the desensitization rate. These phenomena are observed during secretion evoked with elevated K$\sp+$ as well as by a nicotinic agonist. Thus, the effect of phosphorylation is at a post-receptor site. Cells treated with dbcAMP, PDBu, okadaic acid or calyculin A show lowered Na$\sp+$/Ca$\sp{2+}$ exchange activity and prolong cytosolic Ca$\sp{2+}$ transients caused by depolarization. Conversely, H7 enhances Na$\sp+$/Ca$\sp{2+}$ exchange activity. Na$\sp+$/Ca$\sp{2+}$ exchange activity in isolated membrane vesicles is inhibited by PKA and PKC. The results indicate that reversible protein phosphorylation modulates Na$\sp+$/Ca$\sp{2+}$ exchange activity and suggest that modulation of the exchanger may play a role in the regulation of secretion. Four distinct peaks of phosphatase activity were observed in homogenized bovine adrenal medulla cells when fractionated using an HPLC ion exchange DEAE column. Of these phosphatases, peaks II, III and IV show preferential dephosphorylation of the $\alpha$ subunit of phosphorylase kinase relative to the $\beta$ subunit and therefore are classified as protein phosphatases type 2. These phosphatases have broad substrate specificity and distinctly different relative specific activities toward the different substrates. Phosphatase 2A is likely to be the major enzyme in bovine adrenal medulla cells. ATP induced a greater secretory response than did acetylcholine without causing preferential secretion of norepinephrine or epinephrine. These data show that the response to ATP found in cultured cells is not an artifact of cell culture, and that ATP corelease with catecholamines from the storage vesicles has a significant physiological role. Freshly isolated cells were separated on a density gradient; the lower density cells develop a much stronger response to ATP than do the higher density cells.

The oxidation behavior of cholesterol and the relationship between cholesterol and non-cholesterol lipids in oxidation processes were studied in a bovine red blood cell membrane system (RBC Mb). RBC Mb was prepared with a process of hemolysis and hypotonic washing. After preparation, the RBC Mb suspensions were subjected to oxidation treatments of $\rm H\sb2O\sb2$-Fe, UV (254 nm, 312 nm, and 365 nm), and $\gamma$-irradiation. After the treatments, lipids were extracted, and cholesterol, cholesterol oxides and fatty acids were analyzed with GC and GC-MS. Of all the oxidation products, 7-keto-cholesterol was found to be the major oxidation product of cholesterol. Other oxidation products, to a less extent, were 7-hydroxylcholesterol, $\alpha$- and $\beta$-epoxycholesterol, etc. Oxidative destruction of cholesterol was detected together with the destruction of unsaturated and saturated fatty acids, although the destruction rates were different. Model studies indicated that cholesterol and fatty acids in the suspension of RBC Mb lipid extracts were much more susceptible to oxidative destruction than the cholesterol and fatty acids in the native RBC membranes. UV studies showed the complexity of cholesterol behavior in response to UV lights. Although the destruction of solid crystalline cholesterol at 312 nm UV went much faster than that at 254 nm and 365 nm, the fastest cholesterol destruction in RBC Mb aqueous suspensions occurred at 365 nm UV, which was the least powerful of the 3 wavelengths for solid crystalline cholesterol destruction. For 7-keto-cholesterol, the most destructive wavelength was 254 nm. In $\gamma$-irradiation study, there was no distinctive radiolytic product detected in this study. Contrasted with UV and $\rm H\sb2O\sb2$-Fe treatments, the destruction of cholesterol with $\gamma$-irradiation gave rise to far less production of cholesterol oxides which were detectable with the GC-MS procedures.

Embryo growth of oviparous animals depends upon utilization of nutritive proteins, primarily the glycoprotein vitellin (Vt). These proteins are usually extraovarian in origin and accumulate in the oocyte through receptor-mediated endocytosis. This event is well characterized for both insects and vertebrates. In contrast, the mechanisms of yolk protein utilization are not understood. In this study, the German cockroach Blattella germanica was used as a model insect system to explore the components that initiate and regulate Vt processing during early embryo development. In B. germanica, Vt processing is initiated at days 3-4 postoviposition at 30$\sp\circ$C. The yolk of freshly oviposited eggs assayed in vitro for protease was devoid of activity but protease specific activity increased dramatically during embryo development. This activity correlated temporally with Vt processing in vivo suggesting that the protease is necessary for Vt processing. The protease was purified from yolk at day 6 postoviposition by gel filtration and affinity chromatography and classified as a cysteine protease. Its molecular weight, estimated by SDS-PAGE and immunoblotting, was approximately 29 kDa. Its pH optimum was 5.5, within the pH range of yolk granules. The purified protease degraded Vt in vitro yielding peptides of the same apparent molecular weights as Vt processed in vivo. Acidification of day 0 yolk in vitro induced protease activity suggesting that a latent protease is present in eggs early in embryo development. The latent protease activity was purified from yolk at day 0 postoviposition by successive use of gel filtration, Mono Q FPLC, and hydrophobic interaction chromatography. The purified latent protease had a molecular weight of approximately 40 kDa and could be activated in vitro into a cysteine protease of 29 kDa. The conversion depended on acidification and was blocked by the cysteine protease inhibitor E-64, suggesting the activation is autocatalytic. Kinetic studies showed that activation occurred by intermolecular catalysis. The pH-activity and inhibitor sensitivity profile of the in vitro-activated protease matched those of the protease suggesting that the latent protease is the proenzyme of the protease. Active site derivatization of the 40 kDa proprotease revealed that its conversion to the 29 kDa protease in vivo occurs as Vt processing begins, suggesting that Vt processing is regulated through the protease. The proenzyme activation and pH optimum data of the purified protease emphasize that yolk granule acidification is an important cellular factor for the regulation of Vt processing by B. germanica in vivo. A murine polyclonal antibody against purified proprotease was made monospecific by affinity column chromatography. Using this antibody, the proprotease was detected in fat bodies and ovaries of vitellogenic females by immunoblotting. This result is consistent with the hypothesis that the protease precursor is synthesized extraovarily, probably in the fat body.

A comparative analysis of actin localization in Nicotiana pollen tubes has been made using a monoclonal actin antibody and rhodamine-phalloidin (Rh-Ph). The antibody, based on Western blotting of pollen tube extract, labels a polypeptide of 45 kD that comigrates with muscle actin. Immunofluorescence studies reveal a network of axially oriented strands of microfilaments (MFs) that distribute throughout the length of the tube except at the apex, where diffuse staining is usually observed. A similar pattern of MFs is evident after Rh-Ph staining. When pollen tubes are treated with cytochalasin B or D, cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the MF pattern is markedly altered; however, the antibody and Rh-Ph produce different staining patterns. The antibody reveals massive bundles of MFs that distribute throughout the tube length and extend into the very tip. In contrast, Rh-Ph shows mostly a diffuse staining pattern. Immunogold labeling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution (RF-FS) also reveals many MF bundles after cytochalasin treatment. These results thus question the efficacy of Rh-Ph as a faithful F-actin indicator under all conditions. By using two monoclonal myosin antibodies, a myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes The epitopes of these antibodies were found to reside on myosin head and tail portion, and therefore were designated anti-S-1 and anti-LMM, respectively. On Western blots of pollen tube proteins, both antibodies label a polypeptide of approximately 175 kD. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the length of the tube, often with an enrichment in the apical region. These fluorescent spots are believed to represent the vesicles and organelles in the tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope in a certain percentage of cells. The different staining patterns of the nucleus may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and organelles.

The effect of brain microtubule associated proteins and low concentrations of the drug vinblastine have been used to study the modulation of MT dynamic instability in living BSC-1 cells using low-light-level fluorescence microscopy and quantitative MT tracking methods. Heat stable brain MAPs (hs MAPs) and microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine labeled tubulin. Both MAP preparations suppressed MT dynamics in vivo, by reducing the average rate and extent of both growth and shortening events. Both hs MAPs and MAP-2 did not significantly alter frequency of rescue per unit time but significantly increased the frequency of rescue per unit distance. Hs MAPs decreased the catastrophe frequency per unit time by two fold, while MAP-2 reduced this parameter to a lesser extent. When calculated as events per unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. Both MAP-2 and hs MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Hs MAPs increased the average pause duration, decreased the average number of events/min/MT and increased the probability that a paused MT switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual MTs primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the MT length can be detected. The results further suggest that the expression of molecules like MAPs directly contributes to cell type specific MT dynamic behavior. Vinblastine treatment suppresses MT dynamics in vivo, by reducing the average distance and rate of growth and shortening events. Vinblastine treatment increased the average pause duration and the percent time the MTs spent in the paused state; the percent time in shortening was decreased. Vinblastine markedly reduced the frequency of catastrophe measured as events/unit time or unit distance. The frequency of rescue was increased when measured as events/unit distance. The probability of transition from pause to growth phase increased with increasing concentration of vinblastine. These results suggest that low concentration of vinblastine decreases the dynamicity of microtubules in vivo, consistent with the results reported in vitro and indicates that vinblastine might actively cap the ends of the MTs thereby reducing the exchange of tubulin dimers at the ends, consistent with earlier studies (Wilson et al., 1982; Jordan and Wilson, 1990).

The objective of this thesis is to model and analyze the energy consumption in heterogeneous cellular systems and develop techniques to minimize it. First, the energy consumption is modeled and analyzed for multi-layered heterogeneous wireless systems. This work encompasses the characterization of all the energy consumed at the base stations. Then, a novel on-off and cell-association scheme is proposed to minimize the overall network energy consumption while satisfying the spatially- and temporally-varying traffic demands. Second, we exploit the use of multi-stream carrier aggregation not only to improve the energy efficiency, but also to balance it with the conflicting objective of capacity maximization. Third, we analyze the performance of discontinuous reception methods for energy savings within the user equipments. Then, for scenarios that support carrier aggregation, we develop a cross-carrier-aware technique that further enhances such savings with minimum impact on the packet delay. Fourth, the use of small cells as an energy-saving tool and its limitations are analyzed and modeled in OPNET, a high-fidelity simulation and development platform. To bypass such limitations, a novel small cell solution is proposed, modeled, and analyzed in OPNET and then compared against its existing alternative.

A ativação dos fagócitos é crítica para a defesa contra vários patógenos, por isso a importância de estudos que desenvolvam alternativas para a ativação dessas células. Nosso estudo visou investigar os efeitos do BAY 41-2272 na ativação de fagócitos. Para este propósito avaliamos a fagocitose, liberação de superóxido e atividade microbicida. Foram usados PBMC, neutrófilos e a linhagem celular THP-1, cultivadas na presença ou ausência de BAY 41-2272 (1mM ou 3mM) por 1h ou 48h a 37°C. A liberação de superóxido foi avaliada pelo ensaio da redução do citocromo c inibido especificamente pela superóxido dismutase; a fagocitose foi analisada através da co-cultura com partículas de Zymosan e contagem das células com partículas englobadas; e a atividade microbicida foi mensurada por meio da co-cultura com E. coli enteropatogênica seguida pela contagem das CFU formadas pelas bactérias recuperadas dos fagócitos. Além disso, fizemos a dosagem de IL-12, IFN-g e TNF-a e a análise da expressão gênica do gene CYBB. Todos os tipos celulares responderam aos tratamentos. Os PBMC, neutrófilos e THP-1 tratados com BAY 41-2272 produziram significativamente mais superóxido (cerca de 50% mais), e apresentaram significativamente maior atividade fagocítica (cerca de 54% mais), microbicida (cerca do dobro) comparados ao grupo controle não tratado, além de produzir TNFa. Ainda, o tratamento com o fármaco aumentou a expressão do gene da gp91phox nas THP-1 e PBMC. BAY 41-2272, especialmente na dose de 3mM, mostrou um grande potencial na ativação dos fagócitos em vários aspectos. Este potencial pode ser explorado na busca por novas terapias destinadas ao controle de infecções, principalmente no caso das imunodeficiências. / Phagocyte activation is critical role for host defense against several pathogens, so that studies developing alternatives for activating these cell are very important. We investigated the effects of BAY 41-2272 on phagocytes activation. For this purpose we evaluated several aspects indicating cellular activation, as phagocytosis, superoxide release and microbicidal activity. We used PBMC, neutrophils and THP-1 cell lineage cultured or not with BAY 41-2272 (1mM and 3mM) for 1h or 48h at 37°C. Superoxide release was tested by superoxide dismutase-inhibitable cytochrome c reduction assay; phagocytosis was evaluated through co-culture with zymosan particles and counting of ingested particles; and microbicidal activity was measured through co-incubation with enteropathogenic E. coli followed by counting of CFU from recovered bacteria from phagocytes. We analyzed the IL-12, IFN-g and TNF-a cytokine production and gp91phox gene expression. All cell types showed a response to treatments. PBMC, PMN and THP-1 treated with BAY 41-2272 produced significantly more superoxide (about 50% more), and presented significantly more phagocytic (about 54% more), microbicidal (about twice) activity than control group and production more TNF-a than control group. The expression of CYBB gene was more expressed on treated cells than control. BAY 41-2272, especially at 3mM, showed a great potential in activation of phagocytes in several aspects. This potential should be investigated at the light of new therapies seeking infection control, mainly in immunodeficiency.