Background: Mast cells are known for their role in allergy, asthma, and systemic anaphylaxis, and have been shown to play a role in inflammatory disease. Interleukin-10 can regulate inflammatory responses both in vitro and in vivo, and may be a natural regulator of mast cell activation.Objective: To examine Interleukin-10 mediated regulation of FcεRI expression and related downstream signaling molecules, and to determine how this affects mast cell function in vitro and in vivo.Methods: Mast cell FcεRI expression was evaluated with and without IL-10 treatment in human lung and skin mast cells, and on peritoneal mast cells from mice overexpressing IL-10 via injection or a transgenic model. Mast cell function was evaluated by observing responses of IL-10 treated mice to passive systemic anaphylaxis.Results: Interleukin-10 inhibited FcεRI expression on mouse and human mast cells, both in vitro and in vivo. IL-10 also suppressed expression of the key signaling molecules Syk, Fyn, Akt and Stat5. Mice chronically overexpressing IL-10 had a reduced response to passive systemic anaphylaxis, indicating impaired mast cell activation.Conclusion: Interleukin-10 suppresses mast cell FcεRI expression in vitro and in vivo, and reduces IgE-mediated activation. The anti-inflammatory effects of IL-10 may relate to its suppression of critical signaling molecules.Clinical Implications: Interleukin-10 polymorphism is associated with increased IgE levels and incidence of atopic disease; hence IL-10 dysregulation may affect atopic etiology. Further, IL-10 therapy is a possible treatment for atopic allergy and asthma.

Doctor of Philosophy / Department of Biology / Lynn Hancock / Enterococci are the third leading cause of nosocomial infections including urinary tract infections (UTI), surgical site infections (SSI) and blood stream infections. Enterococci are also found in the gastrointestinal tracts of humans, and other mammals. We elucidated the influence of the transcriptional regulator RpoN on enterococcal biofilm formation, virulence potential and cell wall architecture and proposed a potential involvement for carbohydrate metabolism in these processes. Biofilms are held together by matrix (BM) components such as extracellular DNA (eDNA) released by cell death from a sub-population of cells. The rpoN mutant (ΔrpoN) was resistant to autolysis as well as fratricide-mediated cell death and eDNA was not detected in planktonic as well as biofilm cultures. Unlike the parental strain V583, the ΔrpoN mutant formed proteinase K sensitive biofilms, suggesting that protein as well as eDNA serves as an important matrix component. The rabbit model of endocarditis was used to assess the effect of rpoN deletion on enterococcal virulence. Rabbits infected with ΔrpoN had reduced bacterial burden in heart, blood, liver, kidney and vegetation in comparison to the parental strain. The growth defect of ΔrpoN in physiologically relevant glucose levels (5 mM) partially explains the reduced bacterial burdens observed in the virulence study. Microarray analysis of ΔrpoN showed that 10% of the genome is differentially regulated by RpoN. Deletion of rpoN also protects Enterococcus faecalis from lysis in the absence of known modulators of cellular lytic events such as O-acetylation and D-alanylation. Of the four identified enhancer binding proteins in E. faecalis, MptR regulates the RpoN-dependent mannose/glucose uptake system (MptABCD) and the ΔmptR mutant phenocopied the ΔrpoN mutant in the eDNA release and growth assays. Because MptC and MptD have been shown to be the cellular receptors for class IIa and IIc bacteriocins, we are presently testing the hypothesis that these receptors may serve as a global receptor for bacteriocins. In conclusion, our data demonstrates that alterations in the metabolic state of the bacterium, as observed in the ΔrpoN mutant could be responsible for the switch in biofilm matrix composition, and this switch in turn likely influences the virulence potential of the bacterium.

Thesis advisor: Michelle M. Meyer / Bacteria commonly utilize cis-acting mRNA structures that bind specific molecules to control gene expression in response to changing cellular conditions. Examples of these ligand-sensing RNA cis-regulators are found throughout the bacterial world and include riboswitches, which interact with small metabolites to modulate the expression of fundamental metabolic genes, and the RNA structures that bind select ribosomal proteins to regulate entire ribosomal protein operons. Despite advances in both non-coding RNA discovery and validation, many predicted regulatory RNA motifs remain uncharacterized and little work has examined how RNA cis-regulators behave within their physiological context in the cell. Furthermore, it is not well understood how structured RNA regulators emerge and are maintained within bacterial genomes. In this thesis, I validate the biological function of a conserved RNA cis-regulator of ribosomal protein synthesis previously discovered by my group using bioinformatic approaches. I then investigate how bacteria respond to the loss of two different cis-regulatory RNA structures. Using Bacillus subtilis as a model organism, I introduce point mutations into the native loci of the ribosomal protein L20-interacting RNA cis-regulator and the tandem glycine riboswitch and assay the strains for fitness defects. I find that disrupting these regulatory RNA structures results in severe mutant phenotypes, especially under harsh conditions such as low temperatures or high glycine concentrations. Together, this body of work highlights the advantages of examining RNA behavior within its biological context and emphasizes the important role RNA cis-regulators play in overall organismal viability. My studies shed light on the selective pressures that impact structured RNA evolution in vivo and reinforce the potential of cis-regulatory RNAs as novel antimicrobial targets. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Germ cell tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. The chemotherapy burden for patients with malignant germ cell tumours (mGCTs) that require treatment results in substantial longterm side-effects, and, furthermore, poor-risk patients have < 50% survival. Consequently, identifying common molecular changes and novel therapeutic targets in mGCTs is of major clinical importance. MicroRNAs are short, non-protein coding RNAs that regulate gene expression. We previously showed that miR-99a-5p/-100-5p and miR-125b-5p are among the most frequently underexpressed microRNAs in mGCTs, regardless of anatomical site, histological type or patient age. The present study investigates the upstream causes and downstream consequences of such under-expression. The mature form of miR-125b-5p is the product of two genomic loci, which form a cluster with either miR-99a-5p (on chromosome 21q) or miR-100-5p (on chromosome 11q). MiR-99a-5p/- 100-5p share identical 'seed' regions (at nucleotide positions 2-7), which determine their mRNA targets. Cross-reactivity experiment revealed that both miR-99a-5p and miR-100-5p probes were highly cross-reactive to each other's target (from 91% to 95%), indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-99a-5p/-100-5p and miR-125b-5p levels (R2 =0.989) in mGCTs, strongly suggesting co-regulation. Primary microRNA transcripts (pri-miR-99a/-100 and pri-miR-125b), and other genes that colocalise to these miRNA clusters (e.g. BLID on chromosome 11), were quantified by RT-qPCR in four representative cell lines - TCam2, 1411H, 2102Ep, and GCT44 - which were derived from a range of common histological types of mGCTs. A significant down-regulation (p < 0.0001) of all primary transcripts was observed, suggesting transcriptional repression of the entire cluster regions. Treatment of the cell lines with 5'-azacytidine resulted in significant upregulation of all three miRNAs (p < 0.002), as well as BLID (p < 0.02). The methylation status of potential CpG islands at the region of interest on chromosome 11 and chromosome 21 was therefore investigated by Pyrosequencing. Significant hyper methylation was found in 2102Ep, 1411H and GCT44 cell lines, suggesting that the miR-99a-5p/-100-5p and miR-125b-5p clusters are likely transcriptionally silenced by DNA methylation. To assess the functional relevance of these microRNAs in GCT progression, co-transfection of microRNA mimics (8.3 nM miR-99a-5p/-100-5p + 8.3 nM miR-125b-5p) was performed. A significant decrease in cell growth was seen in 1411H (p < 0.01) and TCam2 (p < 0.03) cells. To identify the mimics' downstream mRNA targets, HumanHT-12 v4 Expression Bead Chip (Illumina) mRNA arrays were used and data analysed using Sylamer. This analysis showed that mimic-treated cells were enriched in downregulated genes involved in pro-proliferative mechanisms. Among those, further functional characterisation focussed in particular on TRIM71, FGFR3, E2F7 and LIN28A. Moreover, restoring miR-99a-5p/-100-5p and miR-125b-5p in TCam2 cells also resulted in G0-G1 accumulation, consistent with a cell cycle effect. These data support a functionally important role for miR99a-5p/-100-5p and miR-125b-5p in GCT progression. They also raise the possibility of a therapeutic replenishment approach for treating these, and potentially other, tumours.

Orientador: Izabel Cristina Leite / Banca: Luiz Henrique Carvalho / Banca: Paulo Roberto de Camargo e Castro / Banca: Antonio César Bolonhezi / Banca: Manoel Luiz Ferreira Athayde / Resumo: Com o objetivo de avaliar o efeito de doses de cloreto de mepiquat em cultivares de algodão herbáceo, foi conduzido um experimento no ano agrícola de 2002/2003, na Fazenda de Ensino e Pesquisa da FEIS/UNESP, localizada no município de Selvíria (MS). O delineamento experimental utilizado foi o de blocos ao acaso, com parcelas subdivididas, e quatro repetições. Nas parcelas foram estudados quatro cultivares de algodão (Coodetec 401, IAC 24, DeltaOpal e Deltapine Acala 90) e nas subparcelas, três doses de cloreto de mepiquat (0,0; 50,0 e 100,0 g i.a.ha-1). As parcelas experimentais constituíram-se de seis linhas com 10 m de comprimento e 0,90 m de espaçamento entrelinhas, sendo consideradas úteis as quatro linhas centrais. Foram determinados, em dez plantas de cada parcela, a altura das plantas e o diâmetro do caule. O número de nós no caule, número de ramos e de capulhos por planta foi obtido em cinco plantas amostradas de cada parcela. A produção de algodão em caroço foi determinada através da colheita dos capulhos em quatro metros de cada uma das duas linhas centrais. Amostras de 20 capulhos de cada parcela foram utilizadas para a obtenção da massa média de um capulho, massa de 100 sementes, porcentagem de fibra e características tecnológicas da fibra. A análise de crescimento foi feita determinando-se, a cada 14 dias, a área foliar e a massa seca acumulada nos diferentes órgãos da planta. A partir desses dados foram calculados a área foliar total, razão de área foliar, área foliar específica, razão de massa de folhas, taxa de crescimento relativo e taxa assimilatória líquida. Considerando-se 100% a massa seca total, determinou-se a distribuição em porcentagem da massa seca entre as diferentes estruturas da planta... (Resumo completo, clicar no acesso eletrônico abaixo) / Abstract: The experiment was conducted with the objective of evaluating the effect of doses of mepiquat chloride in cotton cultivars in the agricultural year of 2002/2003, at Teaching and Research Farm of FEIS/UNESP, located in Selvíria, MS, Brazil. The experimental design was complete blocks in split-plot arrangement and four replications. In the plots four cotton cultivars (Coodetec 401, IAC 24, DeltaOpal and Deltapine Acala 90) were studied and in the subplots, three doses of mepiquat chloride (0,0; 50,0 and 100,0 g i.a.ha-1). The experimental plots had six rows with 10 m of length and 0,90 m between rows, being considered useful the central four rows. The height of the plants and the diameter of the stem were measured in ten plants in each plot. The number of nodes in the stem, number of branches and of fruits per plant were obtained in five plants collected from each plot. The cotton yield was determined through the harvested fruits of the two central rows. Samples of 20 fruits of each plot were used to obtain the average mass of one fruit, mass of 100 seeds, fiber percentage and technological characteristics of the fiber. The growth analysis was performed by determining, every 14 days, the leaf area and the dry mass accumulated in the different organs of the plant. From those data, total leaf area, leaf area ratio, specific leaf area, ratio of leaf mass, relative growth rate and net assimilation rate, were calculated. Considering the total dry mass as 100%, the distribution, in percentage, of the dry mass among the different structures of the plant was determined. The obtained results allowed to conclude that 50,0 g i.a.ha-1 of mepiquat chloride caused minor height of the plants in the Coodetec 401, DeltaOpal and Deltapine Acala 90 cultivars while, for 24 IAC the dose of 100,0 g i.a.ha-1 was the best... (Complete abstract, click eletronic address below) / Doutor

The accessory gene regulator (agr) quorum-sensing system is one of the major regulators of virulence factor production in the pathogen Staphylococcus aureus. Activation of the system depends on the production and sensing of a cyclic peptide signal called the autoinducing peptide (AIP). The biosynthesis of AIP depends on the coordinated action of the AgrB integral membrane endopeptidase and SpsB signal peptidase to process the peptide precursor AgrD into the final signal structure. The primary goal of this dissertation was to gain further insight on the role of AgrD and AgrB in the AIP biosynthesis mechanism. Studies in Chapter II were undertaken to better understand the role of AgrD domains in AgrB-mediated processing. A series of truncation and site-directed mutagenesis studies identified key residues in the AgrD C-terminus that were essential for AgrB processing and AIP production. In parallel, genetic manipulation of the N-terminal leader and AIP-encoding sequence revealed a role for these segments in AIP processing. For the first time, a complex of AgrD covalently linked to AgrB was identified, supporting proposals that this intermediate is an important precursor to AIP production. In Chapter III structure-function studies were performed on AgrB to gain further insight into the AIP biosynthetic mechanism. Initially, the agrBD genes were subjected to random mutagenesis and screened for deficiencies in AIP production. Single-site mutations at 20 different residues within AgrB and another 14 in AgrD were isolated. Interestingly, new mutations in the AgrD N-terminal leader were identified that affect AIP biosynthesis at different steps. In AgrB, most of the mutations blocked peptidase activity, but charge alterations to the K129-K131 region were defective in a later pathway step, separating the peptidase function from AIP ring formation and transport. To localize the AgrB mutations, we reevaluated the membrane topology using the substituted cysteine accessibility method. Our new model predicts four transmembrane helices and a reentrant loop, with both termini located outside of the cell. Finally, co-immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. Taken together, these findings provide a better understanding of the functional role of specific AgrD and AgrB regions in AIP biosynthesis.

<p>Controlling the power flow in transmission systems has recently gained increased interest. The difficulties of building new lines and the pressure of having a high utilization of existing assets, makes the flexibility of grid systems increasingly important.</p><p>This master thesis work investigates induction regulators as control devices for active power flow in a transmission system. A small change in angle of the rotor affects both the amplitude and the phase of the voltage. The magnetic coupling in the induction regulator can be controlled by changing the permeability of a thermo magnetic material such as gadolinium and can hence give a second independent controlling parameter. An analytical model and calculations in the</p><p>FEM software AceTripleC together with Matlab, is used to simulate the influence of the regulators connected to a simple grid in case1, a 400 kV scenario and case 2, a 45 kV scenario.</p><p>The analysis was carried out on a small transmission system consisting of two parallel transmission lines connected to source and load. The induction regulators are connected to one of the parallel transmission lines. The regulators modelled in case 1 must be able to control the active power flow in the regulated line to vary between 50 and 150 % of the original power flow through this line.</p><p>This shall be done over a range of 0 to 800 MW transmitted power. The regulators modelled in case 2 must be able to control the active power flow in</p><p>the regulated line to vary between 0 and 30 MW, if this does not cause the power flow in the parallel line to exceed 30 MW. This shall be done over a range of 0 to</p><p>50 MW transmitted power.</p><p>The regulators are designed as small and inexpensive as possible while still fulfilling requirements regarding the active power flow controllability in the grid, current density in windings and maximum flux density in core and gap.</p><p>The results indicate that the size of the 400 kV solution has to be reduced to become competitive whereas for the 45 kV solution the relative difference to existing solution is smaller. Advantages with the proposed design over a phase shifting transformer are mainly a simpler winding scheme and the absence of a tap changer.</p>

The scope of extreme-environment diving defined within this work encompasses diving modes outside of the generally accepted no-decompression, open-circuit, compressed-air diving limits on selfcontained underwater breathing apparatus (scuba) in temperate or warmer waters. Extreme-environment diving is scientifically and politically interesting. The scientific diving operational safety and medical framework is the cornerstone from which diving takes place in the scientific community. From this effective baseline, as evidenced by decades of very low DCS incidence rates, the question of whether compressed air is the best breathing medium under pressure was addressed with findings indicating that in certain depth ranges a higher fraction of oxygen (while not exceeding a PC 2 of 1.6 ATA) and a lower fraction of nitrogen result in extended bottom times and a more efficient decompression. Extremeenvironment diving under ice presents a set of physiological. equipment, training and operational challenges beyond regular diving that have also been met through almost 50 years of experience as an underwater research tool. Diving modes such as mixed-gas, surface-supplied diving with helmets may mitigate risk factors that the diver incurs as a result of depth, inert gas narcosis or gas consumption. A close approximation of inert gas loading and decompression status monitoring is a function met by dive computers, a necessity in particular when the diver ventures outside of the single-dive profile into the realm of multi-level, multi-day repetitive diving or decompression diving. The monitoring of decompression status in extreme environments is now done exclusively through the use of dive computers and evaluations of the performance of regulators under ice have determined the characteristics of the next generation of life-support equipment for extreme-environment diving for science. These polar, deep and contaminated water environments require risk assessment that analyzes hazards such as cold stress, hydration, overheating, narcosis, equipment performance and decompression sickness. Scientific diving is a valuable research tool that has become an integral methodology in the pursuit of scientific questions in extreme environments of polar regions, in contaminated waters, and at depth.

The revolutionary progress that happened recently in the micro-electro mechanicalsystems (MEMS) field and the complementary metal-oxide-semiconductor(CMOS) integrated circuits has made it possible to produce low-cost, low-powerand small size processing circuits. Utilizing wireless communication theory allowsthose circuits to send their data over a network. This wireless sensor network isknown as "Smart Dust". Each wireless sensor node in the network is indicated as "mote". It consistsof several components: sensors, micro-processors, radio transceivers and a powermanagement unit. The power management unit can be divided into several partsincluding battery, power control and regulator. The purpose of the regulator is tosupply a constant reliable voltage to the other parts in the mote as most of thedevices have voltage limits that need to be considered to guarantee producing arobust long-life mote. In this thesis designing a low-power regulator is investigated. The goal of thethesis is to design a regulator that can handle the high-voltage acquired froman energy harvest unit using only 65-nm core transistors. This allows an easierproduction process that results in a low-cost fully-integrated chip. The regulatorarchitecture to be used is a simple linear regulator. The report highlights the theoretical background, the challenges of the analogdesign and presents the results of the simulation that were ran using cadence designsystem software on schematic level.

Automatic voltage regulation is necessary for all power producing synchronous generators to ensure that the produced power have a constant and stable voltage level and to sustain grid stability. The aim of this thesis is to design and build an automatic voltage regulator for a synchronous generator. A six-phase permanent magnet alternator will be used to excite the rotor with solid-state relay controlled rotating bridge rectifier. The field current is regulated by a closed loop control system that is based on a programmable logic controller, PLC. Programing of the PLC is executed in the developing environment CoDeSys, IEC 61131-3, which is the international standard for programing PLC applications. Simulations for predicting the system behavior is done with a web based in-browser tool, circuitlab.com. The results show a good performance of the regulator and the closed loop system although there is room for improvement of the solid-state controlled rectifier system.