Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.

Actin cytoskeletal regulation is of critical importance for a number of diverse cellular functions, including cell motility, endocytosis, cell division and transcription. Tight regulation of actin is critical for many aspects of cancer biology and in particular invasion and metastasis. ADF/cofilins are among the most important actin regulatory proteins. Mammals have three highly conserved members, ADF, CFL1 and CFL2, which regulate actin dynamics by severing and depolymerizing actin filaments. Despite a huge literature on the roles of ADF/cofilins in actin treadmilling and cell migration in vitro and in cancer cell behavior during invasion, very little is known about their collective roles in tissue homeostasis. By employing genetic knock-outs of ADF, in conjunction with conditional depletion of CFL1 using a Cre-LoxP system under the control of the keratin 14 promoter, we were able to study the effects of ADF/CFL1 loss in vivo in the mouse epidermis. Furthermore, by generating ADF-null squamous cell carcinoma (SCC) cell lines and by transiently downregulating CFL1 with RNAi, we were able to investigate further the cellular responses after ADF/CFL1 depletion in vitro. Co-depletion of ADF and CFL1 from the mouse epidermis triggered loss of tissue homeostasis characterized by abnormal thickening of the tissue, actin filament accumulation and nuclear deformation. Loss of ADF/CFL1 in cultured malignant keratinocytes also led to aberrant cell morphology accompanied by unrestrained accumulation of actin stress fibers tethered to enlarged focal adhesions. Enhanced SRF/MAL-mediated transcription fuels this uncontrolled actin polymerization which is also mediated by Arp3. Furthermore, these actin filaments are decorated with phospho-myosin light chain, which indicates their contractile nature. As a consequence, the increased intracellular acto-myosin tension results in nuclear deformation, which is promoted by the deregulated actin filaments tethered to the nuclear envelope via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Overall, we describe new conceptual insight into the cellular functions of ADF/cofilins. We show that their activities are essential for the dynamic regulation of contractile actin filaments that, if left unchecked, lead to loss of cellular homeostasis and cell death promoted by loss of nuclear integrity. Additionally, the critical roles of nuclear actin and actin-associated proteins have recently started being appreciated. Thus, for the first time we set out to investigate new functions of cofilins in the nucleus using proteomics, and identify new cofilin binding partners that implicate them in novel cellular pathways, expanding our knowledge on these small actin-binding proteins.

O principal objetivo desta dissertação é obter um Teorema do Envelope que permita mecanismos não diferenciáveis, preferências arbitrárias e que possa ser aplicado em modelos com múltiplos agentes. Nós alcançamos isto ao expandir a análise de Milgrom e Segal (2002), generalizando seus resultados para espaços de tipos multidimensionais. Dessa forma, continuamos permitindo que a regra de escolha (mecanismo) seja descontínua. Para obter nosso resultado, é necessário o uso do Teorema do Máximo de Berge e, consequentemente, devemos impor compacidade no conjunto de escolha. Inicialmente esta hipótese pode parecer forte, porém argumentamos que em aplicações _e muito improvável termos um conjunto de escolha aberto ou, principalmente, não limitado. Nós também identificamos condições para que a função valor seja absolutamente contínua e mostramos que sua representação integral também é válida para espaços de tipos multidimensionais. Inicialmente propomos uma generalização direta do resultado de Milgrom e Segal (2002), utilizando a hipótese de continuidade absoluta da função de utilidade do agente. Entretanto, esta exigência não possui muito significado econômico e é considerada pouco elegante por parte da literatura. Neste sentido, incorporamos uma hipótese adicional de diferenciabilidade da utilidade em todo o domínio que gera a mesma representação integral e possui uma maior interpretação econômica. Nossos resultados são, em geral, aplicados a modelos com múltiplos agentes, em especial Economia do Setor Público (provisão de bens públicos e taxação ótima) e teoria dos leilões. / The main objective of this dissertation is to obtain an Envelope Theorem that allows non-di erentiable mechanisms, arbitrary preferences, and that can be applied to models with multiple agents. We achieve that by expanding the analysis of Milgrom and Segal (2002) and generalizing their results to multidimensional type spaces. Thus, we continue allowing that the choice rule (mechanism) is discontinuous. For our result, it is necessary to use the Berge's Maximum Theorem and therefore we must impose compactness in the choice set. Initially this assumption may seem strong, but we argue that in applications there is an open or unbounded choice set is very unlikely. We also identify conditions for the value function is absolutely continuous and show that its integral representation is also valid for multidimensional type spaces. Firstly we propose a direct generalization of the Milgrom and Segal (2002)'s result, using the assumption of absolute continuity of the agent's utility function. However, this requirement does not have much economic interpretation and it is considered not very elegant in the literature. In this sense, we incorporate an additional assumption of di erentiability of the utility in all range that generates the same integral representation and it possesses a greater economic interpretation. Our results are generally applied to models with multiple agents, in particular Public Economics (public goods supply and optimal taxation) and auction theory.

Recent evidence suggests that overfishing of large predatory fishes has resulted in substantial population declines and pelagic longline (PLL) fishing is a major contributor. The primary objective of this dissertation is understanding factors that affect the interactions between marine fish PLL fishing. These factors are important to determine vulnerability of bycatch and target species, especially when PLL catch and effort data are used to estimate stock abundance. Chapter 1 reviews 107 publications/reports on this topic. Results indicate that accurate characterization of PLL gear performance requires empirical measurement of horizontal and vertical gear movement; and pop-up satellite tags (PSATs) are best suited for quantifying pelagic fish vertical habitat use if: (i) sampling resolution and data storage are not a function of tracking duration and (ii) substantial monitoring durations are employed. This review documents the current state of knowledge for these factors and guidance for future research. In Chapter 2, hook time-at-depth was monitored for commercial PLL sets targeting swordfish, Xiphias gladius. Temperature-depth recorders (TDRs) were deployed at the hook, systematically along the entire gear length. Results indicated that: (i) hook depth predictions based on catenary geometry drastically overestimated actual fishing depths and (ii) using catenary geometry fails to capture within- and among-set variability, potentially resulting in biased stock assessments. Chapter 3 used temperature-depth data from PSATs on swordfish and blue marlin (Makaira nigricans) and similar data from TDR monitored near-surface and deep PLL fishing to determine the diel probability of these species encountering PLL hooks. Results indicated that blue marlin and swordfish inhabit surface waters at night with similar probability of encountering PLL hooks but may have different vulnerabilities due to temporal separation in feeding with blue marlin preferring daytime and twilight and swordfish preferring nighttime. Therefore, reducing fishing during daylight hours may reduce blue marlin bycatch during PLL targeting swordfish. Chapter 4 alternated non-offset and 10° offset circle hooks during PLL fishing and compared the relative performance on catch rates, percent mortality, and deep hooking percentage. Results indicated that 10° offset circle hooks can reduce fishing efficiency and conservation benefits commonly associated with circle hooks.

Modern communication systems operate with high peak-to-average-power ratio (PAPR) over wide bandwidth. Linearity requirements force operation in a low efficient highly linear back-off region. Then increasing efficiency is becoming critical. One of the most promising technologies to accomplish this is using supply modulation, e.g. envelope tracking (ET) and envelope elimination and restoration (EER). Supply modulated systems have been studied extensively in the past years, but no systems have been presented with flexibility in the envelope amplifier circuit.   In this work the supply modulator amplifiers have been studied. The focus is on hybrid switching amplifier (HSA) as envelope amplifier. Two envelope amplifier prototypes P-I and P-II have been designed. They are both designed for 15W output but P-II has 28V maximum supply voltage and P-I has 15V maximum supply voltage. P-II developed in version A, using silicon (Si) based switching transistor and version B using gallium-nitride (GaN) switching transistor. The efficiency is limited to a maximum 97 % possible by the circuit components.   The linearity was mainly analyzed by AM-AM diagrams. P-I, P-IIA and P-IIB, were analyzed in simulations and measurements. Results show high possibility of improvement with digital processing, i.e. digital pre-distortion (DPD). Linearization will improve the overall performance in the supply modulator (SM) systems, improving the delay issues and distortion produced by the implementation of the system.   The developed flexible board has made it possible to investigate alternative technologies of ET, focused in the hybrid switching amplifier (HSA). This has given the possibility to compare the overall performance for a traditional Si based switch with the novel Ferdinand Braun Institute’s (FBH) GaN-HEMT based switch with regards to bandwidth, efficiency and non-linearities introduced by the envelope tracking amplifier. P-I and P-II show high efficiency (> 60%) in results. For signals with adequate average power levels the efficiency is high, with around 70% efficiency for WCDMA signals. Phase distortions are evident already at a 5 MHz bandwidth.

This thesis aims to implement a linear wireless transmitter based on the envelope-following architecture. A class-E PA is utilized to replace the linear PA used in the traditional envelope-following transmitter for enhancing the average efficiency. The transmitter relies on a digital processor realized by FPGA to generate the baseband IQ signal and corresponding envelope signal. This way can not only achieve more accurate modulation accuracy and wider modulation bandwidth, but also use less analog components for the future convenience of realizing single-chip integration when compared to the traditional envelope-following transmitter. Furthermore, this thesis implements a predistorter in the digital processor to compensate the Vdd/AM distortion of class-E amplifier. Therefore, this transmitter can simultaneously achieve high efficiency and high linearity over a wide input power range. From the results measured in transmitting a QPSK-modulated CDMA2000 1x signal at a chip rate of 1.2288 Mcps, the transmitter incorporating an InGaAs pHEMT class-E PA can achieve 30~44 % in average efficiency (23~38 % in average PAE) with above 44 dBc in ACPR and below 4 % in EVM in the average modulated output power range from 7 to 21 dBm, while the transmitter incorporating a GaAs HBT can achieve 20~40 % in average efficiency (16~35 % in average PAE) with above 43 dBc in ACPR and below 5 % in EVM in the average modulated output power range from 4 to 18.5 dBm.

Using the envelope theory, the mathematical model of the end face profile and the working tool path of a special optical fiber polishing machine is deve- loped in this study. During the polishing process, the polisher is controlled by three parameters including the fiber rotational angle, the height H and the angle between the fiber and polisher. The contact points between the optical fiber and the polishing plate will determine the profile of the fiber end face. The 3-D end face with double-variable curvatures can be fabricated by properly controlling these three parameters. Since the grinding (polishing) material removal rate is related to machining time and normal contact force, the grinding (polishing) tool path and parameters are needed some modification in order to get the precise end profiles. Example of fiber end faces of 2-D elliptical face and 3-D ellipsoid are given to check the developed mathematical model in this study by computer solid modeling.

In the past decade, there has been great motivation to improve the efficiency of power amplifiers (PAs) in handset transmitter systems in order to address critical issues such as poor battery life and excessive heat. Currently, the focus lies on high data rate applications such as wideband code division multiple access (WCDMA) and long term evolution (LTE) standards due to the stringent efficiency and linearity requirements on the PA. This thesis describes a simulation-based study of techniques for enhancing the efficiency of a CMOS power amplifier for WCDMA and LTE applications. The primary goal is to study the concepts of envelope and average power tracking in simulation and to demonstrate the effectiveness of these supply modulation techniques on a CMOS PA design. The P1dB and IMD performance of a Class A/AB CMOS PA has been optimized to operate with high peak-to-average modulation with WCDMA and LTE signals. Behavioral models of envelope and average power tracking are implemented using proposed algorithms, and a system-level analysis is performed. Envelope tracking is seen to offer a peak PAE improvement of 15% for WCDMA, versus a fixed voltage supply, while average power tracking renders a maximum efficiency gain of 9.8%. Better than -33dBc adjacent channel leakage-power ratio (ACLR) at 5MHz offset and EVM below 4% are observed for both supply tracking techniques. For LTE, envelope and average power tracking contribute to a peak PAE enhancement of 15.3% and 7%, respectively. LTE ACLR begins failing the -30dBc specification above 22.5dBm output power during envelope tracking operation in the PA implementation described here. / text

Human immunodeficiency virus (HIV) infection of macrophages in brain and other tissues plays an important role in development of HIV-associated neurological disorders and other aspects of disease pathogenesis. Macrophages express low levels of CD4, and macrophage-tropic HIV strains express envelope glycoproteins (Envs) adapted to overcome this restriction to virus entry by mechanisms that are not well characterized. One mechanism that influences this phenotype is increased exposure of the CD4 or CCR5 binding site, which may increase dissociation of soluble gp120 (sgp120) from Env trimers based on structural models. Little is known about spontaneous sgp120 shedding from primary HIV Envs or its biological significance. In this dissertation, we identify genetic determinants in brain-derived Envs that overcome the restriction imposed by low CD4, examine spontaneous sgp120 shedding by these Envs, and explore the biological significance of these findings. Sequence analysis of the gp120 beta-3 strand of the CCR5-binding site bridging sheet identified D197, which eliminates an N-linked glycosylation site, as a viral determinant associated with brain infection and HIV-associated dementia (HAD), and position 200 as a positively-selected codon in HAD patients. Mutagenesis studies showed that D197 and T/V200 enhance fusion and infection of macrophages and other cells expressing low CD4 by enhancing gp120 binding to CCR5. Sgp120 shedding from primary brain and lymphoid Envs was highly variable within and between patients, representing a spectrum rather than a categorical phenotype. Brain Envs with high sgp120 shedding mediated enhanced fusion and infection with cells expressing low CD4. Furthermore, viruses expressing brain Envs with high sgp120 shedding had an increased capacity to induce lymphocyte activation during PBMC infection, despite similar levels of viral replication. Genetic analysis demonstrated greater entropy and positive selection in Envs with high versus low levels of sgp120 shedding, suggesting that diversifying evolution influences gp120-gp41 association. Finally, we examined V3 loop sequences from dual-tropic brain and lymphoid Envs and found that the frequency of R5X4 HIV-1 is underestimated by most predictive bioinformatic algorithms. Together, these studies provide a better understanding of how neurotropic HIV Envs adapt to target cells expressing low CD4, and possible roles of these viral adaptations in disease pathogenesis.

Over one-third of the world’s population is at risk for infection with dengue virus (DENV), a mosquito-borne virus that can cause a severe febrile disease. There are no specific treatments available for dengue infection, and much remains unknown about how DENV interaction with the host cell leads to a successful infection. This dissertation examines DENV entry using small molecule inhibitors and mutagenesis of the envelope (E) protein, the major protein on the viral surface. This work grew from our initial observation that small molecule GNF-2 is capable of lowering DENV yield when present at two separate points during DENV infection. Treatment of infected cells with GNF-2 post-entry significantly lowered DENV yield, most likely due to GNF-2’s documented activity against Abl kinase. However, we also observed that treatment of virus inocula with GNF-2 prior to cellular infection significantly lowered DENV yield. We discovered that GNF-2 bound directly to the dengue virion and co-localized with DENV envelope protein shortly after cellular infection. Using GNF-2 as a scaffold, we performed a structure-activity relationship study and identified twenty-one compounds that have similar or increased potency as GNF-2 when pre-incubated with virus. Using a subset of compounds from this study, we demonstrated that they block completion of DENV fusion in vitro, suggesting that the compounds inhibit DENV entry by preventing the completion of viral fusion inside cellular endosomes. In experiments complementing the mechanism of action studies, we selected for inhibitor-resistant virus by passaging virus in the presence of small molecules. We identified a single point mutation in the envelope protein located in the domain I/II interface that enhanced viral entry and conferred resistance to virus particles against select compoundsin a single-cycle reporter virus system. Further examination of this E protein “hinge region” found that mutations in this area may affect both release and entry of reporter virus particles. The work presented in this dissertation may inform the design of future small molecule inhibitors of DENV as well as increase our understanding of how point mutations in the DENV E protein can influence viral entry and other steps of the viral life cycle.