Chemical inducers of dimerization (CID) represent an important tool that has been implemented in numerous biological applications namely protein functions, protein stability, signal transduction, gene transcription, etc. Most generally CIDs are defined as bivalent molecules capable of inducing proximity between two targeted proteins. This proximity can in turn promote or disfavor a certain biological activity. Cell permeable small molecules in particular represent a very effective method to induce precise temporal and spatial control over a specific biological target. Our lab has devoted much effort in studying and elucidating the activity and functions of protein kinases, which represent a very attractive therapeutic target for the treatment of cancer and many other disorders. Towards this goal we have developed a general CID enabled three-hybrid split-luciferase methodology for the investigation of kinase-inhibitor interactions in vitro. We demonstrate that by modulating the kinase-ligand affinity of the CID we are able to successfully profile many structurally non-related protein kinases. We also investigate the use of weaker affinity kinase ligands to allow competitive displacement of CID by the selected inhibitor. In addition we report the design, synthesis and applications of novel CID's for the profiling of kinase inhibitors in mammalian cells and we demonstrate the feasibility of the assay to be used as a new platform for the discovery of cell permeable kinase inhibitors. Finally, we report a new ligand-gated split-kinase that can be selectively activated by photocleavable inducers of dimerization. We further prove how the activity of split-proteins can be deactivated with temporal control with use of non DNA damaging UV radiation.

Persistent hypertension is associated with a greater incidence of organ damage and conditions such as stroke, heart failure and endstage kidney disease, which results in increased cardiovascular (CV) morbidity and mortality. Among the patients receiving antihypertensive treatment, the level of adequate blood pressure (BP) control (<140/90 mm Hg) is only 30%-50%. These antihypertensive drugs reduce the risk of CV events only by 20% and stroke by 40%, besides causing adverse effects. The spontaneously hypertensive stroke-prone rat (SHRsp) is a good model of essential hypertension. It starts developing hypertension at 5-6 weeks of age which becomes established hypertension at 12-16 weeks. The SHRsp also develops oxidative stress (characterized by low glutathione levels) and inflammation. We have previously shown that consumption of broccoli sprouts (BSp) decreased oxidative stress, inflammation and blood pressure in both male and female SHRsp. BSp contain sulforaphane glucosinolate (SGS), the precursor of the phase 2 protein inducer sulforaphane. BSp low in SGS did not have these effects. Furthermore, BSp did not have any measurable effects on the normal physiology of Sprague Dawley (SD) rats. The objectives of this study were to determine: (1) the importance of a food matrix in causing these beneficial effects, i.e., can one get the same effects by administration of sulforaphane alone?, (2) whether dietary sulforaphane decreases the level of nitrosylated proteins in the kidneys of SHRsp, (3) whether dietary sulforaphane increases the phase 2 enzyme ã-glutamyl-L-cysteine ligase expression (ã-GCS ), (4) whether dietary sulforaphane increases the phase 2 enzyme glutathione reductase (GRed) expression, (5) whether dietary sulforaphane increases the phase 2 enzyme thioredoxin reductase (TrxR1) expression and (6) whether dietary sulforaphane protects renal artery structure. After 1 week of adaptation, the 4 week old female SHRsp and SD rats were divided into four groups and administered daily by gavage: (i) Corn oil (vehicle) alone (Control); (ii) sulforaphane (5 µmol/kg body weight) in corn oil; (iii) sulforaphane (10 µmol/kg body weight) in corn oil; and (iv) sulforaphane (20 µmol/kg body weight) in corn oil. Systolic BP was determined weekly using a standard tail cuff noninvasive BP measurement system (model 29-SSP; Harvard Apparatus, St. Laurent, QC, Canada). The treatment lasted for 15 weeks. At the end of the treatment period, the animals were anesthetized with isoflurane (3%) and the BP was measured by the intra-arterial catheter method using a BP monitor (MK-2000 instrument; Muromachi Kikai Co., Ltd, Tokyo, Japan). Later, the animals were euthanized and perfused with normal saline, and tissues collected for histology, western blot, gene expression study or measurement of reduced glutathione (GSH). The results of the study showed that chronic administration of sulforaphane in SHRsp significantly increased phase 2 proteins (i.e., significantly increased kidney ã-GCS [0.93. ± 0.07 arbitrary unit (AU)] when compared with SHRsp control [0.36 ± 0.05(AU)] , decreased kidney nitrotyrosine (significantly lowered the levels of nitrotyrosine [0.917± 0.16 AU ] when compared with SHRsp control [1.37± 0.2 AU], protected the arterial structure of small resistance vessels in kidneys, and significantly attenuated the increase in blood pressure by 22-43 mm Hg by the end of the study. In conclusion, the results of this thesis demonstrate that: (i) A minimal change in our diet may have a major impact on our health, (ii) The beneficial health effects previously seen with consumption of BSp are due to the conversion of SGS to sulforaphane and (iii) Long term administration of sulforaphane in SHRsp attenuates the increase in BP and vascular alterations

Persistent hypertension is associated with a greater incidence of organ damage and conditions such as stroke, heart failure and endstage kidney disease, which results in increased cardiovascular (CV) morbidity and mortality. Among the patients receiving antihypertensive treatment, the level of adequate blood pressure (BP) control (<140/90 mm Hg) is only 30%-50%. These antihypertensive drugs reduce the risk of CV events only by 20% and stroke by 40%, besides causing adverse effects. The spontaneously hypertensive stroke-prone rat (SHRsp) is a good model of essential hypertension. It starts developing hypertension at 5-6 weeks of age which becomes established hypertension at 12-16 weeks. The SHRsp also develops oxidative stress (characterized by low glutathione levels) and inflammation. We have previously shown that consumption of broccoli sprouts (BSp) decreased oxidative stress, inflammation and blood pressure in both male and female SHRsp. BSp contain sulforaphane glucosinolate (SGS), the precursor of the phase 2 protein inducer sulforaphane. BSp low in SGS did not have these effects. Furthermore, BSp did not have any measurable effects on the normal physiology of Sprague Dawley (SD) rats. The objectives of this study were to determine: (1) the importance of a food matrix in causing these beneficial effects, i.e., can one get the same effects by administration of sulforaphane alone?, (2) whether dietary sulforaphane decreases the level of nitrosylated proteins in the kidneys of SHRsp, (3) whether dietary sulforaphane increases the phase 2 enzyme ã-glutamyl-L-cysteine ligase expression (ã-GCS ), (4) whether dietary sulforaphane increases the phase 2 enzyme glutathione reductase (GRed) expression, (5) whether dietary sulforaphane increases the phase 2 enzyme thioredoxin reductase (TrxR1) expression and (6) whether dietary sulforaphane protects renal artery structure. After 1 week of adaptation, the 4 week old female SHRsp and SD rats were divided into four groups and administered daily by gavage: (i) Corn oil (vehicle) alone (Control); (ii) sulforaphane (5 µmol/kg body weight) in corn oil; (iii) sulforaphane (10 µmol/kg body weight) in corn oil; and (iv) sulforaphane (20 µmol/kg body weight) in corn oil. Systolic BP was determined weekly using a standard tail cuff noninvasive BP measurement system (model 29-SSP; Harvard Apparatus, St. Laurent, QC, Canada). The treatment lasted for 15 weeks. At the end of the treatment period, the animals were anesthetized with isoflurane (3%) and the BP was measured by the intra-arterial catheter method using a BP monitor (MK-2000 instrument; Muromachi Kikai Co., Ltd, Tokyo, Japan). Later, the animals were euthanized and perfused with normal saline, and tissues collected for histology, western blot, gene expression study or measurement of reduced glutathione (GSH). The results of the study showed that chronic administration of sulforaphane in SHRsp significantly increased phase 2 proteins (i.e., significantly increased kidney ã-GCS [0.93. ± 0.07 arbitrary unit (AU)] when compared with SHRsp control [0.36 ± 0.05(AU)] , decreased kidney nitrotyrosine (significantly lowered the levels of nitrotyrosine [0.917± 0.16 AU ] when compared with SHRsp control [1.37± 0.2 AU], protected the arterial structure of small resistance vessels in kidneys, and significantly attenuated the increase in blood pressure by 22-43 mm Hg by the end of the study. In conclusion, the results of this thesis demonstrate that: (i) A minimal change in our diet may have a major impact on our health, (ii) The beneficial health effects previously seen with consumption of BSp are due to the conversion of SGS to sulforaphane and (iii) Long term administration of sulforaphane in SHRsp attenuates the increase in BP and vascular alterations

Historically inducer pumps have been designed with low inlet diffusion that allows for a gradual pressure rise through the machine that has the ability to slowly collapse any cavitation bubbles that may be present. A novel cavitation control device has been developed by researchers at ConceptsNREC that has been shown in previous experimental work to greatly improve the suction performance of a traditionally designed machine. Computational fluid dynamics (CFD) has been employed to understand the effectiveness of the cavitation control device (CCD) at controlling the conditions that lead to cavitation inception and to determine the impact that the CCD has on the flow. Also the upper limit of design incidence ratio where the CCD is no longer able to control the factors that lead to cavitation inception was to be determined through the CFD approach. All machine geometries and test data were provided by researchers at ConceptsNREC. Two cases were selected for validation work and 32 additional designs were employed in a parametric study where the flow coefficient and design incidence ratio were varied over a typical range of interest for a turbopump application. The results of this computational work show that the CCD is able to control the factors that lead to early cavitation inception. The research shows that the addition of the CCD has an overall stabilizing affect on the flow by significantly decreasing the incidence at the leading edge of the blade. It has been determined that the maximum design incidence ratio where the CCD is able to effectively control the factors that lead to cavitation inception is dependent on the flow coefficient and in general the maximum design incidence ratio decreases as the flow coefficient is increased.

The performance of an inducer with the integration of an inlet cover bleed system known as a stability control device (SCD) is investigated using computational fluid dynamics. Inducers are the first stage of high suction performance pumps and are designed to operate under cavitating conditions. Improvements in design have allowed inducers to operate stably with low inlet head conditions, however, cavitation instabilities ultimately lead to pump failure. It has been shown that inducers that employ an SCD fully suppress cavitation instabilities.The performance of an inducer is explored at both on- and off-design flow coefficients, where the flow coefficient is a normalized flow rate through the inducer. Both the cavitating and non-cavitating performance of the inducer are analyzed. Improved stability is observed when the SCD is implemented, particularly at flow coefficients below the design value. The stabilizing effect of the SCD allows the inducer to operate stably at much lower flow coefficients, which allows for significant improvements in the pumps ability to operate with minimal inlet head. Cavitation instabilities, such as rotating cavitation, are also suppressed when the SCD is implemented.As part of this work, the design space created by the SCD is explored. Variations in the SCD geometry as well as the inlet blade angle of the inducer are explored. High suction performance pumps are required to operate at very low flow coefficients in order to have the best suction performance. Traditionally, only inducers with small inlet blade angles can maintain stable operation at very low flow coefficient. Because of the stabilizing effect of an SCD, inducers with larger inlet blade angles can now operate stably at the low flow rates require for high suction performance pumps. The influence of varying the inlet blade angle is explored in inducers that employ an SCD. This provides a better understanding of the flow physics in inducers that employ an SCD and help to define their design criteria. Stable operation at low flow coefficients is achieved with the larger inlet blade angles, confirming that inducers with larger inlet blade angles that employ an SCD can be used in high suction performance pumps. Modifications to the SCD geometry are considered to better optimize the design. Variations in the SCD geometry have almost no effect on the cavitation breakdown curve for each inducer, however, the stability of the pumps is greatly influenced by the SCD geometry. Some cavitation instabilities are observed in inducers that operate with an SCD. The physics that leads to the generation of these instabilities is unique to an inducer with an SCD. Modifications to the SCD geometry can allow inducers that employ an SCD to suppress traditional cavitation instabilities that occur without an SCD as well as the new instabilities that are observed when an SCD is implemented.

When injected with live P815 tumour cells, DBA/2 mice developed cytotoxic cells reactive to the tumour. In addition, T helper cells from tumour-bearing mice enhanced the iri vitro generation of cytotoxic cells from normal DBA/2 spleen cells. The helper cells had the following properties (1) expression of the Thy-1.2 antigen; (2) resistance to y-radiation; (3) specific enhancement of the cytotoxic response to P815; (4) detectability in P815-bearing mice at the peak of cytotoxic cell activity; (5) activity in the early phase of cytotoxic cell activation. In parallel to the development of helper cell activity, suppressor cells were generated which suppressed the cytotoxic response to P815. These suppressor cells were removed by pre-treating mice with low doses of cyclophosphamide. High doses of cyclophosphamide reduced the cytotoxic response to both P815 and C57B1/6 alloantigens. Cyclophosphamide treatment reduced the frequency of cytotoxic precursor cells directed against P815, and an antigen-reactive helper cell involved in interleukin 2 production. Both interleukin 2 and thymocytes from P815-primed mice, restored the cytotoxic response against P815, to normal levels. Twenty six percent of animals primed with tumour cells cleared a challenge dose of P815 faster than unprimed control mice. Of these, 88% survived longer than the control animals. Eighteen percent of the recipients of cells from tumour-primed mice, cleared a challenge dose of P815 faster than mice injected with normal cells. Of these 53% survived significantly longer than control groups given either normal cells or no cells at all. Cells from mice primed to PPD showed significantly enhanced proliferative responses to soluble and P815-bound PPD, when compared with unprimed animals. However, cells from only a few PPD-primed mice showed enhanced cytotoxicity against P815 tumour cells, and PPD-primed cells either did not alter, or suppressed, the cytotoxic response of normal DBA/2 spleen cells, when stimulated with PPD-coated P815. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

<p>The primary objective of this research was to shed light on the changes in performance observed in a high-speed, centrifugal compressor that occur during the transition from subsonic to transonic operating conditions, using experimental data collected on a research compressor developed by Honeywell Aerospace, as well as results from a numerical model of the compressor.</p> <p> An understanding of the flow behavior in transonic centrifugal compressors is critical as the drive for higher stage pressure ratios while maintaining a compact size results in higher rotational speeds and increased aspect ratios in the inducer of the impeller. Both of these design trends result in higher relative Mach numbers near the impeller leading edge, resulting in the formation of shocks and an increasingly complex flow field. Since it is necessary to maintain high efficiency and adequate surge margin at these conditions—to ensure the compressor is stable across the full operating range—it is important to understand the effects of the transition from subsonic to supersonic flow on performance and stability. Due to the limited availability of research in the open literature regarding transonic centrifugal impellers, especially experimental studies, these behaviors are still not fully understood.</p> <p>Experimental data collected during steady state operation as well as during speed transients, showed a sudden decrease in the variance of the unsteady pressure field throughout the compressor, but most dramatically in the inducer shroud. Analysis of the performance also showed a significant increase in impeller efficiency of approximately 2 points as speed was increased from 80% to 90% of the design speed. Temperature measurements upstream of the impeller leading edge indicated a dramatic reduction in the degree of flow recirculation in the same speed range, indicating the increase in performance is related to a decrease in the blockage near the impeller leading edge. A low pressure region was also observed in the inducer passage, which disappeared upon transition to the transonic operating regime, this coupled with decreased inducer static pressure rise and relative diffusion at lower speeds, strongly indicates that increased loss in the inducer at lower speeds is responsible for the observed performance deficiency during subsonic operation.</p> <p>Analysis of the numerical results revealed that the low pressure region in the inducer may be attributable to the interaction between the inlet shroud boundary layer and the low momentum tip leakage flow in the impeller passage, which at lower speeds, results in the tip leakage flow forming a large recirculation region in the inducer passage. It was also determined that the step change in instability coincides with the inducer shock extending to the shroud and reducing the strength of the interaction between the low momentum regions in the inlet and impeller passage, thereby allowing the tip leakage flow to form into a vortex and preventing the development of the recirculation region in the inducer. </p> <p>This research provides a possible explanation for the observed instability in the compressor, which may allow for further testing of techniques to mitigate the instability caused by the blockage in the inducer, such as casing treatment, bleed, or flow injection into the inducer shroud.</p>

Three-dimensional two-phase unsteady CFD simulations were run on three and four-blade inducers for the purpose of analyzing differences in cavitation stability at design and off-design flow rates. At design flow rates, there were very small differences between the breakdown curves for the three and four-bladed inducers. However, at lower cavitation numbers, the three-bladed inducer exhibited up to three times the rotor forces than the four-bladed inducer. When moving to off-design flow rates, both inducers experienced multiple modes of cavitation instabilities including rotating cavitation, alternate-blade cavitation, and cavitation surge. The four-bladed inducer began experiencing the formation of these modes of instability beginning at a cavitation number of $sigma = 0.047$ whereas the three-bladed inducer began at a cavitation number of $sigma = 0.091$. Additionally, the three-bladed inducer showed rotor forces up to ten times higher than the four-bladed inducer at similar cavitation numbers.Three-dimensional single-phase steady CFD simulations were run on four-blade inducer geometries with $7^{circ}$, $9^{circ}$, $11^{circ}$ and $14^{circ}$ inlet tip blade angles with a stability control device (SCD) installed. The simulations were ran at multiple flow coefficients. Results show interesting flow effects from the SCD. For example, at lower flow coefficients, the incidence angle actually decreases at greater than 70\% span. This is due to a region of accelerated axial flow coming from the recirculation of the SCD which occurs near the shroud. Results also show strong correlations between efficiency and head rise to the local mass flow gain experienced due to the recirculating flow through the SCD. A best fit curve was generated to predict mass flow gain based on the inducer's inlet tip blade angle and flow coefficient. Based on this research, the ability to predict mass flow gain and consequently efficiency and head rise for similarly designed inducers with varying inlet blade angles has been demonstrated.

Osteoporosis is defined as an osteometabolic disease characterized by the reduction of bone mineral density (BMD) and micro-architectural deterioration, leading to enhanced bone fragility and increased susceptibility to fractures. Chronic inflammation affecting bone resorption and/or bone formation is an important mechanism in the hystopathology of osteoporosis. In this regard, several inflammatory markers are associated in special, tumor necrosis factor alpha (TNFα). Recent evidence from the literature pointed out that other members of the TNF superfamily, the tumor necrosis factor TNF-like weak inducer of apoptosis (TWEAK), in its soluble bioactive form (sTWEAK), has been related with diverse endocrine metabolic disarrangements such as diabetes mellitus or metabolic syndrome. Thus, we hypothesized a possible association between sTWEAK plasma levels and bone fractures and diabetes mellitus in post menopausal women. This was a cross sectional study nested in a cohort of 1057 post menopausal women outpatients attending the UBS, Unidades Básicas de Saúde, of Santa Maria. We used data from 52 individuals recruited who reported major bone fractures. For the control group, we aleatory recruited 110 individuals without the history of major bone fractures. All subjects in this study proceeded to a standard questionnaire about clinical data and were submitted to anthropometry evaluation. Blood samples were obtained for the biochemical profile consisting in fast glucose, total cholesterol, TG, HDL, LDL, creatinine, albumin, calcium and phosphorus in serum. sTWEAK concentration was established by ELISA. Assimetric variables were logN transformed. Student T test or Mann-Whitney test were used to comparison of data between two groups. For statistical analysis that form more than two groups were employed ANOVA or Chi quadrate and Fisher s test. An statistical significant reduction (p=0.002) of sTWEAK (log transformed) was observed in diabetes mellitus (DM) subjects in comparison to non-diabetics. There was no differences in the plasma levels of sTWEAK when the history of major bone fractures was analysed. Even when stratified for DM and fractures, Ln sTWEAK remained consistently reduced in the group of individuals with DM without fractures against non-diabetics no-fractures women (p <0.01). Findings from this study indicated that plasma levels of sTWEAK was not related with bone fragility in post menopausal women population. Nevertheless, low plasma levels of this biomarker were associated with diabetes mellitus, as previously described in the literature. / A osteoporose é um transtorno osteometabólico caracterizado pela diminuição da densidade mineral óssea (DMO) e deterioração de sua microarquitetura com o consequente aumento da fragilidade do osso e suscetibilidade à fraturas. A fisiopatologia dessa doença tem a inflamação crônica como importante mecanismo no aumento da reabsorção e redução da formação óssea. Entre os marcadores inflamatórios associados à osteoporose destacam-se aqueles do grupo dos fatores de necrose tumoral, em particular, o fator de necrose tumoral alfa (TNFα). Evidências recentes da literatura observaram que um outro membro da mesma família, o TNFlike weak inducer of apoptosis (TWEAK), em sua forma solúvel (sTWEAK), foi relacionado com a presença de outros distúrbios endócrino metabólicos, tais como diabetes mellitus (DM) e síndrome metabólica. Com base nisso, testamos a hipótese de associação entre sTWEAK e fraturas ósseas maiores, e também DM, em mulheres na pós menopausa. Este é um estudo transversal que partiu de uma coorte de 1057 pacientes na pós menopausa que consultam nas Unidades Básicas de Saúde da cidade de Santa Maria-RS. Foram recrutadas 52 pacientes com histórico de fraturas ósseas maiores e, para o grupo controle, escolhidas aleatoriamente 110 pacientes sem história de fraturas ósseas. Todos os indivíduos do estudo responderam a um questionário padronizado sobre a sua história clínica além de serem submetidos a avaliação antropométrica e exames bioquímicos (glicose de jejum, colesterol total, triglicerídeos, HDL, LDL, creatinina, albumina, cálcio e fósforo). Os níveis plasmáticos de sTWEAK foram avaliados pelo método de ELISA. As variáveis com distribuição assimétrica foram submetidas à transformação para o seu logaritmo natural e então comparadas. Para a análise estatística entre dois grupos foi feita a utilização dos testes T de Student ou Mann-Whitney. Para mais de dois grupos foram empregados ANOVA ou os testes de Chi Quadrado ou Fisher. Foi observada redução significativa (p=0.002) dos valores do sTWEAK (transformado para logN) nos indivíduos com DM em comparação com os sem DM. Não houveram diferenças quando analisado somente o histórico de fraturas. Estratificando a população em subgrupos de acordo com o critério de existência de fratura reportada ou de diagnóstico de DM, permaneceu somente a diferença entre os grupos com DM sem fratura e sem DM sem fratura (p<0.01). Os achados indicam que na população de mulheres na pós menopausa com e sem história de fraturas ósseas maiores, os níveis plasmáticos de sTWEAK não tem associação com a fragilidade óssea. Entretanto, níveis baixos deste biomarcador estão associados com o diagnóstico de DM, como reportado na literatura.

The function of most proteins is regulated by post-translational modifications, of which phosphorylation in particular has been shown to be ubiquitous and of paramount importance to cell signaling. Two enzyme families, protein kinases and phosphatases, regulate phosphorylation, and aberrant activities of family members have been implicated in many diseases such as cancer and neurological disorders. Thus, understanding the function of these enzymes in living cells is important for understanding their biology and for designing new therapies, but a challenging task due to their highly conserved architecture. The major focus of the dissertation is on the development of a new approach to selectively turn-on multiple specific kinases and/or phosphatases using orthogonal ligands as chemical inducers of dimerization (CIDs). Specific kinases or phosphatases were dissected at particular sites into two inactive fragments or split-proteins. The split fragments are attached to interacting protein pairs of CID systems, such that upon addition of the specific ligand they heterodimerize with subsequent reassembly of the split-protein and concomitant activity. We demonstrated the in vitro and in cellulo feasibility of this approach using three orthogonal CIDs, rapamycin, abscisic acid, and gibberellic acid, to turn-on members of the tyrosine kinase group such as Lyn and Src, and of the tyrosine phosphatase group such as PTP1B and SHP1. We have also developed a new synthetic photocleavable di-trimethoprim CID that allows for ligand-gated turn-on of desired kinases in live cells. The new CID can be cleaved or turned-off by UV irradiation which results in a turn-off of kinase activity. Small molecule controlled split-proteins allow for developing logic gates and we demonstrate that the systems we have developed can be used to construct 7 out of the 10 basic, circuit-type Boolean phosphorylation-based logic gates in living cells. These post-translational logic gates may have interesting applications in synthetic biology. Finally, we present an initial approach to use redesigned kinases and redesigned ligands as potential scaffolds for developing new CIDs. Thus, we provide and extend new methodologies that potentially allow for posttranslational control over the activity of user defined split-kinases and split-phosphatases for interrogating and redesigning signaling pathways. The last section of this work focuses on understanding small-molecule selectivity toward protein kinases. We systematically analyzed different reported kinase screens to further understand the reliability of large scale data in the kinome field as the design of selective inhibitors is one the most useful approaches for understanding the function of enzymes or the development of drugs in a natural setting such as a primary cell or an organism.