Small molecule–biopolymer interactions : Ultraviolet–visible and fluorescence spectroscopy and chemometrics

Interactions between small molecules with biopolymers e.g. the bovine serum albumin (BSA protein), are important, and significant information is recorded in the UV–vis and fluorescence spectra of their reaction mixtures. The extraction of this information is difficult conventionally and principally because there is significant overlapping of the spectra of the three analytes in the mixture. The interaction of berberine chloride (BC) and the BSA protein provides an interesting example of such complex systems. UV–vis and fluorescence spectra of BC and BSA mixtures were investigated in pH 7.4 Tris–HCl buffer at 37 °C. Two sample series were measured by each technique: (1) [BSA] was kept constant and the [BC] was varied and (2) [BC] was kept constant and the [BSA] was varied. This produced four spectral data matrices, which were combined into one expanded spectral matrix. This was processed by the multivariate curve resolution–alternating least squares method (MCR–ALS). The results produced: (1) the extracted pure BC, BSA and the BC–BSA complex spectra from the measured heavily overlapping composite responses, (2) the concentration profiles of BC, BSA and the BC–BSA complex, which are difficult to obtain by conventional means, and (3) estimates of the number of binding sites of BC.

Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2-isopropylphenate with bovine serum albumin

The binding interaction of the pesticide Isoprocarb and its degradation product, sodium 2-isopropylphenate, with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both Isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism. The thermodynamic parameters (ΔH°, ΔS° and ΔG°) obtained from the fluorescence data measured at two different temperatures showed that the binding of Isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either Isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide, i.e., the protein is denatured. The sodium 2-isopropylphenate, was estimated to be about 4–5 times more toxic than its parent, Isoprocarb. Synchronous fluorescence spectroscopy and the resolution of the three-way excitation–emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA, Isoprocab and sodium 2-isopropylphenate as a function of the added sodium 2-isopropylphenate. These profiles showed that the degradation product, sodium 2-isopropylphenate, displaced the pesticide in a competitive reaction with the BSA protein.

Investigation of shaft voltage in wind turbine systems with induction generators

This paper presents the analysis of shaft voltage in different configurations of a doubly fed induction generator (DFIG) and an induction generator (IG) with a back-to-back inverter in wind turbine applications. Detailed high frequency model of the proposed systems have been developed based on existing capacitive couplings in IG & DFIG structures and common mode voltage sources. In this research work, several arrangements of DFIG based wind energy conversion systems (WES) are investigated in case of shaft voltage calculation and its mitigation techniques. Placements of an LC line filter in different locations and its effects on shaft voltage elimination are studied via Mathematical analysis and simulations. A pulse width modulation (PWM) technique and a back-to-back inverter with a bidirectional buck converter have been presented to eliminate the shaft voltage in a DFIG wind turbine.

Calculations of capacitive couplings in induction generators to analyse shaft voltage

This paper deals with the analysis of the parameters which are effective in shaft voltage generation of induction generators. It focuses on different parasitic capacitive couplings by mathematical equations, finite element simulations and experiments. The effects of different design parameters have been studied on proposed capacitances and resultant shaft voltage. Some parameters can change proposed capacitive coupling such as: stator slot tooth, the gap between slot tooth and winding, and the height of the slot tooth, as well as the air gap between the rotor and the stator. This analysis can be used in a primary stage of a generator design to reduce motor shaft voltage and avoid additional costs of resultant bearing current mitigation.

Composite load model decomposition : induction motor contribution

In this thesis, a new technique has been developed for determining the composition of a collection of loads including induction motors. The application would be to provide a representation of the dynamic electrical load of Brisbane so that the ability of the power system to survive a given fault can be predicted. Most of the work on load modelling to date has been on post disturbance analysis, not on continuous on-line models for loads. The post disturbance methods are unsuitable for load modelling where the aim is to determine the control action or a safety margin for a specific disturbance. This thesis is based on on-line load models. Dr. Tania Parveen considers 10 induction motors with different power ratings, inertia and torque damping constants to validate the approach, and their composite models are developed with different percentage contributions for each motor. This thesis also shows how measurements of a composite load respond to normal power system variations and this information can be used to continuously decompose the load continuously and to characterize regarding the load into different sizes and amounts of motor loads.

Decomposition of aggregated load : finding induction motor fraction in real load

The main contribution of this paper is decomposition/separation of the compositie induction motors load from measurement at a system bus. In power system transmission buses load is represented by static and dynamic loads. The induction motor is considered as the main dynamic loads and in the practice for major transmission buses there will be many and various induction motors contributing. Particularly at an industrial bus most of the load is dynamic types. Rather than traing to extract models of many machines this paper seeks to identify three groups of induction motors to represent the dynamic loads. Three groups of induction motors used to characterize the load. These are the small groups (4kw to 11kw), the medium groups (15kw to 180kw) and the large groups (above 630kw). At first these groups with different percentage contribution of each group is composite. After that from the composite models, each motor percentage contribution is decomposed by using the least square algorithms. In power system commercial and the residential buses static loads percentage is higher than the dynamic loads percentage. To apply this theory to other types of buses such as residential and commerical it is good practice to represent the total load as a combination of composite motor loads, constant impedence loads and constant power loads. To validate the theory, the 24hrs of Sydney West data is decomposed according to the three groups of motor models.

Effects of pediatric obesity on joint kinematics and kinetics during 2 walking cadences

Objective: To determine whether differences existed in lower-extremity joint biomechanics during self-selected walking cadence (SW) and fast walking cadence (FW) in overweight- and normal-weight children.---------- Design: Survey.---------- Setting: Institutional gait study center.---------- Participants: Participants (N=20; mean age ± SD, 10.4±1.6y) from referred and volunteer samples were classified based on body mass index percentiles and stratified by age and sex. Exclusion criteria were a history of diabetes, neuromuscular disorder, or recent lower-extremity injury.---------- Main Outcome Measures: Sagittal, frontal, and transverse plane angular displacements (degrees) and peak moments (newton meters) at the hip, knee, and ankle joints.---------- Results: The level of significance was set at P less than .008. Compared with normal-weight children, overweight children had greater absolute peak joint moments at the hip (flexor, extensor, abductor, external rotator), the knee (flexor, extensor, abductor, adductor, internal rotator), and the ankle (plantarflexor, inverter, external/internal rotators). After including body weight as a covariate, overweight children had greater peak ankle dorsiflexor moments than normal-weight children. No kinematic differences existed between groups. Greater peak hip extensor moments and less peak ankle inverter moments occurred during FW than SW. There was greater angular displacement during hip flexion as well as less angular displacement at the hip (extension, abduction), knee (flexion, extension), and ankle (plantarflexion, inversion) during FW than SW.---------- Conclusions: Overweight children experienced increased joint moments, which can have long-term orthopedic implications and suggest a need for more nonweight-bearing activities within exercise prescription. The percent of increase in joint moments from SW to FW was not different for overweight and normal-weight children. These findings can be used in developing an exercise prescription that must involve weight-bearing activity.

A quasi-stationary approach to drying kinetics of different shaped food particulates in a heat pump assisted by fluid bed dryer

Experiments were undertaken to study drying kinetics of different shaped moist food particulates during heat pump assisted fluidised bed drying. Three particular geometrical shapes of parallelepiped, cylindrical and spheres were selected from potatoes (aspect ratio = 1:1, 2:1, 3:1), cut beans (length: diameter = 1:1, 2:1, 3:1) and peas respectively. A batch fluidised bed dryer connected to a heat pump system was used for the experimentation. A Heat pump and fluid bed combination was used to increase overall energy efficiency and achieve higher drying rates. Drying kinetics, were evaluated with non-dimensional moisture at three different drying temperatures of 30, 40 and 50o C. Due to complex hydrodynamics of the fluidised beds, drying kinetics are dryer or material specific. Numerous mathematical models can be used to calculate drying kinetics ranging from analytical models with simplified assumptions to empirical models built by regression using experimental data. Empirical models are commonly used for various food materials due to their simpler approach. However problems in accuracy, limits the applications of empirical models. Some limitations of empirical models could be reduced by using semi-empirical models based on heat and mass transfer of the drying operation. One such method is the quasi-stationary approach. In this study, a modified quasi-stationary approach was used to model drying kinetics of the cylindrical food particles at three drying temperatures.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

The application of neural networks to induction machine control

Neural networks (NNs) are discussed in connection with their possible use in induction machine drives. The mathematical model of the NN as well as a commonly used learning algorithm is presented. Possible applications of NNs to induction machine control are discussed. A simulation of an NN successfully identifying the nonlinear multivariable model of an induction-machine stator transfer function is presented. Previously published applications are discussed, and some possible future applications are proposed.