Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine

Grapevine is an extremely important crop worldwide.In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress(WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular net works involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, across-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors.

Trastornos musculoesqueléticos en trabajadores de la industria automotriz: revisión de la literatura 2000 a 2016.

Los trastornos musculoesqueléticos (TME) tienen alta relación con la industria automotriz afectando a los trabajadores en quienes se puede encontrar varias patologías como Síndrome Del Túnel Del Carpo, Epicondilitis, Síndrome del Manguito Rotador, discopatía lumbar y lumbalgias, entre otros. Entre los factores de riesgo asociados a estos trastornos están los movimientos repetitivos, posturas inadecuadas, vibración, uso manual de herramientas, tareas físicas demandantes y el mal levantamiento de pesos. Todas estas patologías son causa de ausentismo laboral en todo el mundo, lo que conlleva a un aumento en el costo económico por incapacidades, ayudas diagnósticas y tratamientos. Se realizó una revisión de la literatura científica de artículos publicados del año 2000 a 2016 con relación a los trastornos musculoesqueléticos en la industria automotriz en las bases de datos de Pubmed, Ebsco Host, ScienceDirect y Embase. La evidencia encontrada sugiere que la patología lumbar es la que presenta mayor prevalencia en la industria, con 65% en la población Europea, 42% en Asia, Norteamérica en un 20% y en América Latina en un 46%. A pesar que en la industria automotriz predominan como fuerza laboral los hombres, se reportó que las mujeres eran las que tenían mayores factores de riesgo para desarrollar un TME y dentro de estos las posturas inadecuadas, movimientos repetitivos, sobrecarga laboral y levantamiento de pesos, sumado al tiempo de exposición que fue un común denominador en cada uno de los estudios analizados. Las conclusiones fueron que la prevalencia de TME en esta industria es elevada y esto amerita la implementación de programas de prevención más enfocados en este tema. Además no se encontró en la literatura la existencia de un método eficiente para análisis postural y de sobrecarga física, lo que habla de una necesidad urgente de realizar más investigaciones enfocadas en este tipo de población.

Prevention of postnatal distress or depression : an evaluation of an intervention at preparation for parenthood classes.

Determined the effectiveness of a psychosocial intervention, provided to expectant couples in routine antenatal classes, on the postpartum psychosocial adjustment of women and men. Preparation for Parenthood programs were randomly allocated to one of three conditions: usual service ('control'), experimental ('empathy'), or non-specific control ('baby-play'). The latter condition controlled for the non-specific effects of the intervention, these being: the provision of an extra class; asking couples to consider the early postpartum weeks; and receiving booster information after the antenatal class, and again shortly after the birth. Women and men were categorised into three levels of self-esteem, as measured antenatally: low, medium and high. 268 participants were recruited antenatally. Interview data and self-report information was collected from 202 of these women at 6 weeks postpartum, and 180 women at 6 months postpartum. The intervention consisted of a session focusing on psychosocial issues related to becoming first-time parents. Participants discussed possible postpartum concerns in separate gender groups for part of the session, and then discussed these issues with their partners

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

Structural behaviour and design of cold-formed steel beams at elevated temperatures

Cold-formed steel members are extensively used in the building construction industry, especially in residential, commercial and industrial buildings. In recent times, fire safety has become important in structural design due to increased fire damage to properties and loss of lives. However, past research into the fire performance of cold-formed steel members has been limited, and was confined to compression members. Therefore a research project was undertaken to investigate the structural behaviour of compact cold-formed steel lipped channel beams subject to inelastic local buckling and yielding, and lateral-torsional buckling effects under simulated fire conditions and associated section and member moment capacities. In the first phase of this research, an experimental study based on tensile coupon tests was undertaken to obtain the mechanical properties of elastic modulus and yield strength and the stress-strain relationship of cold-formed steels at uniform ambient and elevated temperatures up to 700oC. The mechanical properties deteriorated with increasing temperature and are likely to reduce the strength of cold-formed beams under fire conditions. Predictive equations were developed for yield strength and elastic modulus reduction factors while a modification was proposed for the stressstrain model at elevated temperatures. These results were used in the numerical modelling phases investigating the section and member moment capacities. The second phase of this research involved the development and validation of two finite element models to simulate the behaviour of compact cold-formed steel lipped channel beams subject to local buckling and yielding, and lateral-torsional buckling effects. Both models were first validated for elastic buckling. Lateral-torsional buckling tests of compact lipped channel beams were conducted at ambient temperature in order to validate the finite element model in predicting the non-linear ultimate strength behaviour. The results from this experimental study did not agree well with those from the developed experimental finite element model due to some unavoidable problems with testing. However, it highlighted the importance of magnitude and direction of initial geometric imperfection as well as the failure direction, and thus led to further enhancement of the finite element model. The finite element model for lateral-torsional buckling was then validated using the available experimental and numerical ultimate moment capacity results from past research. The third phase based on the validated finite element models included detailed parametric studies of section and member moment capacities of compact lipped channel beams at ambient temperature, and provided the basis for similar studies at elevated temperatures. The results showed the existence of inelastic reserve capacity for compact cold-formed steel beams at ambient temperature. However, full plastic capacity was not achieved by the mono-symmetric cold-formed steel beams. Suitable recommendations were made in relation to the accuracy and suitability of current design rules for section moment capacity. Comparison of member capacity results from finite element analyses with current design rules showed that they do not give accurate predictions of lateral-torsional buckling capacities at ambient temperature and hence new design rules were developed. The fourth phase of this research investigated the section and member moment capacities of compact lipped channel beams at uniform elevated temperatures based on detailed parametric studies using the validated finite element models. The results showed the existence of inelastic reserve capacity at elevated temperatures. Suitable recommendations were made in relation to the accuracy and suitability of current design rules for section moment capacity in fire design codes, ambient temperature design codes as well as those proposed by other researchers. The results showed that lateral-torsional buckling capacities are dependent on the ratio of yield strength and elasticity modulus reduction factors and the level of non-linearity in the stress-strain curves at elevated temperatures in addition to the temperature. Current design rules do not include the effects of non-linear stress-strain relationship and therefore their predictions were found to be inaccurate. Therefore a new design rule that uses a nonlinearity factor, which is defined as the ratio of the limit of proportionality to the yield stress at a given temperature, was developed for cold-formed steel beams subject to lateral-torsional buckling at elevated temperatures. This thesis presents the details and results of the experimental and numerical studies conducted in this research including a comparison of results with predictions using available design rules. It also presents the recommendations made regarding the accuracy of current design rules as well as the new developed design rules for coldformed steel beams both at ambient and elevated temperatures.

Development of a novel rolling-circle amplification technique to detect Banana streak virus which also discriminates between integrated and episomal virus sequences

Bananas are hosts to a large number of banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity amongst BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence non-specific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterisation of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal and/or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV) and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV) and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.

Driving performance impairments due to hypovigilance on monotonous roads

Drivers' ability to react to unpredictable events deteriorates when exposed to highly predictable and uneventful driving tasks. Highway design reduces the driving task mainly to a lane-keeping manoeuvre. Such a task is monotonous, providing little stimulation and this contributes to crashes due to inattention. Research has shown that driver's hypovigilance can be assessed with EEG measurements and that driving performance is impaired during prolonged monotonous driving tasks. This paper aims to show that two dimensions of monotony - namely road design and road side variability - decrease vigilance and impair driving performance. This is the first study correlating hypovigilance and driver performance in varied monotonous conditions, particularly on a short time scale (a few seconds). We induced vigilance decrement as assessed with an EEG during a monotonous driving simulator experiment. Road monotony was varied through both road design and road side variability. The driver's decrease in vigilance occurred due to both road design and road scenery monotony and almost independently of the driver's sensation seeking level. Such impairment was also correlated to observable measurements from the driver, the car and the environment. During periods of hypovigilance, the driving performance impairment affected lane positioning, time to lane crossing, blink frequency, heart rate variability and non-specific electrodermal response rates. This work lays the foundation for the development of an in-vehicle device preventing hypovigilance crashes on monotonous roads.

The effect of emotional and attentional load on attentional startle modulation

The interactive effects of emotion and attention on attentional startle modulation were investigated in two experiments. Participants performed a discrimination and counting task with two visual stimuli during which acoustic eyeblink startle-eliciting probes were presented at long lead intervals. In Experiment 1, this task was combined with aversive Pavlovian conditioning. In Group Attend CS+, the attended stimulus was followed by an aversive unconditional stimulus (US) and the ignored stimulus was presented alone whereas the ignored stimulus was paired with the US in Group Attend CS−. In Experiment 2, a non-aversive reaction time task US replaced the aversive US. Regardless of the conditioning manipulation and consistent with a modality non-specific account of attentional startle modulation, startle magnitude was larger during attended than ignored stimuli in both experiments. Blink latency shortening was differentially affected by the conditioning manipulations suggesting additive effects of conditioning and discrimination and counting task on blink startle.

Machinima

The research field was curatorship of the Machinima genre - a film-making practice that uses real time 3D computer graphics engines to create cinematic productions. The context was the presentation of gallery non-specific work for large-scale exhibition, as an investigation in thinking beyond traditional strategies of white cube. Strongly influenced by the Christiane Paul (Ed) seminal text, 'New Media in the White Cube and Beyond, Curatorial Models for Digital Art', the context was the repositioning of a genre traditionally focussed on delivery through small-screen, indoor, personal spaces, to large exhibition hall spaces. Beyond the core questions of collecting, documenting, expanding and rethinking the place of Machinima within the history of contemporary digital arts, the curatorial premise asked how to best invert the relationship between context of media production within the gaming domain, using novel presentational strategies that might best promote the 'take-home' impulse. The exhibition was used not as the ultimate destination for work but rather as a place to experience, sort and choose from a high volume of possible works for subsequent investigation by audiences within their own game-ready, domestic environments. In pursuit of this core aim, the exhibition intentionally promoted 'sensory overload'. The exhibition also included a gaming lab experience where audiences could begin to learn the DIY concepts of the medium, and be stimulated to revisit, consider and re-make their own relationship to this genre. The research was predominantly practice-led and collaborative (in close concert with the Machinima community), and ethnographic in that it sought to work with, understand and promote the medium in a contemporary art context. This benchmark exhibition, building on the 15-year history of the medium, was warmly received by the global Machinima community as evidenced by the significant debate, feedback and general interest recorded. The exhibition has recently begun an ongoing Australian touring schedule. To date, the exhibition has received critical attention nationally and internationally in Das Superpaper, the Courier Mail, Machinimart, 4ZZZ-FM, the Sydney Morning Herald, Games and Business, Australian Gamer, Kotaku Australia, and the Age.

Behaviour and design of cold-formed steel beams subject to lateral-torsional buckling at elevated temperatures

Cold-formed steel beams are increasingly used as floor joists and bearers in buildings and often their behaviour and moment capacities are influenced by lateral-torsional buckling. With increasing usage of cold-formed steel beams their fire safety design has become an important issue. Fire design rules are commonly based on past research on hot-rolled steel beams. Hence a detailed parametric study was undertaken using validated finite element models to investigate the lateral-torsional buckling behaviour of simply supported cold-formed steel lipped channel beams subjected to uniform bending at uniform elevated temperatures. The moment capacity results were compared with the predictions from the available ambient temperature and fire design rules and suitable recommendations were made. European fire design rules were found to be over-conservative while the ambient temperature design rules could not be used based on single buckling curve. Hence a new design method was proposed that includes the important non-linear stress-strain characteristics observed for cold-formed steels at elevated temperatures. Comparison with numerical moment capacities demonstrated the accuracy of the new design method. This paper presents the details of the parametric study, comparisons with current design rules and the new design rules proposed in this research for lateral-torsional buckling of cold-formed steel lipped channel beams at elevated temperatures.

Physical activity amongst people with chronic back pain : an investigation of perceived barriers and facilitators to inform intervention development

Previous research has indicated people with non-specific low back pain who are physically inactive face a poorer prognosis than people with back pain who participate in low or moderate intensity physical activity. They also face a greater risk of other lifestyle related health conditions, such as diabetes and heart disease. For these reasons, contemporary non-surgical interventions for low back pain aim to incorporate a return to physical activity. However, there is a lack of empirical evidence supporting physical activity interventions for this purpose. It is likely that people with low back pain face additional challenges when trying to commence (or return to) regular physical activity. This exploratory qualitative research aimed to map out perceived barriers and facilitators to undertaking physical activity among people with non-specific low back pain to inform future intervention development.

Tipping the balance : sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.

Letter : anxiety score as a risk factor for radial artery vasospasm during radial interventions : a pilot study

I read with interest the article in Angiology that determined the role of anxiety level on radial artery spasm during transradial coronary angiography.1 As the importance of conducting more randomised controlled trials using anxiolytics to define the relation between anxiety and vasospasm was noted by the authors, I offer the following insights for investigators to consider when conducting such research. While previous research has already identified that moderate procedural sedation and opioid analgesia reduces the incidence of vasospasm,2 the identification of risk factors in the present study is hypothesis generating as to how outcomes might be even further improved. It is possible that selectively applying either even more intensive sedation and analgesia or complementary non-pharmacological stress-reducing therapies, such as music therapy or visualisation and attentive behaviour, to patients ‘at-risk’ of vasospasm (women and those with high levels of anxiety prior to the procedure) might lead to even better patient outcomes...

Spectroscopic studies of the adsorption and reactions of chlorofluorocarbons (CFC-11 and CFC-12) and hydrochlorofluorocarbon (HCFC-22) on oxide surfaces

Raman and Fourier transform infrared (FT-IR) spectroscopy have been applied to a systematic investigation of the adsorption and decomposition of dichlorodifluoromethane (CCl2F2, CFC-12), fluorotrichloromethane (CCl3F, CFC-11), chlorodifluoromethane (CHClF2, HCFC-22) and molecular chlorine on oxide surfaces. Additionally, the effects of heating and ultraviolet photolysis of the CFC and HCFCs adsorbed on the oxide surfaces have been investigated. Spectral features for these species indicated a small wavenumber shift (1-6 cm-1) associated with the adsorbed phase. Some evidence, specifically the appearance of the Raman band at 507 cm-1, is presented to show that chlorine decomposition species are associated with these oxide surfaces. It was concluded that the new spectral feature (at ca. 507 cm-1) related with the decomposition of the CFC and HCFC molecules was an important indicator of the extent to which the reaction between the adsorbed CFC and HCFC and oxide surface has taken place. The extent of CFC-surface interaction has been quantified in terms of a maximum (Raman) frequency shift parameter (AM). Wavenumber shifts suggest both cation-adsorbate and non-specific adsorption interactions are occurring in the internal channels of the zeolites. Slow decomposition of the adsorbed CFCs under ultraviolet-visible photolysis (at ? > 300 nm) and/or thermal treatment was observed spectroscopically. Using FT-IR spectroscopy, the formation of gas-phase products (CO, CO2, HCl) both onyn photolysis and heating was evident. Results of these measurements are compared with the observed atmospheric reactivity of these compounds.