Velocity distributions in a plate heat exchanger channel

The development of a Laser Doppler Anemometer technique to measure the velocity distribution in a commercial plate heat exchanger is described. Detailed velocity profiles are presented and a preliminary investigation is reported on flow behaviour through a single cell in the channel matrix. The objective of the study was to extend previous investigations of plate heat exchanger flow patterns in the laminar range with the eventual aim of establishing the effect of flow patterns on heat transfer performance, thus leading to improved plate heat exchanger design and design methods. Accurate point velocities were obtained by Laser Anemometry in a perspex replica of the metal channel. Oil was used as a circulating liquid with a refractive index matched to that of the perspex so that the laser beams were not distorted. Cell-by-cell velocity measurements over a range of Reynolds number up to ten showed significant liquid mal-distribution. Local cell velocities were found to be as high as twenty seven times average velocity, contrary to the previously held belief of four times. The degree of mal-distribution varied across the channel as well as in the vertical direction, and depended on the upward or downward direction of flow. At Reynolds numbers less than one, flow zig-zagged from one side of the channel to the other in wave form, but increases in Reynolds number improved liquid distribution. A detailed examination of selected cells showed velocity variations in different directions, together with variation within individual cells. Experimental results are also reported on the flow split when passing through a single cell in a section of a channel . These observations were used to explain mal-distribution in the perspex channel itself.

Contextual effects in speed perception may occur at an early stage of processing

How does nearby motion affect the perceived speed of a target region? When a central drifting Gabor patch is surrounded by translating noise, its speed can be misperceived over a fourfold range. Typically, when a surround moves in the same direction, perceived centre speed is reduced; for opposite-direction surrounds it increases. Measuring this illusion for a variety of surround properties reveals that the motion context effects are a saturating function of surround speed (Experiment I) and contrast (Experiment II). Our analyses indicate that the effects are consistent with a subtractive process, rather than with speed being averaged over area. In Experiment III we exploit known properties of the motion system to ask where these surround effects impact. Using 2D plaid stimuli, we find that surround-induced shifts in perceived speed of one plaid component produce substantial shifts in perceived plaid direction. This indicates that surrounds exert their influence early in processing, before pattern motion direction is computed. These findings relate to ongoing investigations of surround suppression for direction discrimination, and are consistent with single-cell findings of direction-tuned suppressive and facilitatory interactions in primary visual cortex (V1).

Analysis and simulation of computer controlled cellular mobile radio systems

Cellular mobile radio systems will be of increasing importance in the future. This thesis describes research work concerned with the teletraffic capacity and the canputer control requirements of such systems. The work involves theoretical analysis and experimental investigations using digital computer simulation. New formulas are derived for the congestion in single-cell systems in which there are both land-to-mobile and mobile-to-mobile calls and in which mobile-to-mobile calls go via the base station. Two approaches are used, the first yields modified forms of the familiar Erlang and Engset formulas, while the second gives more complicated but more accurate formulas. The results of computer simulations to establish the accuracy of the formulas are described. New teletraffic formulas are also derived for the congestion in multi -cell systems. Fixed, dynamic and hybrid channel assignments are considered. The formulas agree with previously published simulation results. Simulation programs are described for the evaluation of the speech traffic of mobiles and for the investigation of a possible computer network for the control of the speech traffic. The programs were developed according to the structured progranming approach leading to programs of modular construction. Two simulation methods are used for the speech traffic: the roulette method and the time-true method. The first is economical but has some restriction, while the second is expensive but gives comprehensive answers. The proposed control network operates at three hierarchical levels performing various control functions which include: the setting-up and clearing-down of calls, the hand-over of calls between cells and the address-changing of mobiles travelling between cities. The results demonstrate the feasibility of the control netwvork and indicate that small mini -computers inter-connected via voice grade data channels would be capable of providing satisfactory control

Contextual modulation involves suppression and facilitation from the center and the surround

In psychophysics, cross-orientation suppression (XOS) and cross-orientation facilitation (XOF) have been measured by investigating mask configuration on the detection threshold of a centrally placed patch of sine-wave grating. Much of the evidence for XOS and XOF comes from studies using low and high spatial frequencies, respectively, where the interactions are thought to arise from within (XOS) and outside (XOF) the footprint of the classical receptive field. We address the relation between these processes here by measuring the effects of various sizes of superimposed and annular cross-oriented masks on detection thresholds at two spatial scales (1 and 7 c/deg) and on contrast increment thresholds at 7 c/deg. A functional model of our results indicates the following (1) XOS and XOF both occur for superimposed and annular masks. (2) XOS declines with spatial frequency but XOF does not. (3) The spatial extent of the interactions does not scale with spatial frequency, meaning that surround-effects are seen primarily at high spatial frequencies. (4) There are two distinct processes involved in XOS: direct divisive suppression and modulation of self-suppression. (5) Whether XOS or XOF wins out depends upon their relative weights and mask contrast. These results prompt enquiry into the effect of spatial frequency at the single-cell level and place new constraints on image-processing models of early visual processing. © ARVO.

Immunological and biochemical techniques in the analysis of tear proteins

This study is concerned with the analysis of tear proteins, paying particular attention to the state of the tears (e.g. non-stimulated, reflex, closed), created during sampling, and to assess their interactions with hydrogel contact lenses. The work has involved the use of a variety of biochemical and immunological analytical techniques for the measurement of proteins, (a), in tears, (b), on the contact lens, and (c), in the eluate of extracted lenses. Although a diverse range of tear components may contribute to contact lens spoilation, proteins were of particular interest in this study because of their theoretical potential for producing immunological reactions. Although normal host proteins in their natural state are generally not treated as dangerous or non-self, those which undergo denaturation or suffer a conformational change may provoke an excessive and unnecessary immune response. A novel on-lens cell based assay has been developed and exploited in order to study the role of the ubiquitous cell adhesion glycoprotein, vitronectin, in tears and contact lens wear under various parameters. Vitronectin, whose levels are known to increase in the closed eye environment and shown here to increase during contact lens wear, is an important immunoregulatory protein and may be a prominent marker of inflammatory activity. Immunodiffusion assays were developed and optimised for use in tear analysis, and in a series of subsequent studies used for example in the measurement of albumin, lactoferrin, IgA and IgG. The immunodiffusion assays were then applied in the estimation of the closed eye environment; an environment which has been described as sustaining a state of sub-clinical inflammation. The role and presence of a lesser understood and investigated protein, kininogen, was also estimated, in particular, in relation to contact lens wear. Difficulties arise when attempting to extract proteins from the contact lens in order to examine the individual nature of the proteins involved. These problems were partly alleviated with the use of the on-lens cell assay and a UV spectrophotometry assay, which can analyse the lens surface and bulk respectively, the latter yielding only total protein values. Various lens extraction methods were investigated to remove protein from the lens and the most efficient was employed in the analysis of lens extracts. Counter immunoelectrophoresis, an immunodiffusion assay, was then applied to the analysis of albumin, lactoferrin, IgA and IgG in the resultant eluates.

Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

Disturbing visual working memory:electrophysiological evidence for a role of the prefrontal cortex in recovery from interference

Single cell recordings in monkeys support the notion that the lateral prefrontal cortex (PFC) controls reactivation of visual working memory representations when rehearsal is disrupted. In contrast, recent fMRI findings yielded a double dissociation for PFC and the medial temporal lobe (MTL) in a letter working memory task. PFC was engaged in interference protection during reactivation while MTL was prominently involved in the retrieval of the letter representations. We present event-related potential data (ERP) that support PFC involvement in the top-down control of reactivation during a visual working memory task with endogenously triggered recovery after visual interference. A differentiating view is proposed for the role of PFC in working memory with respect to endogenous/exogenous control and to stimulus type. General implications for binding and retention mechanisms are discussed.

Characterization of microvesicles released from human red blood cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

A micro-opto-electro-mechanical system (MOEMS) for microstructure manipulation

Microstructure manipulation is a fundamental process to the study of biology and medicine, as well as to advance micro- and nano-system applications. Manipulation of microstructures has been achieved through various microgripper devices developed recently, which lead to advances in micromachine assembly, and single cell manipulation, among others. Only two kinds of integrated feedback have been demonstrated so far, force sensing and optical binary feedback. As a result, the physical, mechanical, optical, and chemical information about the microstructure under study must be extracted from macroscopic instrumentation, such as confocal fluorescence microscopy and Raman spectroscopy. In this research work, novel Micro-Opto-Electro-Mechanical-System (MOEMS) microgrippers are presented. These devices utilize flexible optical waveguides as gripping arms, which provide the physical means for grasping a microobject, while simultaneously enabling light to be delivered and collected. This unique capability allows extensive optical characterization of the structure being held such as transmission, reflection, or fluorescence. The microgrippers require external actuation which was accomplished by two methods: initially with a micrometer screw, and later with a piezoelectric actuator. Thanks to a novel actuation mechanism, the "fishbone", the gripping facets remain parallel within 1 degree. The design, simulation, fabrication, and characterization are systematically presented. The devices mechanical operation was verified by means of 3D finite element analysis simulations. Also, the optical performance and losses were simulated by the 3D-to-2D effective index (finite difference time domain FDTD) method as well as 3D Beam Propagation Method (3D-BPM). The microgrippers were designed to manipulate structures from submicron dimensions up to approximately 100 μm. The devices were implemented in SU-8 due to its suitable optical and mechanical properties. This work demonstrates two practical applications: the manipulation of single SKOV-3 human ovarian carcinoma cells, and the detection and identification of microparts tagged with a fluorescent "barcode" implemented with quantum dots. The novel devices presented open up new possibilities in the field of micromanipulation at the microscale, scalable to the nano-domain.

Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts

Background Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). Conclusions While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

Analisi della propagazione elettrica nel tessuto cardiaco mediante simulazione numerica implementata su GPU

Il segnale elettrico si propaga nel tessuto cardiaco attraverso gap-junctions che si trovano tra i miociti cardiaci e in ciascuno di essi si avvia un processo chiamato potenziale d'azione (PA). In questa tesi prenderò in considerazione il modello Luo-Rudy 1991 e il difetto oggetto di studio sono le Early Afterdepolarizations (EADs). Si analizzerà la propagazione del potenziale d’azione in un cavo di 300 cellule. Dopo alcune simulazioni preliminari è emersa l’utilità di trovare una soluzione che permettesse di ridurre i tempi di calcolo, il modello è stato quindi implementato in CUDA. Il lavoro è stato sviluppato nei seguenti step: 1) l’impiego dell’ambiente di calcolo MATLAB per implementare il modello, descrivendo ogni cellula attraverso il modello Luo-Rudy 1991 e l’interazione elettrica inter-cellulare, considerando un cavo di 300 cellule; 2) individuazione dei parametri che, adeguatamente modificati, sono in grado di indurre EADs a livello single cell; 3) implementazione del modello in CUDA, creando uno strumento che potrà essere utilizzato per aumentare notevolmente il numero delle simulazioni nell’unità di tempo; 4) messa a punto di un criterio per valutare in modo conciso la bontà (safety factor) della relazione source-sink. L’utilità di un simile criterio è quella di valutare, sia nel caso di propagazione di AP che in quello di eventuale propagazione di EADs, la propensione alla propagazione in un tessuto. Il primo capitolo descriverà il potenziale d’azione, il modello usato e la teoria del cavo. Il secondo capitolo discuterà l’implementazione del modello usato, descriverà CUDA e come il modello sia stato implementato. Il terzo capitolo riguarderà i primi risultati ottenuti dalle simulazioni e come la variazione dei parametri influisce sulla forma delle EADs. L’ultimo capitolo approfondirà i requisiti necessari per far avvenire una propagazione in un cavo.

Transcriptome and Functional Analysis of Carotid Body Glomus Cells

The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited.

To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response.

Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.

Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

Opening Basement Membrane Gaps During C. elegans Uterine-Vulval Attachment

The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.