Melanoma cell colony expansion parameters revealed by approximate Bayesian computation

In vitro studies and mathematical models are now being widely used to study the underlying mechanisms driving the expansion of cell colonies. This can improve our understanding of cancer formation and progression. Although much progress has been made in terms of developing and analysing mathematical models, far less progress has been made in terms of understanding how to estimate model parameters using experimental in vitro image-based data. To address this issue, a new approximate Bayesian computation (ABC) algorithm is proposed to estimate key parameters governing the expansion of melanoma cell (MM127) colonies, including cell diffusivity, D, cell proliferation rate, λ, and cell-to-cell adhesion, q, in two experimental scenarios, namely with and without a chemical treatment to suppress cell proliferation. Even when little prior biological knowledge about the parameters is assumed, all parameters are precisely inferred with a small posterior coefficient of variation, approximately 2–12%. The ABC analyses reveal that the posterior distributions of D and q depend on the experimental elapsed time, whereas the posterior distribution of λ does not. The posterior mean values of D and q are in the ranges 226–268 µm2h−1, 311–351 µm2h−1 and 0.23–0.39, 0.32–0.61 for the experimental periods of 0–24 h and 24–48 h, respectively. Furthermore, we found that the posterior distribution of q also depends on the initial cell density, whereas the posterior distributions of D and λ do not. The ABC approach also enables information from the two experiments to be combined, resulting in greater precision for all estimates of D and λ.

Mathematical models for collective cell spreading

Collective cell spreading is frequently observed in development, tissue repair and disease progression. Mathematical modelling used in conjunction with experimental investigation can provide key insights into the mechanisms driving the spread of cell populations. In this study, we investigated how experimental and modelling frameworks can be used to identify several key features underlying collective cell spreading. In particular, we were able to independently quantify the roles of cell motility and cell proliferation in a spreading cell population, and investigate how these roles are influenced by factors such as the initial cell density, type of cell population and the assay geometry.

Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate “real time” gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1

Migraine is a common episodic neurological disorder, typically presenting with recurrent attacks of severe headache and autonomic dysfunction. Apart from rare monogenic subtypes, no genetic or molecular markers for migraine have been convincingly established. We identified the minor allele of rs1835740 on chromosome 8q22.1 to be associated with migraine (P = 5.38 x 10(-)(9), odds ratio = 1.23, 95% CI 1.150-1.324) in a genome-wide association study of 2,731 migraine cases ascertained from three European headache clinics and 10,747 population-matched controls. The association was replicated in 3,202 cases and 40,062 controls for an overall meta-analysis P value of 1.69 x 10(-)(1)(1) (odds ratio = 1.18, 95% CI 1.127-1.244). rs1835740 is located between MTDH (astrocyte elevated gene 1, also known as AEG-1) and PGCP (encoding plasma glutamate carboxypeptidase). In an expression quantitative trait study in lymphoblastoid cell lines, transcript levels of the MTDH were found to have a significant correlation to rs1835740 (P = 3.96 x 10(-)(5), permuted threshold for genome-wide significance 7.7 x 10(-)(5). To our knowledge, our data establish rs1835740 as the first genetic risk factor for migraine.

Fabrication and characterization of plasma polymer thin films from monoterpene alcohols for applications in organic electronics and biotechnology

After more than twenty years of basic and applied research, the use of nanotechnology in the design and manufacture of nanoscale materials is rapidly increasing, particularly in commercial applications that span from electronics across renewable energy areas, and biomedical devices. Novel polymers are attracting significant attention for they promise to provide a low−cost high−performance alternative to existing materials. Furthermore, these polymers have the potential to overcome limitations imposed by currently available materials thus enabling the development of new technologies and applications that are currently beyond our reach. This work focuses on the development of a range of new low−cost environmentally−friendly polymer materials for applications in areas of organic (flexible) electronics, optics, and biomaterials. The choice of the monomer reflects the environmentally−conscious focus of this project. Terpinen−4−ol is a major constituent of Australian grown Melaleuca alternifolia (tea tree) oil, attributed with the oil's antimicrobial and anti−inflammatory properties. Plasma polymerisation was chosen as a deposition technique for it requires minimal use of harmful chemicals and produces no hazardous by−products. Polymer thin films were fabricated under varied process conditions to attain materials with distinct physico−chemical, optoelectrical, biological and degradation characteristics. The resultant materials, named polyterpenol, were extensively characterised using a number of well−accepted and novel techniques, and their fundamental properties were defined. Polyterpenol films were demonstrated to be hydrocarbon rich, with variable content of oxygen moieties, primarily in the form of hydroxyl and carboxyl functionalities. The level of preservation of original monomer functionality was shown to be strongly dependent on the deposition energy, with higher applied power increasing the molecular fragmentation and substrate temperature. Polyterpenol water contact angle contact angle increased from 62.7° for the 10 W samples to 76.3° for the films deposited at 100 W. Polymers were determined to resist solubilisation by water, due to the extensive intermolecular and intramolecular hydrogen bonds present, and other solvents commonly employed in electronics and biomedical processing. Independent of deposition power, the surface topography of the polymers was shown to be smooth (Rq <0.5 nm), uniform and defect free. Hardness of polyterpenol coatings increased from 0.33 GPa for 10 W to 0.51 GPa for 100 W (at 500 μN load). Coatings deposited at higher input RF powers showed less mechanical deformation during nanoscratch testing, with no considerable damage, cracking or delamination observed. Independent of the substrate, the quality of film adhesion improved with RF power, suggesting these coatings are likely to be more stable and less susceptible to wear. Independent of fabrication conditions, polyterpenol thin films were optically transparent, with refractive index approximating that of glass. Refractive index increased slightly with deposition power, from 1.54 (10 W) to 1.56 (100 W) at 500 nm. The optical band gap values declined with increasing power, from 2.95 eV to 2.64 eV, placing the material within the range for semiconductors. Introduction of iodine impurity reduced the band gap of polyterpenol, from 2.8 eV to 1.64 eV, by extending the density of states more into the visible region of the electromagnetic spectrum. Doping decreased the transparency and increased the refractive index from 1.54 to 1.70 (at 500 nm). At optical frequencies, the real part of permittivity (k) was determined to be between 2.34 and 2.65, indicating a potential low-k material. These permittivity values were confirmed at microwave frequencies, where permittivity increased with input RF energy – from 2.32 to 2.53 (at 10 GHz ) and from 2.65 to 2.83 (at 20 GHz). At low frequencies, the dielectric constant was determined from current−voltage characteristics of Al−polyterpenol−Al devices. At frequencies below 100 kHz, the dielectric constant varied with RF power, from 3.86 to 4.42 at 1 kHz. For all samples, the resistivity was in order of 10⁸−10⁹ _m (at 6 V), confirming the insulating nature of polyterpenol material. In situ iodine doping was demonstrated to increase the conductivity of polyterpenol, from 5.05 × 10⁻⁸ S/cm to 1.20 × 10⁻⁶ S/cm (at 20 V). Exposed to ambient conditions over extended period of time, polyterpenol thin films were demonstrated to be optically, physically and chemically stable. The bulk of ageing occurred within first 150 h after deposition and was attributed to oxidation and volumetric relaxation. Thermal ageing studies indicated thermal stability increased for the films manufactured at higher RF powers, with degradation onset temperature associated with weight loss shifting from 150 ºC to 205 ºC for 10 W and 100 W polyterpenol, respectively. Annealing the films to 405 °C resulted in full dissociation of the polymer, with minimal residue. Given the outcomes of the fundamental characterisation, a number of potential applications for polyterpenol have been identified. Flexibility, tunable permittivity and loss tangent properties of polyterpenol suggest the material can be used as an insulating layer in plastic electronics. Implementation of polyterpenol as a surface modification of the gate insulator in pentacene-based Field Effect Transistor resulted in significant improvements, shifting the threshold voltage from + 20 V to –3 V, enhancing the effective mobility from 0.012 to 0.021 cm²/Vs, and improving the switching property of the device from 10⁷ to 10⁴. Polyterpenol was demonstrated to have a hole transport electron blocking property, with potential applications in many organic devices, such as organic light emitting diodes. Encapsulation of biomedical devices is also proposed, given that under favourable conditions, the original chemical and biological functionality of terpinen−4−ol molecule can be preserved. Films deposited at low RF power were shown to successfully prevent adhesion and retention of several important human pathogens, including P. aeruginosa, S. aureus, and S. epidermidis, whereas films deposited at higher RF power promoted bacterial cell adhesion and biofilm formation. Preliminary investigations into in vitro biocompatibility of polyterpenol demonstrated the coating to be non−toxic for several types of eukaryotic cells, including Balb/c mice macrophage and human monocyte type (HTP−1 non-adherent) cells. Applied to magnesium substrates, polyterpenol encapsulating layer significantly slowed down in vitro biodegradation of the metal, thus increasing the viability and growth of HTP−1 cells. Recently, applied to varied nanostructured titanium surfaces, polyterpenol thin films successfully reduced attachment, growth, and viability of P. aeruginosa and S. aureus.

Reduction of Secondary Degeneration after Spinal Cord Injury by Acute Delivery of Proinflammatory Growth Factors

INTRODUCTION Inflammation is a protective attempt to facilitate the removal of damaged tissue and to initiate the healing response in other tissues. However, after spinal cord injury (SCI), this response is prolonged leading to secondary degeneration and glial scarring. Here, we investigate the potential of sustained delivery of pro-inflammatory factors vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) to increase early inflammatory events and promote inflammatory resolution. Method Animal ethics approval was obtained from the Queensland University of Technology. Adult Wistar-Kyoto rats (12-16 weeks old) were subjected to laminectomies and T10 hemisections. Animals were then randomised to treatment (implantation of osmotic pump (Alzet) loaded with 5ug VEGF & 5 ug PDGF) or control groups (lesion control or lesion plus pump delivering PBS). Rats were sacrificed at one month and the spinal cords were harvested and examined by immunohistology, using anti-neurofilament-200(NF200) and anti- ionized calcium binding adapter molecule 1 (Iba1). One way ANOVA was used for statistic analysis. Results At 1 month, active pump-treated cords showed a high level of axonal filament throughout the defects as compared to the control groups. The mean lesion size, as measured by NF200, was 0.47mm2 for the lesion control, 0.39mm2 for the vehicle control and 0.078mm2 for the active pump group. Significant differences were detected between the active pump group and the two control groups (AP vs LC p= 0.017 AG vs VC p= 0.004). Iba-1 staining also showed significant differences in the post-injury inflammatory response. Discussion We have shown that axons and activated microglia are co-located in the lesion of the treated cord. We hypothesise the delivery of VEGF/PDGF increases the local vessel permeability to inflammatory cells and activates these along with the resident microglia to threshold population, which ultimately resolved the prolonged inflammation. Here, we have shown that maintaining the inflammatory signals for at least 7 days improved the morphology of the injured cord. Conclusion This study has shown that boosting inflammation, by delivery VEGF/PDGF, in the early phase of SCI helps to reduce secondary degeneration and may promote inflammation resolution. This treatment may provide a platform for other neuro-regenrative therapies.

The relationship between subjective appetite sensations, markers of inflammation and appetite in dialysis patients

Background: Poor appetite is a marker of morbidity and mortality in hemodialysis patients, making it an important area for research. Visual analog scales (VAS) can capture a range of subjective sensations related to appetite (such as hunger, desire to eat or fullness), but have not been commonly used to measure appetite in dialysis patients. The aim of this study was to explore the association between retrospective ratings of appetite using VAS and a range of clinical variables as well as biomarkers of appetite in hemodialysis patients.----- Methods: 28 hemodialysis patients (mean age 61±17y, 50% male, median dialysis vintage 19.5(4-101) months) rated their appetite using VAS for hunger, fullness and desire to eat and a 5-point categorical scale measuring general appetite. Blood levels of the appetite peptides leptin, ghrelin and peptide YY were also measured.----- Results: Hunger ratings measured by VAS were significantly (p<0.05) correlated with a range of clinical, nutritional and inflammatory markers: age (r=-0.376), co-morbidities, (r=-0.380) PG-SGA score (r=-0.451), weight (r=-0.375), fat-free mass (r=-0.435), C-Reactive Protein (CRP) (r=-0.383) and Intercellular adhesion molecule (sICAM-1) (r=-0.387). There was a consistent relationship between VAS and appetite on a 5-point categorical scale for questions of hunger, and a similar trend for desire to eat, but not for fullness. Neither method of measuring subjective appetite correlated with appetite peptides.----- Conclusions: Retrospective ratings of hunger on a VAS are associated with a range of clinical variables and further studies are warranted to support their use as a method of measuring appetite in dialysis patients.

A review of rapid prototyping techniques for tissue engineering purposes

Rapid prototyping (RP) is a common name for several techniques, which read in data from computer-aided design (CAD) drawings and manufacture automatically threedimensional objects layer-by-layer according to the virtual design. The utilization of RP in tissue engineering enables the production of three-dimensional scaffolds with complex geometries and very fine structures. Adding micro- and nanometer details into the scaffolds improves the mechanical properties of the scaffold and ensures better cell adhesion to the scaffold surface. Thus, tissue engineering constructs can be customized according to the data acquired from the medical scans to match the each patient’s individual needs. In addition RP enables the control of the scaffold porosity making it possible to fabricate applications with desired structural integrity. Unfortunately, every RP process has its own unique disadvantages in building tissue engineering scaffolds. Hence, the future research should be focused into the development of RP machines designed specifically for fabrication of tissue engineering scaffolds, although RP methods already can serve as a link between tissue and engineering.

Mechanical properties of advanced tissue engineering scaffold architectures

In tissue engineering, porous scaffolds are used as a temporal support for tissue regeneration through cell adhesion, proliferation and differentiation. Besides applying a suitable material that is both biocompatible and biodegradable, the architectural design of the porous scaffold can be of essential for successful tissue regeneration. The architecture is of great influence on mechanical properties and transport properties of nutrients and metabolites1.

Modelling cell separation during plant organ abscission

A simple mathematical model is presented to describe the cell separation process that plants undertake in order to deliberately shed organs. The focus here is on modelling the production of the enzyme polygalacturonase, which breaks down pectin that provides natural cell-to-cell adhesion in the localised abscission zone. A coupled system of three ordinary differential equations is given for a single cell, and then extended to hold for a layer of cells in the abscission zone. Simple observations are made based on the results of this preliminary model and, furthermore, a number of opportunities for applied mathematicians to make contributions in this subject area are discussed.

Cell sourcing for bone tissue engineering : amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem (AFS) cells and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-e-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.

Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared with adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression, respectively. This links BRN2 as an activator, and conversely, MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense-ablated cell lines decreased the melanoma sphere-forming capability, cell adhesion during 3D-spheroid formation and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere-culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than the growth as adherent melanoma cells.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.