Plant-based immunocontraceptive control of wildlife - "potentials, limitations, and possums"

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immuno-contraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests. (c) 2004 Elsevier Ltd. All rights reserved.

Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro

Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 mum-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells. (C) 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.

Gravity spun polycaprolactone fibers for applications in vascular tissue engineering:proliferation and function of human vascular endothelial cells

Poly(ε-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under low-shear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 × 104 cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering. © Mary Ann Liebert, Inc.

The breakdown of plant cell biomass by fungi

Three species of fungi Sporotrichum thermophile, Botrytis cinerea and Trichoderma viride were assessed for their ability to utilize a variety of plant cell substrates (methanol extracted), Catharanthus roseus, Daucus carota, re-autoclaved C. roseus, re-autoclaved D. carota) which preliminary studies had indicated contained the necessary nutrients for fungal growth. Incubated in a suitable manner all three fungal species were able to grow on C. roseus and D. carota plant cell biomass in addition to material which had undergone methanol extraction or a re-autoclaving process to remove soluble components. Fungal biomass yields were markedly influenced by substrate, with each fungal species demonstrating a preference for particular plant cell material. Incubation conditions i.e. static or shaken and temperature also proved important. Release of glucose (i.e. values higher than Day 0) promoted by fungal breakdown of plant cell biomass was only noted with methanol extracted, re-autoclaved C. roseus and re-autoclaved D. carota material. A re-autoclaved substrate was also generally associated with high fungal C1, Cx, B-glucosidase and endo-polygalacturonase activity. In addition for each enzyme highest values were usually obtained from a particular fungal species. Buffering cultures at pH 3 or 5 further influenced enzyme activity, however in a majority of cases when flasks were unbuffered and the pH rose naturally to alkaline values higher enzyme activity was recorded. Likewise Tween 80 addition had only a limited beneficial effect. Finally filtrates containing glucose produced both from the re-autoclaving process and through fungal activity on plant cell biomass were utilized for Fusarium oxysporum, Saccharomyces cerevisiae and C. roseus plant cell culture. Although reasonable fungal biomass was obtained the use of such filtrates proved unsuitable for plant cell growth.

Novel biodegradable fibres for applications in tissue engineering and drug delivery

The preparation and characterisation of novel biodegradable polymer fibres for application in tissue engineering and drug delivery are reported. Poly(e-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. The tensile strength and stiffness of as-spun fibres were highly dependent on the concentration of the spinning solution. Use of a 6% w/v solution resulted in fibres having strength and stiffness of 1.8 MPa and 0.01 GPa respectively, whereas these values increased to 9.9 MPa and 0.1 GPa when fibres were produced from 20% w/v solutions. Cold drawing to an extension of 500% resulted in further increases in fibre strength (up to 50 MPa) and stiffness (0.3 GPa). Hot drawing to 500% further increased the fibre strength (up to 81 MPa) and stiffness (0.5 GPa). The surface morphology of as-spun fibres was modified, to yield a directional grooved pattern by drying in contact with a mandrel having a machined topography characterised by a peak-peak separation of 91 mm and a peak height of 30 mm. Differential scanning calorimetery (DSC) analysis of as-spun fibres revealed the characteristic melting point of PCL at around 58°C and a % crystallinity of approximately 60%. The biocompatibility of as-spun fibres was assessed using cell culture. The number of attached 3T3 Swiss mouse fibroblasts, C2C12 mouse myoblasts and human umbilical vein endothelial cells (HUVECs) on as-spun, 500% cold drawn, and gelatin coated PCL fibres were observed. The results showed that the fibres promoted cell proliferation for 9 days in cell culture and was slightly lower than on tissue culture plastic. The morphology of all cell lines was assessed on the various PCL fibres using scanning electron microscopy. The cell function of HUVECs growing on the as-spun PCL fibres was evaluated. The ability HUVECs to induce an immune response when stimulated with lipopolysaccaride (LPS) and thereby to increase the amount of cell surface receptors was assessed by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that PCL fibres did not inhibit this function compared to TCP. As-spun PCL fibres were loaded with 1 % ovine albumin (OVA) powder, 1% OVA nanoparticles and 5% OVA nanoparticles by weight and the protein release was assessed in vitro. PCL fibres loaded with 1 % OVA powder released 70%, 1% OVA nanoparticle released 60% and the 5% OVA nanoparticle released 25% of their protein content over 28 days. These release figures did not alter when the fibres were subjected to lipase enzymatic degradation. The OVA released was examined for structural integrity by SDS-PAGE. This showed that the protein molecular weight was not altered after incorporation into the fibres. The bioactivity of progesterone was assessed following incorporation into PCL fibres. Results showed that the progesterone released had a pronounced effect on MCF-7 breast epithelial cells, inhibiting their proliferation. The PCL fibres display high fibre compliance, a potential for controlling the fibre surface architecture to promote contact guidance effects, favorable proliferation rate of fibroblasts, myoblasts and HUVECs and the ability to release pharmaceuticals. These properties recommended their use for 3-D scaffold production in soft tissue engineering and the fibres could also be exploited for controlled presentation and release of biopharmaceuticals such as growth factors.

Osmotic dehydration in plant tissues

The primary aim of the thesis is to provide a comprehensive investigation of the osmotic dehydration processes in plant tissue. Effort has been concentrated on the modelling for simulating the processes. Two mathematical models for simulating the mass transfer during osmotic dehydration processes in plant tissues are developed and verified using existing experimental data. Both models are based on the mechanism of diffusion and convection of any mobile material that can transport in plant tissues. The mass balance equation for the transport of each constituent is established separately for intracellular and extra-cellular volumes with taking into account the mass transfer across the cell membrane the intracellular and extra-cellular volumes and the shrinkage of the whole tissue. The contribution from turgor pressure is considered in both models. Model two uses Darcy’s law to build the relation between shrinkage velocity and hydrostatic pressure in each volume because the plant tissue can be considered as the porous medium. Moreover, it has been extended to solve the multi-dimensional problems. A lot of efforts have been made to the parameter study and the sensitivity analyses. The parameters investigated including the concentration of the osmotic solution, diffusion coefficient, permeability of the cell membrane, elastic modulus of the cell wall, critical cell volume etc. The models allow us to quantitatively simulate the time evolution of intracellular and extra-cellular volumes as well as the time evolution of concentrations in each cross-section.

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range.

Functional studies of lignin biosynthesis genes and putative flowering gene in Miscanthus x giganteus and studies on indolyl glucosinolate biosynthesis and translocation in Brassica oleracea

The first topic area of this thesis involved studies on the accumulation and translocation of glucosinolates (GSs), bioactive secondary plant compounds, in broccoli plants. Changes in GS accumulation and gene expression levels in response to exogeneous methyl jasmonate (MeJA) treatment were analyzed in different tissue types at different developmental stages of broccoli. Greater accumulation of GSs with MeJA treatment was observed in apical leaves of broccoli seedlings and florets of plants at harvest maturity. Increases in indolyl GS in apical leaves of seedlings and florets were coupled with the up-regulation of indolyl GS biosynthesis genes. The accumulation of indolyl GSs appears to be modulated by MeJA treatment in an organ-specific manner for optimal distribution of defense substances in the plant. Metabolic profiling of hydrophilic metabolites using GC-MS demonstrated increased accumulation of various phenolics, ascorbates and amino acids in broccoli tissues after MeJA treatment. Distinct changes in carbohydrate levels observed between different tissues (vegetative leaves and floret tissues) of broccoli plants after treatment suggest that carbon metabolism is differentially modulated by MeJA treatment in different tissue types depending on sink-source relationships. Reduced levels of hexose sugars and tricarboxylic acid intermediates after MeJA treatment may reflect the increased requirement for carbon and energy needed to drive secondary product biosynthesis to accumulate metabolites for defense against insects and other herbivores. Substantial increases of indolyl and aromatic GSs after exogenous treatment with MeJA in stem and petioles of seedlings and the existence of intact indolyl-GS forms in phloem exudates suggest enhanced de novo synthesis in combination with active transport. Indoly GSs share structural similarities with the auxin, IAA, and may interact with components of the auxin transport system for intra- and extra-cellular transport or translocation. Application of the auxin efflux inhibitor, 1-naphthylphthalamic acid (NPA) reduced MeJA-mediated accumulation of indolyl GSs in broccoli florets and seedling tissues. NPA did not inhibit expression of indolyl GS biosynthesis genes shown to be upregulated by MeJA treatment or the accumulation of tryptophan, the amino acid precursor of indolyl GSs. Exogenous application of benzyl GS to Arabidopsis roots induced ectopic expression of the PIN1 protein associated with the auxin transport system similar to treatment with NPA, again suggesting GS interaction with the auxin efflux carrier system. The inhibitory effect of NPA on MeJA-mediated accumulation of GS may be due to competitive binding of NPA to auxin efflux carrier components and that GS transport is mediated by the auxin transport system. The inhibitory effect of NPA on indolyl and aromatic GS accumulation and the bioactivity of exogenous treatment of these GS compounds in PIN1 localization, Arabidopsis root growth, and gravitrophic response suggest that indolyl and aromatic GSs may be antagonistic to IAA transport and biosynthesis. Indolyl and aromatic GSs can also be potentially converted into IAA by hydrolysis. This intrinsic feature of GSs may be the part of a sophisticated regulatory process where the metabolic pathways in the plant shift from active growth to a reversible defense posture in response to biotic or abiotic stress. It seems likely that indolyl and aromatic GSs are important compounds that provide connections between jasmonate and auxin signaling. Further studies are required to reveal the regulatory mechanism for crosstalk between the two hormones. The third part of this research was to investigate effect of selenium fertilization and MeJA treatment on accumulation of GSs in broccoli florets. Increasing dietary intake of the element selenium (Se) has been shown to reduce the risk of cancer. Simultaneous enhancement of both Se and GS concentrations in broccoli floret tissue were conducted through the combined treatment of MeJA with Se fertilization. A low level of Se fertilization (concentration) with MeJA treatment displayed no significant changes in total aliphatic GS concentrations with 90% and 50% increases in indolyl and total GSs concentrations, respectively. This result suggests that Se- and GS-enriched broccoli with improved health-promoting properties can be generated by this combined treatment. The second topic of this thesis was conducted to provide basic information required to improve biomass quality and productivity and develop tools for gene transformation in Miscanthus x giganteus. The perennial rhizomatous grass, Miscanthus x giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. x giganteus must be propagated vegetatively by mechanicalling divided rhizomes or from micropropagated plantlets. The effect of callus type, age and culture methods on regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. x giganteus propagation. Seven lignin biosynthesis genes and one putative flowering gene were isolated from M. x giganteus by PCR reactions using maize othologous sequences. Southern hybridization and nuclear DNA content analysis indicated that the genes isolated from M. x giganteus exist in the genome of other Miscanthus species as multiple copies. Analysis of lignin content and histological staining of lignin deposition indicated that higher lignin content is found in mature stem node tissues compared to young leaves and apical stem nodal tissues. Cell wall lignification is associated with increasing tissue maturity in Miscanthus species. RNAi and antisense constructs harboring sequences of these genes were developed to generate Miscanthus transgenic plants with suppressed of lignin biosynthesis and delayed flowering.

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved e.g. through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots including physical and chemical ones such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates or media (reviewed by Ragonezi et al. 2010). The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant- growth-promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests.

Establishment of a protocol for the cryopreservation of cell sheets of adipose tissue stem cells

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Germinação de sementes e otimização de técnicas de micropropagação de umbuzeiro (spondias tuberosa, arr.) anacardiaceae

The brazilian-plum (Spondias tuberosa, His) is a tropical fruit tree that has been consolidated in the market for agribusiness processing, due to its characteristic flavor of fruit. Accordingly, studies to optimize the propagation of plants are necessary for production of seedlings with agronomic and quality assurance measures. This study aimed at determining the efficient techniques for uniform seed germination, as brazilian-plum seed present mechanical dormancy, and establish optimal culture media for multiplication of shoots from the in vitro micropropagation. Firstly, in a greenhouse at the Universidade Federal do Rio Grande do Norte, was evaluated the influence of different methods of breaking dormancy in the emergence of seedlings of brazilian-plum and speed of germination (IVG) of seeds. After 60 days of cultivation, it was found that splay in the distal portion of the seed was the best treatment, with rates of 85.33% in germinability and 3.415 of IVG, compared with the treatment of seed-soaking in water for 12h + humus and the control group. Subsequently, new sources of seedling explants were obtained in studies of tissue culture. Laboratory of Plant Biotechnology that the university, was used stem apex, nodal segments and internodes in search of decontamination with various concentrations of calcium hypochlorite [Ca(OCl)2] and micropropagation, inoculating them in half WPM (1980) with various concentrations of 6-benzylaminopurine (BAP). We used 10 sample units with three replications for different concentrations of [Ca(OCl)2], BAP and explants type. After thirty days, which was observed for the control of contamination, during the establishment in vitro, concentrations of [Ca(OCl)2] between 0.5% and 2.0% were effective in combating exogenous contamination of the apex. In nodal segments and internodes, concentrations of [Ca(OCl)2] between 1.0% and 2.0% and 1.5% and 2.0% were respectively, sufficient to reduce the percentage of losses in these infestations explants. For micropropagation, the culture medium supplemented with 0.1 mg.L-1 BAP promotes better development of multiple shoots per explants from nodal segment. However, success does not get to shoot training in internodal segment

Regulation of bone marrow stem cells through oscillatory shear stresses - A heart valve tissue engineering perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range. ^

The distribution and spread of citrus canker in Emerald, Australia

Citrus canker is a disease of citrus and closely related species, caused by the bacterium Xanthomonas citri subsp. citri. This disease, previously exotic to Australia, was detected on a single farm [infested premise-1, (IP1). IP is the terminology used in official biosecurity protocols to describe a locality at which an exotic plant pest has been confirmed or is presumed to exist. IP are numbered sequentially as they are detected] in Emerald, Queensland in July 2004. During the following 10 months the disease was subsequently detected on two other farms (IP2 and IP3) within the same area and studies indicated the disease first occurred on IP1 and spread to IP2 and IP3. The oldest, naturally infected plant tissue observed on any of these farms indicated the disease was present on IP1 for several months before detection and established on IP2 and IP3 during the second quarter (i.e. autumn) 2004. Transect studies on some IP1 blocks showed disease incidences ranged between 52 and 100% (trees infected). This contrasted to very low disease incidence, less than 4% of trees within a block, on IP2 and IP3. The mechanisms proposed for disease spread within blocks include weather-assisted dispersal of the bacterium (e.g. wind-driven rain) and movement of contaminated farm equipment, in particular by pivot irrigator towers via mechanical damage in combination with abundant water. Spread between blocks on IP2 was attributed to movement of contaminated farm equipment and/or people. Epidemiology results suggest: (i) successive surveillance rounds increase the likelihood of disease detection; (ii) surveillance sensitivity is affected by tree size; and (iii) individual destruction zones (for the purpose of eradication) could be determined using disease incidence and severity data rather than a predefined set area.