The effects of overexpression of lamin a and two mutants associated with premature aging on stem cell differentiation and proliferation.

The nuclear lamina is composed of A and B-type lamins and performs a number of functions within the nucleus. Mutations within lamins give rise to a group of diseases called laminopathies including autosomal dominant Emery-Dreifuss muscular dystrophy and lipodystrophy. Hutchinson Gilford Progeria Syndrome (HOPS), or so-called premature aging, is a rare and devastating laminopathy disorder caused by mutations in LMNA that leads to the production of a truncated mutant form of prelamin A, called progerin or LAA50, that cannot undergo proteolytic processing to yield mature lamin A. In an atypical form of HOPS, a point mutation at amino acid 644 alters the Zmpste24 endoproteolytic cleavage site, causing the production of mutant prelamin A. Why and how these prelamin A mutants cause such devastating phenotypes is not folly understood. It has been suggested that the mutant prelamin A may affect the ability of adult stem cells to self-renewal and differentiate, which are essential processes in order to replace damage of old tissues, critical for organismal longevity. Herein, expression plasmids containing two mutations found in classical and atypical HOPS were transiently overexpressed in clonal rat dermal papilla (DP) 9 cells, which, in vivo, reside at the base of the hair follicle and play an important role in hair follicle cycling. DP9 cells showed an increased number of nuclear abnormalities compared to control cells, which have previously been shown to be characteristic of cells from HGPS patients. DP9 cell lines stably overexpressing either FLAG-prelamin A， FLAG-prelamin ΑΔ50 or FLAG-prelamm A(R644C) were created. To assess the ability of the stem cells to self-renew, stable cell lines were routinely passaged and counted. Results indicate that stable overexpression of FLAG-prelamin ΑΔ50 and FLAG-prelamin A(R644C) inhibits the ability of stem cells to self-renew in vitro. Previous reports have shown that DP9 cells may be directed towards both adipogenic and osteogenic lineages. Therefore, in this study, stable DP9 cell lines expressing FLAG- prelamin A， FLAG-prelamin ΑΔ50 or FLAG-prelamin A(R644C) were exposed to an adipogenic medium for six days. Results showed that overexpression of FLAG-prelamin A, FLAG-prelamm ΑΔ50 and FLAG-prelamm A(R644C) inhibited the accumulation of intracellular lipids, reflecting a decreased ability to differentiate in vitro. Taken together, these results suggest the lipodystrophy and alopecia associated with HGPS may be due to the failure of adult stem cell populations within each tissue to both self-renew and differentiate, and may underpin the disease pathogenesis. Furthermore, these results have implications for other laminopathies that produce mutant prelamin A

The multiple roles of A-type lamins in cellular aging, cell cycle progression and the DNA damage response.

A-type lamins are a group of type V intermediate filaments whose main members are lamin A and C. Lamins A/C are components of the nuclear lamina and are encoded by the LMNA gene. Lamins A/C have a variety of cellular functions, including maintaining the structural integrity of the nucleus and the regulation of signal transduction pathways, transcription factors and DNA replication. Mutations in LMNA give rise to a diverse spectrum of diseases, termed laminopathies, which include premature aging syndromes. In Chapter 3, I sought to understand the role of wild type lamin A in normal cellular aging. Lamin A C-terminal cysteine residues were irreversibly oxidized during the in vitro aging of human dermal fibroblasts (HDFs), which impaired the ability of lamin A to form disulfide bonds, causing loss of function. Furthermore, loss of these cysteine residues induced premature senescence, suggesting that these cysteine residues are important for lamin A function during cellular aging. In Chapter 4, I extended previous findings implicating A-type lamins in the control of cell cycle progression. Loss of A-type lamins or its nucleoplasmic binding partner, LAP2α, caused delayed G1/S-phase progression, reduced cellular proliferation and cell cycle exit. Proliferative defects could not be rescued via treatment with anti-oxidants. In Chapters 5 and 6, I addressed the role of wild type mature lamin A/C in the DNA damage response (DDR). A-type lamins interact with the DDR mediator protein 53BP1 via its Tudor domain. Loss of LMNA caused endogenous DNA damage and loss of 53BP1 protein levels. Furthermore, loss of LMNA resulted in defective DNA repair that ultimately led to increased sensitivity to DNA damage. Together, the data presented here extends previous findings implicating A-type lamins in cell cycle progression and provides novel insights into the cellular roles of A-type lamins in cellular aging and the DNA damage response

Field pea in the great southern

Semi-leafless field pea like Kaspa have lifted the benchmark set by the previous conventional varieties such as Parafield and Dunwa. Semi-leafless field pea have the same traits as conventional field pea: adaptation to a range of soil types; wide selection of weed control options (including crop topping); nitrogen input; disease break; and reduction in root lesion nematodes. Semi-leafless pea is easier to harvest and is less prone to pod shatter

Tools for decoding ubiquitin signaling in DNA repair

The maintenance of genome stability requires dedicated DNA repair processes and pathways that are essential for the faithful duplication and propagation of chromosomes. These DNA repair mechanisms counteract the potentially deleterious impact of the frequent genotoxic challenges faced by cells from both exogenous and endogenous agents. Intrinsic to these mechanisms, cells have an arsenal of protein factors that can be utilised to promote repair processes in response to DNA lesions. Orchestration of the protein factors within the various cellular DNA repair pathways is performed, in part, by post-translational modifications, such as phosphorylation, ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs). In this review, we firstly explore recent advances in the tools for identifying factors involved in both DNA repair and ubiquitin signaling pathways. We then expand on this by evaluating the growing repertoire of proteomic, biochemical and structural techniques available to further understand the mechanistic basis by which these complex modifications regulate DNA repair. Together, we provide a snapshot of the range of methods now available to investigate and decode how ubiquitin signaling can promote DNA repair and maintain genome stability in mammalian cells

Nanowire and microband arrays for enhanced electrochemical sensing

Water quality monitoring is crucial to ensure that (i) water is safe to be consumed by humans, and (ii) that it is of adequate quality for aquatic life. The European Union has dedicated two of its seventeen sustainable development goals for 2030 to water quality. While many methods are available to monitor water quality, these typically involve the use of additional reagents, or require specialist equipment or personnel to carry out the required analysis. In this thesis, electrochemical methods are explored and developed such that they allow for reagent-free, point-of-care testing without the need for specialist training. The main goal of this thesis was to develop interference free measurements that could be applied to remote analysis. To do this, interdigitated micro-electrode arrays were designed and fabricated wherein each comb of electrodes could be biased at different potentials allowing for unique analysis opportunities. The first section deals with the detection of residual free chlorine, the most common disinfectant in water. To do this a pH control method using the interdigitated electrodes was developed that forced the free chlorine in solution to convert to hypochlorous acid, which can be easily detected. This approach prevents loss of signal due to presence of hypochlorite, which is less active in the potential window investigated. Concentrations as low as 0.35 ppm were measured and this method was applied to both buffered water samples and tap water samples. Following from this, the pH control method was applied to the detection of monochloramine. This is the second most common disinfectant for water, typically used in North America. It is also a by-product of water chlorination, so it also needs to be monitored in systems where it is not used as the primary disinfectant. The pH control method was refined in this section and resulted in a sensor that could detect monochloramine by converting it to dichloramine. Monochloramine detection is hindered by the presence of dissolved oxygen, while dichloramine is free from oxygen interference. A detection limit of 0.03 ppm was calculated and this method was applied to water distribution networks even those with high conductivity and alkalinity. Finally, a reagent free method of dissolved oxygen quantification was developed. Dissolved oxygen is a measure of water quality typically used to determine how well the water supports aquatic life. Low dissolved oxygen concentrations can also indicate the presence of bacteria or contaminants like nitrates and nitrites. The detection method developed herein involves reduction of dissolved oxygen to produce hydrogen peroxide, which is subsequently detected at the other comb of electrodes free from interference. Gold and platinum mixed arrays were used to take advantage of both materials properties for oxygen reduction and peroxide oxidation. Oxygen concentrations between 0 and 10 ppm were detected using this method (benchmarked against commercial gold standards), and this approach was applied to real water samples taken from farm run off. This water typically contains high concentrations of fertilizer, yet accurate oxygen measurements were obtained

Use of sap flow measuring techniques to estimate water-use of multi-stem plants

In the UK, coppice willow is a potentially high yielding energy crop which if widely planted could have significant effects of hydrology. This is one reason why researchers are interested in developing reliable techniques for estimating whole-plant water use of such trees. In recent years various sap flow measuring techniques have become commercially available. The aim of this thesis was to evaluate the reliability sap flow measurement and to consider issues related to best practice when used on multi-stemmed woody plants. The thesis starts by reviewing methodologies for estimating tree water-use with particular focus on sap flow gauges. Subsequently over three years, experiments were undertaken using a commercially available Stem Heat Balance (SHB) sap flow gauges, manufactured by Dynamax Inc, Houston, Texas, on coppice willow grown in lysimeters. Plant responses were monitored through a range of soil-water conditions from flood to drought. Over three years a methodology for deriving an accurate assessment of total plant water-use using a lysimeter water balance (LWB) was developed. Whole-plant water-use, estimated from scaled up sap flow measurements from individual stems were compared against LWB values. Both, stem basal area and leaf area were used as scalars to derive values of plant water-use. In the final experiment, four out of eight, different sized sap flow gauges, with the appropriate scalar, gave estimates similar (±7%) to LWB values of whole-plant water use over a period of ‘unstressed’ growing conditions. Variation in the accuracy of estimates was considered to be a function of a) error inherent to the SHB technique, b) error in scalar values used to derive whole-plant estimates, and c) apparently autonomous responses of individual stems to changes in soil water status. In non-water stressed conditions and where sap flow rates are high, errors from the technique were minimised by selecting Targe’ stems (16-19 mm diameter). Under extreme water stress conditions, reductions in leaf area can result in errors if the estimate is based on stem diameter. Where individual stem flow rates were perturbed by changes in soil-water conditions, selecting an ‘intermediate’ sized stem (in this case 10-13 mm diameter) appeared to minimise errors.MPhi