Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Subject: search.filters.subject.Gene regulation

Search Results

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Stochastic simulation of multiscale complex systems with PISKaS: A rule-based approach
    (Elsevier Inc, 2018-03-29) Perez-Acle T; Fuenzalida I; Martin AJM; Santibañez R; Avaria R; Bernardin A; Bustos AM; Garrido D; Dushoff J; Liu JH
    Computational simulation is a widely employed methodology to study the dynamic behavior of complex systems. Although common approaches are based either on ordinary differential equations or stochastic differential equations, these techniques make several assumptions which, when it comes to biological processes, could often lead to unrealistic models. Among others, model approaches based on differential equations entangle kinetics and causality, failing when complexity increases, separating knowledge from models, and assuming that the average behavior of the population encompasses any individual deviation. To overcome these limitations, simulations based on the Stochastic Simulation Algorithm (SSA) appear as a suitable approach to model complex biological systems. In this work, we review three different models executed in PISKaS: a rule-based framework to produce multiscale stochastic simulations of complex systems. These models span multiple time and spatial scales ranging from gene regulation up to Game Theory. In the first example, we describe a model of the core regulatory network of gene expression in Escherichia coli highlighting the continuous model improvement capacities of PISKaS. The second example describes a hypothetical outbreak of the Ebola virus occurring in a compartmentalized environment resembling cities and highways. Finally, in the last example, we illustrate a stochastic model for the prisoner's dilemma; a common approach from social sciences describing complex interactions involving trust within human populations. As whole, these models demonstrate the capabilities of PISKaS providing fertile scenarios where to explore the dynamics of complex systems.
  • Thumbnail Image
    Item
    Regulation of the carotenoid biosynthetic pathway in petals of California poppy (Eschscholzia californica) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Zhou, Jun
    Carotenoids are essential plant pigments. They function in a wide range of processes including light harvesting in the photosynthetic apparatus, photoprotection against light damage, and pigmentation in flowers and fruits to attract pollinators and seed-dispersal herbivores. Carotenogenesis has been studied extensively in the last century in both photosynthetic and non-photosynthetic tissues of many plant species. Although most of the enzymes and their metabolites of the pathway have been identified, little is still known about how carotenoid production is regulated. Previous studies have proposed that regulation of the carotenoid pathway is through metabolite feedback occurring at both transcriptional and post transcriptional levels. This thesis examines the evidence for carotenogenesis gene transcription being feedback regulated by changes in carotenoid metabolites in petals of California poppy (Eschscholzia californica), and if so, by which metabolite(s). Virus-induced gene silencing (VIGS) was used to silence carotenoid biosynthetic genes in the petals of orange California poppy. High efficacy of silencing was achieved by first infiltrating and then drenching the California poppy seedlings with the Agrobacterium tumefaciens strain GV3101 containing the VIGS vectors. The VIGS vectors included portions of carotenoid gene fragments isolated from California poppy. qRT-PCR confirmed that transcript abundance of the targeted carotenogenesis genes EcaPDS, EcaZDS, EcaLCYb, EcaCHYb and EcaZEP was significantly reduced in the flower petals. Reduced transcript abundance of all genes apart from EcaLCYb altered flower colour. HPLC analyses revealed that the colour altered flower petals with knocked-down expression of each targeted gene resulted in a reduction of total carotenoid content and an altered profile of carotenoids. This manifested as an accumulation of higher amounts of intermediates including phytofluene, ζ-carotene, β-carotene and zeaxanthin, some of which are not usually seen in the flowers, and a reduction of the end products such as retro-carotene-triol and eschscholtzxanthin. However, these alterations in carotenoid profiles were not associated with any dramatic changes in transcript abundance of the non-TRV-targeted endogenous genes in the pathway. Therefore, little evidence was found for metabolite feedback regulation of transcriptional activity in the carotenoid biosynthetic pathway from this study. Other possible mechanisms for controlling carotenogenesis are discussed.
  • Thumbnail Image
    Item
    Development of a tetracycline-inducible lentiviral vector with an instant regulatory system : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2013) Yang, Tian
    Lentiviral vectors, originally derived from human immunodeficiency virus, provide highly efficient viral gene delivery vehicles. Lentiviral vectors often use a constitutive promoter to drive the expression of a therapeutic gene. To regulate the expression of a therapeutic gene, a regulatory system such as Tet-On needs to be established in the target cell lines to produce a regulatory protein, reverse Tet-responsive transcriptional activator (rtTA). The expressed rtTA binds to the tetracycline responsive element (TRE) in the promoter in response to doxycycline and activates transcription of gene of interest. A hypothesis in this study is based on the speculation that a basal leaky expression of rtTA in the bi-directional TRE vectors allows instantly inducible expression of a gene of interest and thereby avoids the time-consuming procedures for generating Tet-On cell lines. Based on this hypothesis, a novel lentiviral vector has been developed to examine an instant induction of PP2Cβ as a target gene. Three instantly inducible bicistronic lentiviral vectors [pLenti-Bi-TRE-Tet-on (V), pLenti-Bi-TRE-Tet-on-PP2Cβ WT (WT), pLenti- Bi-TRE-Tet-on-PP2Cβ MUT (MUT)] were constructed and characterised to assess the usefulness of these vectors. Transient transfection of both WT and MUT vectors into HEK293T cells showed a great induction of PP2Cβ expression upon 24 h of 1 μM doxycycline treatment. The result promises the use of these vectors as a mammalian expression plasmid with a feature of inducible target gene expression. However, viral infection studies involving lentiviral packaging and infection procedures did not show a reproducible expression of rtTA or PP2Cβ in HEK293T cells. Therefore, the inducibility of viral transduction needs to be improved for the future studies of PP2Cβ in primary cells.
  • Thumbnail Image
    Item
    Eukaryotic signature proteins : guides to pathogenic eukaryotic parasites : a thesis presented in partial fulfilment of the requirements of the degree of PhD in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Han, Jian
    Eukaryotic Signature Proteins (ESPs) are proteins that delineate the eukaryotes from the archaea and bacteria. They have no homologues in any prokaryotic genome, but their homologues are present in all main branches of eukaryotes. ESPs are thus likely to have descended from ancient proteins that have existed since the first eukaryotic cell. This project looks at ESPs of some eukaryotic parasites and human (Homo sapiens) as their host organism and focuses on Giardia lamblia, a fresh water pathogenic basal eukaryote. The ESP datasets from Giardia and two other parasites, Trichomonas vaginalis and Plasmodium falciparum, as well as the host human were calculated in light of available genomic data and the datasets contained a range of proteins associated with membrane, cytoskeleton, nucleus and protein synthesis. ESPs have great potential in phylogenetic studies since these proteins are present in all eukaryotes and are expected to have a slow and constant rate of evolution. Phylogenetic analyses were performed on the 18 eukaryotic organisms including some basal eukaryotes, and also for mammals, using orthologues of the all ESPs from these organisms. Strategies such as concatenating sequences and constructing consensus networks were tested to evaluate their potential with large numbers of ESP alignments. The results were promising, and ESPs hold great potential for their use in future phylogenetic analyses of eukaryotes. RNA interference is hypothesised to be an ancient mechanism for gene regulation and like the ESPs, it is typically found in all main branches of eukaryotes. High throughput sequencing data from Giardia and Trichomonas small RNAs (15-29mers) were re-analysed showing two length peaks for Giardia RNAs: a “larger peak” and an “ultra small peak”, the former of which is likely to be the product of the enzyme Dicer, which processes miRNA. The “ultra small peak” but not the “larger peak” was also found in Trichomonas. The two peaks possibly represent two different mechanisms of RNA interference (RNAi) in these parasites, but analysis of potential target sites from the Dicer-processed RNAs has not yet shown any indication that ESPs are regulated any differently from other parasite proteins. Sugar metabolic pathways including glycolysis and citric acid cycle were searched for ESPs, this was done to determine the relationship between the conservation of eukaryotic metabolic pathways and conservation of individual proteins. However no ESPs were identified from these pathways because Giardia has enzymes that show more similarity to those from prokaryotes than eukaryotes. These enzymes are significantly different from that of the host‟s, and these alternative enzymes offer potential as novel drug targets. In addition, ESPs that are present from host but lost in some parasites were analysed, and these ESPs are involved in many understudied pathways. It is these differences which can provide a guide in determining which pathways we should examine when designing drug targets. Overall, numerous proteomic similarities and differences in ESPs were identified between host and parasite. These proteins show potential for future evolutionary studies, and will guide future directions in ancestral eukaryotic regulation and metabolism.
  • Thumbnail Image
    Item
    How the pigment stripes form in snapdragon (Antirrhinum majus) flowers : a study of the molecular mechanism of venation pigmentation patterning in flowers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2006) Shang, Yongjin
    Floral stripes are a common pigmentation pattern in plants. Defining the molecular mechanisms of the striped pattern formation will aid understanding of how a gene can be differentially regulated across a population of similar cells. In the venation phenotype of Antirrhinum majus, the anthocyanin pigment is typically confined to the adaxial epidermal cells overlaying the petal veins. To explore how this pattern forms this study focused on the expression and regulation of Venosa, a Myb regulator of anthocyanin biosynthesis. Pigment complementation experiments demonstrated that the lack of a MYB factor caused the lack of pigment in the cells outside the venation pigmentation domain. An allele of Venosa was isolated and identified. It was a mutant version of functional Venosa due to the central part being replaced by a transposon. Phenotype / genotype analysis indicated that the venation pigmentation patterning was due to the functional Venosa. In situ mRNA hybridisation showed that Venosa was expressed from the xylem to the adaxial epidermis, and was controlled spatially and quantitatively by a signal associated with the petal veins. Venosa expression provided the longitudinal axis for venation pigmentation stripes, and determined the location and intensity of the pigmented cells. Because another factor required for pigmentation, a bHLH factor, is specifically expressed in epidermal cells and it provides the transverse axis. The pigmented stripes are the cross expression domain of these two kinds of factors. The transcriptional controlling property of a 2.4 kb (relative to the ATG) promoter region of the Venosa gene was analysed. The -900 bp fragment was characterised in detail using 5'-end deletion mutagenesis. A heterologous host, tobacco, was used for analysis in stable transgenics. The homologous host, Antirrhinum, was used for transient assays. The efficacy and efficiency of different reporter genes (intron-containing GUS, GFP, Venosa cDNA and genomic Venosa) and enhancement systems (transcriptional enhancer, translational enhancer, inhibitor of post transcriptional gene silencing and a two-step signaling amplification system) for the detection of low-level reporter gene expression were also tested. The strength of expression correlated to the length of the promoter fragment, and expression was detected using deletions down to -500 bp, although only weak expression was found. This expression was flower specific but not vein related in both plant hosts. No expression was detected in petals of either host with fragments shorter than -500 bp. The results suggest that the fragment from -380 bp to -900 bp positively affected Venosa expression at the transcriptional level, but might not be sufficient to define venation. A possibility is that the venation controlling property is negatively controlled at the epigenetic level, such as DNA methylation status and / or chromatin structure. The role of gibberellin and sugar in the pigment and venation patterning formation of Antirrhinum was studied. The results suggest that gibberellin is not required for pigmentation or venation patterning. Convincing evidence on the role of sugar signaling could not be obtained from the experiments, due to the difficulty in separating the impact on pigmentation from other functions of sugars in petal development. In addition, the in situ analysis detected the expression of a gene probably related to aurone biosynthesis that may be a regulatory gene of this biosynthetic pathway.