School of BioSciences - Research Publications
Permanent URI for this collection
Search Results
1 - 10 of 1532
-
ItemPI3K Activation in Neural Stem Cells Drives Tumorigenesis which can be Ameliorated by Targeting the cAMP Response Element Binding (CREB) Protein(Oxford University Press, 2018-10)BACKGROUND: Hyperactivation of phosphoinositide 3-kinase (PI3K) signaling is common in cancers, but the precise role of the pathway in glioma biology remains to be determined. Some understanding of PI3K signaling mechanisms in brain cancer comes from studies on neural stem/progenitor cells (NSPCs), where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate critical cell functions. METHODS: To investigate the role of the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a PIK3CAH1047A oncogenic mutant and deletion of the PI3K negative regulator, phosphatase and tensin homolog (PTEN), to NSPCs. RESULTS: Expression of a Pik3caH1047A was sufficient to generate tumors with oligodendroglial features, but simultaneous loss of PTEN was required for the development of invasive, high-grade glioma. Pik3caH1047A-PTEN mutant NSPCs exhibited enhanced neurosphere formation which correlated with increased Wnt signaling, while loss of cAMP response element binding protein (CREB) in Pik3caH1047A-Pten mutant tumors led to longer symptom-free survival in mice. CONCLUSION: Taken together, our findings present a novel mouse model for glioma demonstrating that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.
-
ItemInvestigating Neural Stem Cell and Glioma Stem Cell Self-renewal Potential Using Extreme Limiting Dilution Analysis (ELDA)(BIO-PROTOCOL, 2018-09-05)Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in vitro. We further show how ELDA can be used on cells, before and after forced differentiation to amplify inherent differences in sphere-forming potential between mutant and control NSCs. Ultimately, ELDA exploits a difference in the ability of a single or a few seeded stem cells to self-renew, divide and form neurospheres. Importantly, the assay also allows a comparison between genetically distinct cells or between the same cells under different conditions, where the impact of target-specific drugs or other novel cancer stem cell therapies can be tested.
-
ItemNo Preview AvailableHBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells(American Society for Microbiology, 2020-02-01)HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.
-
ItemAssessment and management of reproduction in Australian monotremes and marsupials(CSIRO Publishing, 2019)
-
ItemMales evolve to be more harmful under increased sexual conflict intensity in a seed beetle(OXFORD UNIV PRESS INC, 2020-01-28)One conspicuous manifestation of sexual conflict is traumatic mating, in which male genitalia damage the female during copulation. The penis of the seed beetle, Callosobruchus maculatus, is covered in spines that damage the female reproductive tract. Females kick males ostensibly to shorten these harmful copulations. How these iconic conflict behaviors coevolve in response to sexual conflict intensity can provide insight into the economics of these traits. We examined whether male harm and female resistance coevolved in response to elevated sexual conflict. We quantified copulation behavior and female reproductive tract damage of individuals from replicated populations evolving for 32 generations under low or high sexual conflict (female- and male-biased treatments, respectively). First, we permitted females ad libitum matings with males from either sex-ratio treatment, recording her tract damage and longevity. Second, we performed a full-factorial cross of matings by males and females from each of the replicate populations, recording mating and kicking duration and reproductive output. We found manipulation of sexual conflict intensity led to the evolution of male harmfulness, but not female resistance to harm. We also demonstrate that female kicking does not respond to sexual conflict intensity, suggesting it does not function to mitigate male harm in this species. Our findings demonstrate the complexities of behavioral and morphological coevolutionary responses to sexual conflict intensity in an important model species.
-
ItemSocially cued anticipatory adjustment of female signalling effort in a moth(The Royal Society Publishing, 2020-12-23)Juvenile population density has profound effects on subsequent adult development, morphology and reproductive investment. Yet, little is known about how the juvenile social environment affects adult investment into chemical sexual signalling. Male gumleaf skeletonizer moths, Uraba lugens, facultatively increase investment into antennae (pheromone receiving structures) when reared at low juvenile population densities, but whether there is comparable adjustment by females into pheromone investment is not known. We investigate how juvenile population density influences the ‘calling' (pheromone-releasing) behaviour of females and the attractiveness of their pheromones. Female U. lugens adjust their calling behaviour in response to socio-sexual cues: adult females reared in high juvenile population densities called earlier and for longer than those from low juvenile densities. Juvenile density also affected female pheromonal attractiveness: Y-maze olfactometer assays revealed that males prefer pheromones produced by females reared at high juvenile densities. This strategic investment in calling behaviour by females, based on juvenile cues that anticipate the future socio-sexual environment, likely reflects a response to avoid mating failure through competition with neighbouring signallers.
-
ItemCharacterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis(ELSEVIER, 2020-02-07)Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.
-
ItemConserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019-06-28)Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein.
-
ItemUsing decision support tools in emergency animal disease planning and response: Foot and mouth disease (CEBRA Project 1404D), Technical Report prepared for the Department of Agriculture and Water Resources(University of Melbourne, 2016)Modelling studies both in Australia and overseas have shown that vaccination can be very effective in reducing the size and duration of an FMD outbreak. Vaccination is most effective in reducing the duration and size of an outbreak when used early and is less effective the longer you delay. However, a decision to vaccinate early in the outbreak may result in using vaccination in situations where it is not actually required, with consequent implications for post-outbreak surveillance, the management of vaccinated animals and the ability to regain FMD-free status and access to markets. Overall, the choice of control measure to adopt in an FMD outbreak will thus depend on the variable and potentially conflicting objectives of the control program. As an important component of disease planning and preparedness for the department, the project will report on key information that could be used in an FMD outbreak to infer the potential scale of an outbreak and information to support disease management decision-making.
-
ItemIncorporating economic components in Australia's FMD modelling capability and evaluating post-outbreak management to support return to trade (CEBRA project 1608D), Technical Report for the Department of Agriculture, Water and Environment(University of Melbourne, 2017)Following an outbreak of FMD, surveillance will be required to demonstrate that infection has been eradicated from the population and enable any remaining movement restrictions to be lifted within the country. Proof of freedom will also be needed to satisfy trading partners and regain access to international markets. Although vaccination is increasingly being recognised as an important tool to assist in containing and eradicating FMD outbreaks, it will make achieving recognition of free status more difficult—keeping vaccinated animals in the population will delay the period until FMD-free status is regained under the World Organisation for Animal Health (OIE) guidelines and add additional complications to the postoutbreak surveillance program. There is no agreed approach to post-outbreak management of vaccinated animals in AUSVETPLAN with the options being to: (1) allow vaccinated animals to remain in the population to live out their normal commercial lives (vaccinate-to-live); (2) remove all vaccinated animals from the population (vaccinateand- remove). Under option 2, vaccinated animals could be subject to (a) slaughter to waste i.e. remove and dispose of vaccinated animals; or (b) slaughter and salvage i.e. attempt to sell either raw or processed product from vaccinated animals. For (b) there may be some residual value of products that could offset some of the costs. The project will bring together epidemiological and economic expertise from the Department, the Australian National University, and CEBRA to formally explore and establish a science-based and cost effective approach to regaining free-status after an FMD outbreak as expeditiously as possible. The project will expand the Department’s modelling capability as well as providing insights into postoutbreak FMD management and contribute to Australia’s FMD preparedness.