Phylogeography of southern New Zealand: Implications of Pliocene and Pleistocene Processes and Evolutionary Concordance Across Independent Loci

The endemic fauna of the South Island has proven to be an ideal taxonomic group to examine the impact of climatic and geological processes on the evolution of New Zealand's biota since the Pliocene. This thesis examines the phylogeography of McCann's skink (Oligosoma maccanni) in order to provide insight into the relative contribution of Pliocene and Pleistocene processes on patterns of genetic structure in South Island biota. This thesis also investigates the phylogeography of the brown skink (O. zelandicum) to examine whether Cook Strait landbridges facilitated gene flow between the North and South Island in the late-Pleistocene. This thesis also investigates the presence of genealogical concordance across independent loci for the endemic alpine stick insect, Niveaphasma. I obtained mitochondrial DNA (mtDNA) sequence data (ND2 and ND4; 1284 bp) from across the range of both skink species and mtDNA (COI; 762 bp) and nuclear sequence data (EF1 ; 590 bp) from across the range of Niveaphasma. I used DGGE in order to resolve nuclear EF1 alleles and examined phylogeographic patterns in each species using Neighbour-Joining, Maximum Likelihood and Bayesian methods. Substantial phylogeographic structure was found within O. maccanni, with divergences among clades estimated to have occurred during the Pliocene. Populations in the Otago/Southland region formed a well-supported lineage within O. maccanni. A genetic break was evident between populations in east and west Otago, while north-south genetic breaks were evident within the Canterbury region. There was relatively minor phylogeographic structure within O. zelandicum. Our genetic data supports a single colonization of the North Island by O. zelandicum from the South Island, with the estimated timing of this event (0.46 Mya) consistent with the initial formation of Cook Strait. There was substantial genetic structuring identified within Niveaphasma, with a well-supported lineage present in the Otago/Southland region. There was also a genetic break between populations in Canterbury and eastern Otago with those in central Otago and Southland. The genetic data provided strong genealogical concordance between mtDNA haplotypes and nuclear alleles suggesting an accurate depiction of the historical isolation identified between the major clades of Niveaphasma. This finding offers compelling evidence for the use of nuclear gene phylogeography alongside mtDNA for future evolutionary studies within New Zealand

The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation

The DNA damage-responsive protein kinases ATM and ATR phosphorylate SQ/TQ motifs that lie in clusters in most of their in vivo targets. Budding yeast Cdc13p contains two clusters of SQ/TQ motifs, suggesting that it might be a target of Mec1p/Tel1p (yeast ATR/ATM). Here we demonstrated that the telomerase recruitment domain of Cdc13p is phosphorylated by Mec1p and Tel1p. Gel analysis showed that Cdc13p contains a Mec1/Tel1-dependent post-translational modification. Using an immunoprecipitate (IP)-kinase assay, we showed that Mec1p phosphorylates Cdc13p on serine 225, 249, 255 and 306, and Tel1p phosphorylates Cdc13p on serine 225, 249 and 255 in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13p SQ motifs phosphorylated by Mec1p and Tel1p caused multiple telomere and growth defects. In addition, normal telomere length and growth could be restored by expressing a Cdc13–Est1p hybrid protein. These results demonstrate the telomerase recruitment domain of Cdc13p as an important new telomere-specific target of Mec1p/Tel1p

Inhibition of the inositol kinase Itpkb augments calcium signaling in lymphocytes and reveals a novel strategy to treat autoimmune disease

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease

Targeting Cx43 and N-Cadherin, Which Are Abnormally Upregulated in Venous Leg Ulcers, Influences Migration, Adhesion and Activation of Rho GTPases

Venous leg ulcers can be very hard to heal and represent a significant medical need with no effective therapeutic treatment currently available

Numt-Mediated Double-Strand Break Repair Mitigates Deletions during Primate Genome Evolution

Non-homologous end joining (NHEJ) is the major mechanism of double-strand break repair (DSBR) in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs). Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler DNA, prevents nuclear processing of DSBs that could result in deleterious repair

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Phylogeography of southern New Zealand: Implications of Pliocene and Pleistocene Processes and Evolutionary Concordance Across Independent Loci

The endemic fauna of the South Island has proven to be an ideal taxonomic group to examine the impact of climatic and geological processes on the evolution of New Zealand's biota since the Pliocene. This thesis examines the phylogeography of McCann's skink (Oligosoma maccanni) in order to provide insight into the relative contribution of Pliocene and Pleistocene processes on patterns of genetic structure in South Island biota. This thesis also investigates the phylogeography of the brown skink (O. zelandicum) to examine whether Cook Strait landbridges facilitated gene flow between the North and South Island in the late-Pleistocene. This thesis also investigates the presence of genealogical concordance across independent loci for the endemic alpine stick insect, Niveaphasma. I obtained mitochondrial DNA (mtDNA) sequence data (ND2 and ND4; 1284 bp) from across the range of both skink species and mtDNA (COI; 762 bp) and nuclear sequence data (EF1 ; 590 bp) from across the range of Niveaphasma. I used DGGE in order to resolve nuclear EF1 alleles and examined phylogeographic patterns in each species using Neighbour-Joining, Maximum Likelihood and Bayesian methods. Substantial phylogeographic structure was found within O. maccanni, with divergences among clades estimated to have occurred during the Pliocene. Populations in the Otago/Southland region formed a well-supported lineage within O. maccanni. A genetic break was evident between populations in east and west Otago, while north-south genetic breaks were evident within the Canterbury region. There was relatively minor phylogeographic structure within O. zelandicum. Our genetic data supports a single colonization of the North Island by O. zelandicum from the South Island, with the estimated timing of this event (0.46 Mya) consistent with the initial formation of Cook Strait. There was substantial genetic structuring identified within Niveaphasma, with a well-supported lineage present in the Otago/Southland region. There was also a genetic break between populations in Canterbury and eastern Otago with those in central Otago and Southland. The genetic data provided strong genealogical concordance between mtDNA haplotypes and nuclear alleles suggesting an accurate depiction of the historical isolation identified between the major clades of Niveaphasma. This finding offers compelling evidence for the use of nuclear gene phylogeography alongside mtDNA for future evolutionary studies within New Zealand