Decolonizing Environmental Education: A Resource Guide for Non-Indigenous Educators

The field of environmental education is shaped by pervasive narratives and ideologies of settler colonialism. Many scholars cite the need for Indigenous voices and perspectives to be centered in education in order to move towards a process of decolonization, reconciliation, and support for ongoing Indigenous resurgence. In order for non-Indigenous educators to be able to guide students towards critical understanding of the history and context of land in environmental education, they must first investigate their own identity and assumptions within settler colonial society and education. This capstone project provides a resource guide in the form of a website for non-Indigenous environmental educators seeking to critically examine their own role in settler colonial systems and engage in co-resistance and Indigenous resurgence by centering Indigenous voices in their teaching. Using transformative learning and social justice education frameworks, the resource guide includes self-reflective practices and resources for critical analysis to decolonize curriculum and center Indigenous perspectives, as well as teaching resources exclusively created by Indigenous authors for use with students in the classroom or field. Intended for use by both formal and informal environmental educators, the resource guide is publically accessible and self-paced. Annotated resources allow readers to choose the most appropriate tools to further investigate internal biases and develop a personal path towards self-decolonization in order to facilitate similar critical and transformative learning experiences for students. (224 words

Kinetics and specificity of human mitochondrial DNA polymerase gamma and HIV-1 reverse transcriptase

textThe human mitochondrial DNA (mtDNA) genome must be faithfully maintained by the mitochondrial DNA replication machinery. Deficiencies in mtDNA maintenance result in the accumulation of mutations and deletions, which have been associated with a number of neuromuscular degenerative disorders including, mtDNA depletion syndrome, Alpers syndrome, progressive external opthalmoplegia (PEO), and sensory ataxic neuropathy, dysarthria, and opthalmoparesis (SANDO). The mtDNA replication machinery is comprised of a nuclearly-encoded DNA polymerase gamma (Pol γ), single-stranded DNA binding protein (mtSSB), and a hexameric mtDNA helicase. In this work, we employed quantitative pre-steady state kinetic techniques to establish the mechanisms responsible for the replication of the human mitochondrial DNA by Pol γ and explored the effects of point mutations that are observed in heritable diseases. With our biochemical characterization of mutants of Pol γ, we have shown unique characteristics that would lead to profound physiological consequences over time. Additionally, we have made significant progress towards reconstitution of the mitochondrial DNA replisome by monitoring DNA polymerization that is dependent on helicase unwinding of double stranded DNA. Overall, this work provides a better understanding of the mechanism of mtDNA replication and has important implications toward understanding the role of mitochondrial DNA replication in mitochondrial disease, ageing and cancer. In addition to the work on the mtDNA replisome, we have applied pre-steady state kinetic techniques to better understand the mechanism of RNA-dependent DNA polymerization by HIV reverse transcriptase (HIV-RT). This enzyme is responsible for the replication of the viral genome in HIV and is a common target for anti-HIV drugs. We have characterized the role of enzyme conformational changes in the kinetics of incorporation of correct nucleotide and the Nucleotide Reverse Transcriptase Inhibitor (NRTI) AZT by wild-type enzyme, as well as a mutant with clinical resistance to AZT. This work provides a better understanding of the complete mechanism of RNA-dependent DNA polymerization, the changes in the mechanism in the presence of inhibitor and the development of resistance to this nucleoside analog; and thereby this work contributes to the long-term goal of designing more effective drugs that can possibly deter resistance and be used successfully for treatment of HIV.Cellular and Molecular Biolog

Eesti noorte rahvusvaheline vabatahtlik teenistus laste ja noortega

http://www.ester.ee/record=b4427638~S1*es

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

ABSTRACT Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity. IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease

KarS § 217² ehk usalduse kuritarvitamise ning § 201 lg 1 alt 2 ehk võõra vara omastamise koosseisude vaheteo analüüs

http://www.ester.ee/record=b5143085*es

TRANSLATION REGULATION DURING HCMV INFECTION

During viral infection competition for resources is inevitable. One of the fiercest areas of competition is mRNA translation. Host ribosomes are utilized by both host and viral mRNAs, since no virus encodes its own ribosome. Human Cytomegalovirus (HCMV) is a ubiquitous human pathogen that causes significant disease in newborns and immunocompromised individuals. While most viruses suppress host mRNA translation to limit competition for ribosomes, HCMV stimulates host mRNA translation. The goal of this dissertation is to investigate the mechanisms that allow HCMV mRNAs to efficiently compete for access to host ribosomes. mRNA translation begins with translation initiation, which is the rate limiting step of mRNA translation. To determine how viral mRNAs undergo translation initiation we investigated whether the host eIF4F translation initiation complex controlled HCMV mRNA translation. We found that despite eIF4F disruption that suppressed host mRNA translation, HCMV mRNAs continue to translated in the absence of eIF4F. To identify factors that may promote HCMV mRNA translation independent of eIF4F we performed a mass spectrometry screen for mRNA m7G cap binding proteins in HCMV infected cells. Two viral proteins were identified in our screen, pTRS1 and pIRS1. In addition to binding mRNA during infection, pTRS1 associates with the host translation machinery. We also demonstrated that pTRS1 is sufficient to preferentially stimulate translation of luciferase reporters containing viral sequences outside the context of infection. pTRS1 is a known inhibitor of the host antiviral kinase PKR. However, pTRS1 was able to stimulate translation in cells lacking PKR, indicating that pTRS1 has functions other than antagonizing PKR. Finally, we evaluated HCMV replication in the absence of pTRS1 or its redundant homolog pIRS1. Using a novel infection model we confirmed that either pTRS1 or pIRS1 is necessary to antagonize PKR for efficient HCMV replication. We also show that pTRS1 is necessary and sufficient to prevent stress granule formation. In fact pTRS1 can also prevent stress granule formation as a result of HRI activation suggesting that pTRS1 may antagonize HRI in addition to PKR. Using our novel infection system we also demonstrate the necessity of the PKR binding domain of pTRS1 for efficient HCMV replication.Doctor of Philosoph

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics

An unbiased proteomics approach to identify human cytomegalovirus RNA-associated proteins

AbstractPost-transcriptional events regulate herpesvirus gene expression, yet few herpesvirus RNA-binding proteins have been identified. We used an unbiased approach coupling oligo(dT) affinity capture with proteomics to identify viral RNA-associated proteins during infection. Using this approach, we identified and confirmed changes in the abundance or activity of two host RNA-associated proteins, DHX9 and DDX3, in cells infected with human cytomegalovirus (HCMV). We also identified and confirmed previously unreported activities for the HCMV US22 and pp71 proteins as RNA-associated viral proteins and confirmed that a known viral RNA-binding protein, pTRS1, associates with RNA in infected cells. Further, we found that HCMV pp71 co-sedimented with polysomes, associated with host and viral RNAs, and stimulated the overall rate of protein synthesis. These results demonstrate that oligo(dT) affinity capture coupled with proteomics provides a rapid and straightforward means to identify RNA-associated viral proteins during infection that may participate in the post-transcriptional control of gene expression

Internetbasierte Reaktionen auf Firmenaktivitäten : ein Beitrag zur Entwicklung innovativer Dienstleistungen

Gegenstand der vorliegenden Arbeit ist die Untersuchung einzelner Produkte und Tools zur Beobachtung und Analyse von Internet-Publikationen und die Beschreibung des theoretischen Hintergrunds solcher Dienstleistungen. Zu Anfang wird ein allgemeiner Überblick über das Medium Internet mit seinen zahlreichen Diensten und seine wachsende Verbreitung gegeben. Aufgrund dieser zunehmenden Bedeutung wird es für Unternehmen immer wichtiger, neben Botschaften in klassischen Medien, wie Print- Medien, TV- und Hörfunkkanälen, auch Botschaften in Online-Medien wahrzunehmen und auszuwerten. Der erste Schritt ist die Recherche geeigneter Publikationen im Internet. Hierfür gibt es zahlreiche Instrumente, von denen allerdings nicht alle für zielgenaue Recherchen geeignet sind. Nach der Recherche folgt die Medienresonanzanalyse, die insbesondere für Internet-Publikationen einige Besonderheiten aufweist. Der praktische Teil enthält eine Übersicht ausgewählter Firmen und Produkte, die bereits entsprechende Tools anbieten. Sowohl allgemeine Firmendaten als auch Informationen zu den Produkten der Unternehmen werden beschrieben und bewertet. Eine Checkliste zur Gestaltung eines „idealen“ Dienstes fasst die gesammelten Informationen noch einmal zusammen

Human Cytomegalovirus pTRS1 and pIRS1 Antagonize Protein Kinase R To Facilitate Virus Replication

ABSTRACT Human cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication. IMPORTANCE To successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease