A Room Full of Pigeons and Three Spectators

My work explores themes of identity politics, domesticity, land and memory access - in relation to my own lived experiences as a Palestinian refugee, a daughter of a missing war casualty and an artist. In this thesis book, I present a cumulation of thoughts, emotions and findings, along with a selection of works from the last two years. During my time at the Rhode Island School of Design, messenger pigeons took over my studio, channeling physical and mental realms of grief and remembrance. By looking closely at the bird’s aesthetics and behaviorisms in my first semester, their resting deceased bodies in the second and third, I developed a better understanding of my relationship to mark-making, displacement and trauma. In my final thesis and closing of a life chapter, these birds and all the meaning they carry, land and disappear into a deep dark immersive environment; a water body that drowns yet grounds me firmly in my continual search of inaccessible connections

A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

BACKGROUND: The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. RESULTS: Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. CONCLUSIONS: Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags

Specific binding of a hexanucleotide to HIV-1 reverse transcriptase: a novel class of bioactive molecules

Short oligonucleotides below 8–10 nt in length adopt relatively simple structures. Accordingly, they represent interesting and so far unexplored lead compounds as molecular tools and, potentially, for drug development as a rational improvement of efficacy seem to be less complex than for other classes of longer oligomeric nucleic acid. As a ‘proof of concept’, we describe the highly specific binding of the hexanucleotide UCGUGU (Hex-S3) to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) as a model target. Ultraviolet (UV) cross-linking studies and competition experiments with primer/template substrates and a RT-directed aptamer suggest site-specific binding of Hex-S3 to the large subunit (p66) of the viral enzyme. The affinity of 5.3 μM is related to hexanucleotide-specific suppression of HIV-1 replication in human cells by up to three orders of magnitude indicating that Hex-S3 exerts specific and biologically relevant activity. Experimental evidence described here further suggests a systematic hexamer array-based search for new tools for molecular biology and novel lead compounds in nucleic acid-based drug development

The cellular source for APOBEC3G's incorporation into HIV-1

<p>Abstract</p> <p>Background</p> <p>Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear.</p> <p>Results</p> <p>Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.</p> <p>Conclusions</p> <p>Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.</p

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells

Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain

It is now well accepted that the structural protein Pr55Gag is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55Gag is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55Gag sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55Gag-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development

Generation and characterization of a defective HIV-1 Virus as an immunogen for a therapeutic vaccine

BACKGROUND: The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. RESULTS: Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. CONCLUSIONS: We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles.This study was supported by grants FIS PI050265, FIS PI040503, FIS PI070291, FIS Intrasalud 080752, FIS PS09/01297, FIS PI10/02984, SAF2006-26667-E, FIT 09-010-205-9, FIPSE 36780/08, Fundación Mútua Madrileña, TRA-094, EC10-153, ISCIII-RETIC RD06/0006, HIVACAT–HIV Development Program in Catalonia, FIPSE 36630/07, UE Program Health 2009 CHAARM. Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes

Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the in vitro impact of established inhibitors of mitochondrial respiratory chain complex IV or cytochrome c oxidase on oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes as well as on differentiation capacity of OPCs from P0 rat. Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders. © 2010 Wiley-Liss, Inc

The role of Pr160gag-pol in HIV-1 assembly /

The ultimate goal of retroviral infection is to transfer the RNA genome from one cell to another. This requires that the viral genome be assembled within the structure of an infectious particle. The Gag precursor in HIV-1 is alone sufficient to produce viral particles. In order to obtain interactions required for assembly, the Gag molecules must first be concentrated at a cellular site. Membrane and RNA have been proposed to act as intracellular scaffolds for aligning Gag molecules and facilitating their interaction with each other. In vitro studies using truncated Gag molecules have indicated that RNA is important in facilitating a membrane-independent interaction between Gag molecules, and that this RNA need not be viral. During HIV-1 assembly, Gag-Pol is also incorporated into the virus. Gag-Pol has also been shown also responsible for the selective packaging of tRNALys3, the primer for reverse transcriptase. The factors facilitating the interaction between Gag and Gag-Pol, as well as the role of Gag-Po1 in the assembly process have been less studied. It has been assumed, however, that these molecules interact with each other through similar sequences involved in Gag/Gag interactions. Evidence for this includes the fact that unmyristylated Gag or Gag-Pol molecules can be rescued into assembly complexes by myristylated Gag.In this work, we used COS7 cells transfected with wild type and mutant HIV-1 proviral DNA to investigate the role of Gag-Pol in viral assembly. Experiments conducted on lysates of transfected cells revealed an RNA requirement for Gag/Gag-Pol interaction. Analysis of the effect of mutations within the nucleocapsid region in either Gag or Gag-Pol precursors indicated that RNA-facilitated Gag polymerization is required for Gag/Gag-Pol interaction, and not a direct association of Gag-Pol with RNA. We further examined the selective packaging of tRNALys3 into viral particles during viral assembly. The effects of various mutations in Pr160gag-pol upon the incorporation of tRNALys were studied. We determined that a 54 amino acid sequence in the thumb region of RT within Gag-Pol is required for tRNA Lys incorporation into the virus, and may contribute to the tRNA Lys binding site in Pr160gag-pol. These results demonstrate an important role for Gag-Pol in the assembly process