Of the 12 million people who inject drugs worldwide, 13% are chronically infected with Human Immunodeficiency Virus (HIV), i.e., they live with HIV. Chronic opioid use affects the host immune system and increases an individual’s susceptibility to HIV infection. However, it is unclear how opioid use changes the course of HIV pathogenesis. Particularly, there is a gap in understanding how opioids impact HIV latency. Latency results in a reservoir of infected quiescent cells that evade antiviral immune responses, are not targeted by antiretroviral therapy (ART), and allow HIV viremia to rebound upon treatment interruption. While in vitro studies show that opioids modulate the activity of transcription factors involved in T-cell activation and HIV transcription, few studies have investigated whether opioid use impacts HIV latency in vivo in HIV-infected people. In this research, peripheral blood mononuclear cells (PBMCs) were utilized from People with HIV (PWH) with or without recent opioid use or opioid use disorder (OUD) who were enrolled in the Linking Infection and Narcology Care-Part II (LINC-II) and Studying Partial Agonists for Ethanol and Tobacco Elimination in Russians with HIV (St PETER HIV ARCH) studies conducted in St. Petersburg, Russia. Intact proviral DNA digital droplet PCR (ddPCR) assays were performed on PBMCs from antiretroviral treated PWH, with (n=8) or without (n=11) current OUD, to quantify intact and defective proviral genomes. Samples from ART-treated PWH with OUD compared to those without OUD had similar levels of intact and defective proviruses. To evaluate latency reversal, PBMCs from ART-treated PWH with or without OUD, were activated with anti-CD3/28 beads and RT-ddPCR assays were performed to measure HIV LTR-gag RNA. A variable response in PWH without OUD was seen where half of the samples showed an increase in HIV RNA upon activation. Interestingly, only 1 of 8 samples from PWH with OUD showed an increase in HIV transcription. However, no suppression of HIV reactivation was found in vitro from latent cells generated using a primary CD4+ T-cell latency model in the presence or absence of morphine. Similarly, no differences in HIV integration and transcription in vitro were observed between morphine and control conditions. Additionally, expression of opioid receptors was not detected in primary PBMCs, CD4 T cells, or macrophages. These results show that PWH with OUD have a pool of persistent HIV proviruses that are refractive to reactivation, although opioids did not affect HIV replication and latency reactivation in vitro. The discrepancy in these in vitro and in vivo results and the lack of expression of opioid receptors in immune cells suggests that while opioids do not directly impact HIV replication, latency, and reactivation in target CD4+ cells, opioids could indirectly shape the HIV reservoir in vivo by modulating general immune functions, neuroderived factors or other cells that are responsive to opioids. Eradication of the latent HIV reservoir is necessary to achieve a cure for HIV/AIDS. One approach for latency eradication is the “shock and kill” approach that entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. An alternative approach is to permanently repress HIV expression precluding viral rebound after ART discontinuation. Here, a nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB) was used to epigenetically silence the proviral DNA. I show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, which are histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies. The research presented here provides evidence of opioid-mediated modulation of HIV-1 latency reactivation in PWH with opioid dependency. Additionally, we show that HIV-1 reactivation can be suppressed by epigenetic remodeling of the HIV-1 promoter using a repurposed CRISPR/Cas9 system.

Host cell reactivation (HCR) of viruses has been used in the past to assess the DNA repair capacities of various mammalian cell types. In this study, a PCR-based HCR technique was developed for determining DNA repair capacity of mammalian cells. Many DNA lesions, including UV photoproducts, block DNA amplification by Taq polymerase, and the exponential nature of PCR imparts a tremendous potential for quantifying the remaining non-adducted DNA templates from small samples. Ad5HCMVspl ZacZ is a recombinant nonreplicating adenovirus (Ad) containing the lacZ reporter gene under the control of the human cytomegalovirus (HCMV) immediate early promoter inserted into the deleted El region of the viral genome. This virus is unable to replicate, but it can efficiently express the reporter gene in many types of mammalian cells, including human fibroblasts. Using quantitative PCR, the induction and repair of UV photoproducts was measured in a 2.6 kb region of the lacZ reporter gene inserted into the deleted El region of Ad5HCMVspllacZ and in a 2.8 kb region of the endogenous E4 region of the virus. Primers flanking the regions were added to equal amounts of DNA extracted from cells infected with unirradiated or UV-irradiated Ad5HCMVspl lacZ and each sample was amplified by PCR using radiolabelled nucleotides as substrates. PCR products were separated by agarose gel electrophoresis and quantified using a phosphorimaging system. Results show a simple exponential decrease in PCR product with increasing UV fluence to the virus. There was a significant removal of UV photoproducts by 24 hours after infection of normal human fibroblasts. A reduced capacity for lesion removal was detected after infection of nucleotide excision repair deficient fibroblasts derived from patients with xeroderma pigmentosum (XP) and Cockayne syndrome (CS). Previous work from our lab using a P-gal reporter gene assay has shown that both UV light and heat shock treatment of cells prior to infection with UV-damaged Ad5HCMVspl lacZ enhances HCR. Application of the quantitative PCR technique to the study of inducible repair shows there is an enhancement in the rate of lesion removal from both regions of the vector in UV-irradiated normal lung fibroblast cells, compared to unirradiated cells. This demonstrates that previous reports of enhanced host cell reactivation are indicative of a genuine enhancement of DNA repair. Also, the P-gal reporter gene assay was used to investigate inducibility of UV lesion repair by ionising radiation; no significant increase in HCR of p-gal activity was found in cells treated with y-rays compared to untreated cells. / Thesis / Master of Science (MS)

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Why are some memories easy to retrieve, while others are more difficult to access? Here, we tested whether we could bias memory replay, a process whereby newly learned information is reinforced by reinstating the neuronal patterns of activation that were present during learning, towards particular memory traces. The goal of this biasing is to strengthen some memory traces, making them more easily retrieved. To test this, participants were scanned during interleaved periods of encoding and rest. Throughout the encoding runs, participants learned triplets of images that were paired with semantically related sound cues. During two of the three rest periods, novel, irrelevant sounds were played. During one critical rest period, however, the sound cues learned in the preceding encoding period were played in an effort to preferentially increase reactivation of the associated visual images, a manipulation known as targeted memory reactivation. Representational similarity analyses were used to compare multi-voxel patterns of hippocampal activation across encoding and rest periods. Our index of reactivation was selectively enhanced for memory traces that were targeted for preferential reactivation during offline rest, both compared to information that was not targeted for preferential reactivation and compared to a baseline rest period. Importantly, this neural effect of targeted reactivation was related to the difference in delayed order memory for information that was cued versus uncued, suggesting that preferential replay may be a mechanism by which specific memory traces can be selectively strengthened for enhanced subsequent memory retrieval. We also found partial evidence of discrimination of unique temporal sequences within the hippocampus. Over time, multi-voxel patterns associated with a given triplet sequence became more dissimilar to the patterns associated with the other sequences. Furthermore, this neural marker of sequence preservation was correlated with the difference in delayed order memory for cued versus uncued triplets, signifying that the ability to reactivate particular temporal sequences within the hippocampus may be related to enhanced temporal order memory for the cued information. Taken together, these findings support the claim that awake replay can be biased towards preferential reactivation of particular memory traces and also suggest that this preferential reactivation, as well as representations of reactivated temporal sequences, can be detected within patterns of hippocampal activation. / Psychology

In the bustling heart of Chicago's LaSalle Street, the 111 West Monroe site presents a unique narrative of adaptive reuse, encapsulating the thesis of 'Multiplicity of Experiences.' This thesis ventures beyond the conventional restoration, aspiring to reinvigorate and repurpose two distinct architectural forms — a robust brick structure and its transparent steel and glass counterpart — through strategic cuts that foster gathering and community interaction. The manipulation of light at varying intensities becomes the silent choreographer of space, delineating zones of activity and orchestrating the human experience within these revitalized edifices. The old brick building, a testament to historical solidity, now breathes anew as student housing, offering challenges turned opportunities through its dense facade and muted interiors. In contrast, the steel and glass structure, with its morning light and open vistas, evolves into a sanctuary for families, children, and the elderly, promoting a sense of openness and interconnectivity. This architectural duality harmonizes to form a living, dynamic tableau that captures the multiplicity of human experiences, enabled by horizontal and vertical incisions that carve out communal and private spheres. The groundwork of the thesis is the concept of gathering — initiating with the public's allure into the retail and amenity spaces on the lower levels, ascending to the private residences above, and culminating in the shared terraces that bridge different age groups and backgrounds. This thesis demonstrates how spatial intervention, particularly through the nuanced application of light and the deliberate choreography of openings, can script diverse experiences from serendipitous encounters to orchestrated communal activities. It presents a model of adaptive reuse that not only conserves the structural essence but also injects a new pulse into the urban fabric, championing a symbiosis between the old and the new, the individual and the collective, the built and the unbuilt. Through the transformative act of cutting and layering, this study sheds light on the potency of architectural intervention in shaping human and spatial experiences. The result is a rich mosaic of life, where every stratum, every incision, every ray of light adds a verse to the narrative of gathering, from dawn's first light to the tranquil hum of evening, offering a blueprint for reactivating spaces that reach beyond their walls to touch the human soul. / Master of Architecture / This thesis explores adaptive reuse at 111 West Monroe, using architectural cuts to manipulate light and space, thereby creating a multiplicity of experiences that foster community interaction and rejuvenate urban life across diverse generations.

The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...

The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...