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

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...

A Esclerose Múltipla (EM) é uma doença autoimune caracterizada por um processo neuroinflamatório com degeneração axonal progressiva. O medicamento Natalizumab (Biogen Idec, NC, USA) representa hoje um dos tratamentos mais promissores para EM. Entretanto, pacientes sob esse tratamento possuem maiores chances de desenvolver Leucoencefalopatia Multifocal Progressiva (LEMP), em decorrência de uma possível reativação do poliomavírus JC (VJC). Além do VJC, o poliomavírus BK (VBK) pode representar uma preocupação adicional para tais pacientes, uma vez que também apresenta capacidade de causar encefalopatias. Apesar do Natalizumab ser uma ótima ferramenta contra a EM, o fato de interagir de alguma maneira com os poliomavírus, em especial o VJC, impede que seja utilizado em larga escala. Dessa forma,o objetivo desse trabalho foi investigar os padrões de excreção e reativação dos VJC e VBK em pacientes com EM durante o tratamento com Natalizumab e comparar aos padrões observados em pacientes que se encontram sob outros tratamentos. Amostras seriadas de sangue e urina foram coletadas e submetidas a testes de biologia molecular para detecção do vírus e caracterização molecular. Foram analisados 97 pacientes em diferentes tempos de acompanhamento. Não foi observada presença de poliomavírus no sangue de nenhum dos indivíduos analisados. Entretanto, 36% excretavam poliomavírus na urina em pelo menos uma das coletas, sendo que 21,7% excretavam VJC, 9,3% excretavam VBK e 5,1% excretavam ambos os poliomavírus. Não foi observada diferença entre as taxas de excreção urinária de poliomavírus entre pacientes que tratavam com Natalizumab (38,9%) e pacientes que sob outros tratamentos (34,5%), sendo que para o Grupo Controle (GC); 21,3%, 8,2% e 4,9% excretavam VJC, VBK e ambos os vírus, respectivamente e para o grupo Grupo Natalizumab (GN) 22,2%, 11,1% e 5,6% excretavam VJC, VBK e ambos os vírus, respectivamente. As análises moleculares da Região Regulatória do VJC revelaram sequências de característica arquetípica. A reconstrução filogenética de sequências do gene VP1 do VJC revelou predominância do genótipo 3 e do genótipo 1 para o VBK. Não foi observada diferença estatística da carga viral do VJC e do VBK entre os dois grupos. Foi detectada uma mutação (E29G) na VP1 de uma paciente que apresentou alta carga viral do VJC. Do grupo GN, 14 apresentaram anticorpos para VJC, sendo que desses 58% apresentou excreção de VJC, 42% não apresentou excreção urinária, interessantemente uma paciente não apresentou anticorpos contra o VJC, mas apresentou excreção de VJC. Pode-se concluir principalmente que a detecção de anticorpos, concomitantemente com a investigação molecular do VJC poderá contribuir para uma melhor determinação da estratificação do risco de desenvolvimento de LEMP em indivíduos com EM sob tratamento com Natalizumab. / Multiple sclerosis (MS) is an autoimmune disease characterized by neuronal inflamatory process with progressive axonal degeneration. The drug Natalizumab (Biogen Idec, NC, USA) is today one of the most promising treatments for MS. However, patients undergoing this treatment have higher chances of developing progressive multifocal leukoencephalopathy (PML), due to a possible reactivation of the polyomavirus JC (VJC). Besides VJC, the BK polyomavirus (VBK) may represent an additional concern for such patients, since it also has ability to cause encephalopathies. Despite Natalizumab be a great tool against MS, the fact that drug some way may interact with polyomavirus, especially VJC, prevents it from being used on a large scale. Thus, aim of this study was to investigate the patterns of excretion and reactivation of VJC and VBK in MS patients during treatment with Natalizumab and compare the patterns observed in patients who are under other treatments. Serial blood samples and urine were collected and submitted to molecular biology tests for virus detection and molecular characterization. Ninety seven patients were analyzed at different follow-up times. There was no polyomavirus DNA in the blood of none subjects analyzed. However, 36% of patients excreted polyomavirus in the urine in at least one of the samples, of those 21.7%, 9.3% and 5.1% excreted VJC, VBK and both polyomavirus, respectively. No difference was observed between the rates of urinary excretion of polyomavirus patients treated with Natalizumab (38.9%) and patients treated with other drugs (34.5%), for the Control Group (GC); 21,3%, 8,2% and 4,9 shed VJC, VBK and both viruses, respectevely and for the Natalizumab Group (GN) 22,2%, 11,1% and 5,6% shed VJC, VBK and both viruses, respectvely. Molecular analysis of the Regulatory Region of VJC revealed sequences similar to the archetype form of VJC. A phylogenetic reconstruction of the VP1 gene sequences revealed VJC predominance of genotype 3 and genotype 1 for VBK. There was no statistical difference in the viral load VJC and VBK between the two groups. It was detected a mutation (E29G) in VP1 of a patient who had a high viral load VJC, however the mutation disappeared after a few months of monitoring. Fourteen patients of GN had antibodies to VJC, and of these 58% had excretion VJC, 42% showed no urinary excretion, interestingly one patient had no antibodies against VJC but showed excretion of VJC. It can be concluded that mostly anti-VJC antibodies detection, concurrently with the VJC molecular research may contribute to a better determination of risk stratification for development of PML in patients with MS undergoing treatment with Natalizumab.