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.

Future communication systems require low latency Fast Fourier transform (FFT)computation with a small cost of area. In this study, a memory-based FFT processorwith low latency is designed. To reduce latency and maintain constant outputsample rate, a scheduling method suitable for input sample rate and clock rateis used in the radix-2 butterfly processing elements. The scheduling scheme employsa combination of ASAP and ALAP scheduling strategies. A mathematicalexpression that models FFT’s latency is given. The size of FFT, the input samplerate, and the number of processing elements are the input parameters of the expression.The effect of using pipelined processing element is also studied. Lastly,the proposed low latency design is compared with other low-latency FFT designs.The result shows that, in the 4G LTE application scenario, our memory-based designcan do the FFT computations faster with a small area.

Equine herpesvirus type 1 (EHV-1) is a common pathogen of horses that typically causes upper respiratory disease, however is also associated with late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can recrudesce during times of stress, which can lead to the initiation of devastating outbreaks. Some variants of EHV-1 have been associated with more severe disease outcomes. Appropriate outbreak management focuses on minimizing the movement of potentially exposed horses. This approach lacks a strategy for prevention at the level of latency largely due to a knowledge paucity in regards to carriage rate of latent EHV-1. Biosecurity decisions are also dependent on awaiting currently-available diagnostic testing that often take several days for results. Thus, our work has been focused on understanding the carriage rate of the latent virus in different geographic regions as well as improving diagnostic efficiency, both of which are essential for improving the management of EHV-1 disease. Loop mediated isothermal amplification (LAMP) is a method that amplifies nucleic acid rapidly at a constant temperature and is minimally affected by inhibitors that are often found in clinical samples. This procedure can be followed by multiple detection methods. A new, efficient sequencing method, called nanopore sequencing, has been developed in a handheld device, called MinION, that provides thorough output in a timely manner. When combined with LAMP, it has been referred to as LAMPore. The first objective of our work was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes from horses in Virginia. Our second objective was to perform direct DNA sequencing of EHV-1 using the mobile MinION sequencer in combination with LAMP viral enrichment. Our findings demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia as well as successful detection and identification of EHV-1 in equine nasal swab samples using LAMPore sequencing. / Doctor of Philosophy / Horses can develop disease from a virus called equine herpesvirus type 1 (EHV-1). Symptoms can vary from mild respiratory signs to the inability to rise leading to death or euthanasia. Horses transmit this virus to other nearby horses; however, the virus also becomes dormant once a horse is infected, meaning the virus is not infectious but is present within the animal. When the horse undergoes stress, such as during travel or competition, the virus can become active again, leading to the spread to other horses. This results in outbreaks, many of which are devastating to the equine industry. In order to minimize the risks of this virus spreading and causing disease, management is currently focused on minimizing the movement of horses that may have been exposed to the virus. There is little information regarding the number of horses that harbor the dormant virus and the current methods to detect the infectious virus can take multiple days for results. These limit decision-making during the management of an outbreak. Our work seeks to determine the number of horses in a region that harbor EHV-1 and also to test a new, efficient diagnostic method to identify the virus in samples from horses. Our findings showed a low number of horses in Virginia harbor dormant EHV-1 in the lymph nodes under their mandible, a common site of dormancy. Further, we found that our new method of detection was effective in identifying the virus in samples from nasal secretions from horse.

5G is an enabler to several new use cases. To support all of them, the network infrastructure must be flexible and it should adapt to the different situations. This feature is powered by SDN, NFV, and Automation, three of the main pillars on which the 5G network is built.Traditional network management approaches may not be suitable for the 5G Core Network User Plane, which holds strict requirements in terms of latency and throughput. Therefore, Artificial Intelligence agents have been proposed to manage the 5G in a more efficient manner, delivering a more optimized allocation of the resources. This approach requires real-time monitoring of the data passing by the Core Network, a feature not standardized by the current protocols. In this thesis, the design of a monitoring protocol for the 5G Core Network User Plane has been studied, focusing on precise measurement of latencies. Then, a In-band Network Telemetry (INT) framework has been implemented on top of a User Plane Function prototype. The prototype is built on top of a novel User Plane implementation, based on chaining of atomic functions called micro-UPFs (µUPFs).While the main focus of this work has been latency measurement, packet counters, byte counters and Inter Packet Gap values can be collected from the framework, proving the main KPIs of a 5G User Plane. The INT framework has been implemented through two new µUPFs, one for updating the INT metadata and one for collecting them. These metadata are attached to the user packets as GTP-U extended header, maintaining compatibility with the standard protocol. Moreover, the implemented framework allows high flexibility through dynamic tuning of the parameters, providing mechanisms to reduce the amount of telemetry data generated and, thus, the system overhead.The framework has been tested on a physical setup of four server machines, abstracting a Core Network User Plane, connected with 10 Gbps NICs. In all the tests performed, the performances of the User Plane are affected by the new functionalities only when INT metadata are inserted very frequently. The results show that is possible to monitor the three main KPIs of a 5G User Plane without heavily limiting the system performances. / 5G är en möjliggörare för flera nya användningsfall: för att stödja dem alla måste nätverksinfrastrukturen vara flexibel och den ska anpassa sig till de olika situationerna. Denna funktion drivs av SDN, NFV och Automation, tre av de viktigaste pelarna som 5G-nätverket är byggt på.Traditionella nätverkshanteringsstrategier kanske inte passar för 5G Core Network, som har strikta krav när det gäller latens och genomströmning. Därför har Artificial Intelligence-agenter föreslagits att hantera 5G på ett mer effektivt sätt, vilket ger en mer optimerad fördelning av resurserna. Detta tillvägagångssätt kräver realtidsövervakning av data som passerar via Core Network, en funktion som inte standardiseras med de aktuella protokollen.I denna avhandling har utformningen av ett övervakningsprotokoll för 5G Core Network User Plane studerats med fokus på exakt mätning av latenser. Sedan har ett in-band Network Telemetry (INT) -ramverk implementerats ovanpå en prototyp för User Plane Function. Denna prototyp utnyttjade Chain Controllerarkitekturen, en ny användarplan-implementering baserad på kedjan av atomfunktioner som kallas µUPF.Medan huvudfokuset för detta arbete har varit latensmätning, kan paketräknare, byttäknare och Inter Packet Gap-värden samlas in från ramverket, vilket bevisar de viktigaste KPI: erna i ett 5G-nätverk. INT-ramverket har implementerats genom två nya µUPF, en för att uppdatera INT-metadata och en för att samla dem. Dessa metadata är anslutna till användarpaketen som GTP-U utökad rubrik, bibehållande kompatibilitet med standardprotokollet. Dessutom tillåter det implementerade ramverket hög flexibilitet som tillåter dynamisk inställning av parametrarna, tillhandahåller mekanismer för att minska mängden telemetri-data som genereras och därmed systemomkostnaderna.Ramverket har testats på en fysisk installation av fyra servermaskiner som abstraherar ett Core Network User Plane, anslutet med 10 Gbps NIC. I samtliga tester påverkas testbäddens prestationer av de nya funktionerna först när INT-metadata sätts in mycket ofta. Resultaten visar att det är möjligt att övervaka de tre huvudsakliga KPI: erna i ett 5G-nätverk utan att starkt begränsa systemprestanda.

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