Elucidating the role of effector caspases in immune development using lentiviral RNAi

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Includes bibliographical references.Caspases play an important role in apoptosis, or programmed cell death. In particular, three highly related effector caspases, caspases-3, -6, and -7, translate upstream death signals into the physical manifestations of apoptosis by proteolytically cleaving structural and enzymatic targets. However, it is not clear what specific role each individual caspase plays in apoptosis or whether interaction between them is important. We used RNA interference (RNAi) to examine their roles in immune cells. RNAi has revolutionized the field of mammalian genetics by expediting the interrogation of gene function. Endogenous genes are targeted for silencing by the introduction of double stranded RNAs, known as short interfering RNAs (siRNAs), through a mechanism that is well conserved across many species. While this technique has been used successfully in tissue culture experiments, our studies focused on extending the use of RNAi into immune cells. Our initial experiments demonstrated that primary T cells were capable of RNAi-based gene silencing, but were difficult to introduce siRNAs into. Therefore, more robust techniques for the stable and efficient introduction of siRNAs into primary immune cells and animal models were required.(cont.) Viral vectors, which can infect a wide variety of cell types and drive consistent transgene expression, provide a potential delivery vehicle for short hairpin RNAs (shRNAs), an alternative form of double stranded RNA produced within the target cell. Thus, we designed a lentiviral vector system for delivering shRNAs and used the vector to generate transgenic knockdown animals. Further experiments enhanced this vector system by enabling tissue- or temporal-specific transgene or shRNA expression as well as reducing variegated viral expression. Using these lentiviral RNAi vectors, we began to assess the role of effector caspases in the immune system. We generated T cell lines in which the effector caspases were ablated individually or simultaneously by RNAi and tested whether these cells were resistant to apoptosis. Of the three effector caspases, only silencing of caspase-3 protected against cell death in T cells, whereas simultaneous knockdown of caspase-6 or caspase-7 with caspase-3 provided no additional protective effect against apoptosis. We also generated transgenic caspase-7 knockdown animals and found that this caspase might influence B cell development.by Christopher P. Dillon.Ph.D

A multicomponent holistic care pathway for people who use drugs in Tayside, Scotland

Background: People Who Use Drugs (PWUD) are at high risk of non-fatal overdose and other drug-related harms. The United Kingdom drugs policy landscape makes it challenging to support those at risk. Tayside, in East Scotland, has a sizeable population at risk of drug-related harms. In 2021, the National Health Service implemented a care pathway for PWUD to provide multidimensional healthcare interventions. We aimed to quantify drug-related harms; assess wider health and well-being; and understand substance use trends and behaviours, among those engaged in the pathway.Methods: Existing community-embedded blood-borne virus pathways were adapted to provide multiple healthcare assessments over three visits. We undertook an observational cohort study to analyse uptake and outcomes for the initial cohort of PWUD engaged at appointment one.Results: From August 2021–September 2022, 150 PWUD engaged with the pathway. Median age was 39 (34–42) years, 108 (72%) were male, and 124 (83%) lived in deprived areas. Seventy (47%) had been disengaged from healthcare for over a year. Polysubstance use was reported by 124 (83%), 42 (28%) disclosed injecting daily, and 54 (36%) shared equipment. Fifty-four (36%) experienced recent non-fatal overdose, and there were six overdose fatalities (4.1 [1.5–9.0] per 100PY). The offer of take-home naloxone was accepted by 108 (72%). Fourteen (9%) were diagnosed with Hepatitis C and two (1%) with HIV. Renal, hepatological, and endocrine impairment were observed among 30 (20%), 23 (15%), and 11 (7%), people respectively. Ninety-six (65%) had high risk of clinical depression. Forty-eight (32%) declined Covid-19 vaccination.Conclusion: The pathway engaged PWUD with high exposure to recent non-fatal overdose and other drug-related harms, alongside co-morbid health issues. Our results suggest multi-dimensional health assessments coupled with harm reduction in community settings, with appropriate linkage to care, are warranted for PWUD. Service commissioners should seek to integrate these assessments where possible

Precision Electron-Beam Polarimetry using Compton Scattering at 1 GeV

We report on the highest precision yet achieved in the measurement of the polarization of a low energy, $\mathcal{O}$(1 GeV), electron beam, accomplished using a new polarimeter based on electron-photon scattering, in Hall~C at Jefferson Lab. A number of technical innovations were necessary, including a novel method for precise control of the laser polarization in a cavity and a novel diamond micro-strip detector which was able to capture most of the spectrum of scattered electrons. The data analysis technique exploited track finding, the high granularity of the detector and its large acceptance. The polarization of the $180~\mu$A, $1.16$~GeV electron beam was measured with a statistical precision of $<$~1\% per hour and a systematic uncertainty of 0.59\%. This exceeds the level of precision required by the \qweak experiment, a measurement of the vector weak charge of the proton. Proposed future low-energy experiments require polarization uncertainty $<$~0.4\%, and this result represents an important demonstration of that possibility. This measurement is also the first use of diamond detectors for particle tracking in an experiment.Comment: 9 pages, 7 figures, published in PR

Mechanical Properties of Molybdenum Disulfide and the Effect of Doping: An in Situ TEM Study

Direct observations on nanopillars composed of molybdenum disulfide (MoS₂) and chromium-doped MoS₂ and their response to compressive stress have been made. Time-resolved transmission electron microscopy (TEM) during compression of the submicrometer diameter pillars of MoS₂- and Cr-doped MoS₂ (Cr: 0, 10, and 50 at %) allow the deformation process of the material to be observed and can be directly correlated with mechanical response to applied load. The addition of chromium to the MoS₂ changed the failure mode from plastic deformation to catastrophic brittle fracture, an effect that was more pronounced as chromium content increased

Local host-dependent persistence of the entomopathogenic nematode Steinernema carpocapsae used to control the large pine weevil Hylobius abietis

Entomopathogenic nematodes (EPN) applied inundatively to suppress insect pests are more likely to persist and establish in stable agroecosystems than in annual crops. We investigated a system of intermediate stability: three stumps harbouring the large pine weevil (Hylobius abietis L.; Coleoptera: Curculionidae), a major European forestry pest. We tested whether persistence of EPN Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) applied around stumps is maintained by recycling of EPN through pine weevils developing within stumps. Steinernema carpocapsae was detected in soil around and under the bark of treated tree stumps up to two years, but not 4–5 years after application. Differences in nematode presence between sites were better explained by tree species (pine or spruce) than soil type (mineral or peat). Presence of S. carpocapsae in soil was positively correlated with the number of H. abietis emerging from untreated stumps the previous year, which was greater for pine stumps than spruce stumps

Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA

'To live and die [for] Dixie': Irish civilians and the Confederate States of America

Around 20,000 Irishmen served in the Confederate army in the Civil War. As a result, they left behind, in various Southern towns and cities, large numbers of friends, family, and community leaders. As with native-born Confederates, Irish civilian support was crucial to Irish participation in the Confederate military effort. Also, Irish civilians served in various supporting roles: in factories and hospitals, on railroads and diplomatic missions, and as boosters for the cause. They also, however, suffered in bombardments, sieges, and the blockade. Usually poorer than their native neighbours, they could not afford to become 'refugees' and move away from the centres of conflict. This essay, based on research from manuscript collections, contemporary newspapers, British Consular records, and Federal military records, will examine the role of Irish civilians in the Confederacy, and assess the role this activity had on their integration into Southern communities. It will also look at Irish civilians in the defeat of the Confederacy, particularly when they came under Union occupation. Initial research shows that Irish civilians were not as upset as other whites in the South about Union victory. They welcomed a return to normalcy, and often 'collaborated' with Union authorities. Also, Irish desertion rates in the Confederate army were particularly high, and I will attempt to gauge whether Irish civilians played a role in this. All of the research in this paper will thus be put in the context of the Drew Gilpin Faust/Gary Gallagher debate on the influence of the Confederate homefront on military performance. By studying the Irish civilian experience one can assess how strong the Confederate national experiment was. Was it a nation without a nationalism