SARS-CoV-2, the coronavirus responsible for the CoVID-19 syndrome, continues to be a major public health crisis worldwide. While the ongoing vaccination development and deployment efforts are a critical first line of defense, small-molecule therapeutics are needed to treat those who are infected and in desperate need of medical attention. Perhaps the most studied coronaviral target is the 3CL protease enzyme responsible for the proteolytic processing of the viral polyproteins pp1a and pp1ab which is essential for viral replication. We evaluated a series of five new compounds with four containing an (acyloxy)-methyl ketone reactive group including clinical candidate Pf-00835231 for their inhibitor potencies against SARS-CoV23CL protease. All five compounds exhibit remarkable potencies with Kivalues in the high picomolar to low nanomolar range against SARS-CoV-2. The X-ray structures of all five compounds were determined in complexes with SARS-CoV-2 3CLpro to between 1.4 Å and 1.6 Å resolution. All five compounds are observed to form a covalent bond with catalytic Cysteine 145 with four compounds forming adducts with the expected tetrahedral geometry. Compound 4 however, which contains an (acyloxy)-methyl ketone warhead, was found to form an adduct with bond geometries similar to an episulfonium cation. Despite possessing similar chemistry and scaffolds, inhibitor binding to the SARS-CoV-2 3CLpro induced a variety of subtle active site conformational differences, particularly in the S2/S4 separating strand and connecting strands.<div><br></div><div>Understanding substrate specificity in coronaviral main protease is essential for designing competitive inhibitors. While first principles are already established, including a Q/X cleavage-site (where X is either Alanine or Serine), differences exist between α, β, γand δ clades that are important for recognition, and ultimately inhibitor design. Covalent complexes of SARS-CoV-2 and avian infectious bronchitis virus (IBV) covalently bound, in trans, to the C-terminus of the same enzyme have been crystallized and modeled at 2.6 and 2.2 Å, respectively. The similarities and differences in their binding are described in chapter 6.<br></div><div><br></div><div>Middle-East Respiratory Syndrome Coronavirus is a re-emergent zoonotic pathogen with a 30% mortality rate in humans. The positive-sense single-stranded RNA genome is translated by the into polyproteins 1a (pp1a) and polyprotein 1ab (pp1ab). Pp1ab contains the constituents of the viral RNA-dependent RNA polymerase complex. These must be cleaved by 3CLpro, which is contained within this pair of polyproteins, before viral replication and transcription may occur. Attempts to drug this cysteine protease have proven difficult since competitive inhibitors activate the Wild-Type protease at low concentrations via a non-competitive binding mechanism. This mechanism is mediated by non-conserved residues in regions that are distal to the catalytic site. During the viral life-cycle these residues modulate recognition This study focuses on determining the identities of these residues and their effects on the dose-response curves of established competitive inhibitors of Coronaviral 3CLpro. It also explores how the residues in these regions synergize to effect intrinsic kinetic parameters of this family of enzymes including turnover (kcat) and dimer dissociation constant (KD). Additionally, a rapid-equilibrium kinetic model was developed to rationalize this unique phenomenon where competitive inhibitors cause significant activation of the enzyme’s activity.<br></div>

Thesis (M.S.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Despite evidence that Human Immunodeficiency Virus (HIV) is not transmitted through casual contact, people continue to demonstrate behaviors which indicate a fear of casual-contact transmission. This has been identified as contributing to the social isolation experienced by people with AIDS. This study was undertaken to determine the relationships among AIDS Knowledge (AK), Perceived Susceptibility (PS) to casual-contact transmission of HIV and Social Distance (SO), or the degree of hesitancy to interact with people with AIDS in casual-contact situations. The study was conducted in a Vermont community; an area with a low incidence of AIDS. Sixty eight subjects, from a random, digit-dialing sampling technique, were interviewed by telephone. Instrumentation consisted of: an AIDS Knowledge Tool, a six-item tool to measure PS to casual-contact transmission of HIV, a six-item SD scale to measure the level of casual-contact at which the individual would hesitate to interact with a person with AIDS, and demographic information. The scales measuring PS and SD were developed by the investigator. A negative, significant relationship was observed between AK and PS, and a positive, significant relationship was found between PS and SD. The relationship between AK and SD did not reach significance levels. A subscale of 9 Transmission-Related AIDS Knowledge items was developed and was found to have a stronger, negative relationship with PS, and a negative, significant relationship with SD. It was concluded that the findings, despite the methodological limitations, warranted further study with a larger, more diverse sample and to further develop instrumentation. The association between TransmissionRelated AK and both PS and SD indicates that education programs, specifically related to transmission may impact people's fears of casual contact transmission and their distancing behavior. / 2031-01-01

Malaria is an infectious disease, where the parasite, Plasmodium, infects mosquitos, and then infects humans through a mosquito bite. In 2019, there were over 229 million cases of malaria worldwide with children under the age of 5 being the most vulnerable. Risk factors for children under five include not sleeping underneath a mosquito net, having a lower education, and the type of household. Most cases and deaths happened in the sub-Saharan region of Africa, including Sierra Leone. The aim of this project was to study the prevalence and malarial factors that are associated with children under the age of five sleeping underneath mosquito nets at night in Sierra Leone in 2019. Data was extracted from the Demographic and Health Surveys (DHS) Program in the Standard DHS for Children’s recode 2019. SPSS Statistics was used to conduct analysis. This cross-sectional study focused on the prevalence of children under five sleeping underneath a mosquito net, which is the outcome variable. The explanatory variables included type of mosquito net slept under, region, place of residence, and fever in the last two weeks. The first part of the results focused on descriptive tests, finding the frequency and percentage of each variable. Then chi-squared tests were performed between the outcome and explanatory variables, determining the test statistic, p-value, and degrees of freedom. The sample size was 9,899 responses. For the frequency results of the explanatory variables, with missing values excluded, majority of responses lived in the Southern region (n=2,591; 25.5%), lived in a rural area (n=6,873; 69.4%), had children under five sleep under treated nets (n=5,271; 63.4%), and did not have a fever in the last two weeks (n=7,418; 81.8%). The frequency results of the outcome variable were that majority of responses had all their children under five sleep under a mosquito net (n=5,278; 56.6%). There was significant association between children under 5 sleeping underneath a mosquito net with type of net (p<0.001), region (p<0.001), place of residence (p<0.0001), and fever in the last two weeks (p=0.006). One limitation of this study was that it doesn’t ask participants if they had malaria, therefore, variables were chosen based on that they can be used as a surrogate for malaria. Even though, the whole country is endemic to malaria, the Southern region could potentially have the highest cases of malaria, since most responses lived in that region and had many children sleeping under nets. Also, the majority of responses did not have a fever in the last two weeks, and this could be due to having a mosquito net in place to reduce the risk of malaria. In conclusion, studying malaria variables and the possible relationship with children under the age of five on whether they slept under a mosquito net is beneficial to help better understand malaria and the mosquito nets, which is a useful prevention technique.

Multisystem Inflammatory Syndrome in Children (MISC-C) is a systemic inflammatory disorder associated with novel COVID-19. Children diagnosed with COVID-19 typically experience mild viral symptoms, however, in rare instances more severe disease will develop. Current epidemiology states <1% of children diagnosed with COVID will develop complications of MIS-C. Symptoms of MIS-C are varying, but in general include abnormal vital signs (tachycardia, tachypnea), Kawasaki-like symptoms, respiratory distress, cardiac involvement, and shock like symptoms. Abdominal pain, features of acute kidney injury, coagulopathy, and neurologic dysfunction have also been reported. It is currently recommended for children with any combination of the above symptoms to be hospitalized and treated for MIS-C, given the potential for severe consequences. This case presentation aims to highlight the neurologic symptoms that can occur with the diagnosis of MIS-C. Current estimations predict 20% of children with MIS-C will develop more severe central nervous system symptoms. Our patient presented with neuropsychiatric symptoms, which has little documentation in current medical literature. Initial presentation, differential diagnosis, hospital workup, and treatment of disease will be discussed. Our case is a previously healthy 8yo male with past medical history of ADHD. He presented to the Emergency room with chief complaints of fever, confusion, and visual and motor hallucinations 3 weeks after receiving a positive COVID test. Prior to the date of presentation, the patient’s only symptom of COVID 19 was rhinorrhea. ED workup revealed tachycardia, pallor and erythema/swelling of fingers, facial rash, abdominal pain, and leukocytosis. Initial differential diagnosis included sepsis secondary to urinary tract infection versus encephalopathic MIS-C. Labs at presentation showed CRP and D-dimer within normal limits, and treatment for UTI was initiated. For the first few days of hospital stay, patients condition was unchanged and inflammatory marker levels rose. The patient also showed sustained tachycardia, fever, widened pulse pressures, nocturnal incontinence, and one episode of bigeminy. Treatment targeting MIS-C with, IV ceftriaxone, vancomycin, IVIG, methylprednisone, and aspirin was begun. Inflammatory markers subsequently began to downtrend and patients condition began to improve. This case study highlights central nervous system features as a potential primary presenting symptom of MIS-C. Currently, there is little literature on COVID 19 associated psychosis. Further research is needed to assess the underlying potential inflammatory mechanisms responsible for our patient’s visual and motor hallucinations resulting from MIS-C.

Many lines of evidence indicate that CD8+ T cells are important in the control of HIV-1 infection and this has led to much vaccine research focused at eliciting virus specific CTL. However to date, the few large-scale trials of HIV-1 vaccines designed to elicit CD8+ T cells have produced disappointing results. This has highlighted our incomplete knowledge of the factors that determine if such cells are capable of viral control. The aim of this thesis is to further characterise qualitative aspects of HIV-1 specific CTL that are associated with both good and bad anti-viral activity. HIV-1 specific CTL responses were investigated in three ways. Firstly, by longitudinally analysing an immunodominant HLA-B*08 restricted CD8+ T cell response in a single rapid progression patient. Secondly, HLA-B*40 restricted CTL responses to HIV-1 where characterised within a Chinese slow progressor cohort. Lastly, factors that affect the processing and presentation of certain overlapping HIV-1 specific CD8+ T cell epitopes were examined. The results of these studies reveal that subtle variations in both host and viral proteins can have a substantial impact on virus specific CTL and in turn may impact on the outcome of disease. The generation of HIV-1 specific CD8+ T cells is a complex process affected by many variables including the viral sequence of epitope flanking regions as well as polymorphisms in the proteins involved in antigen processing and presentation. To add a further layer of complexity, it appears that HIV-1 virus specific CTL can modulate their functionality throughout the course of infection. Such factors should therefore be taken into account during HIV-1 vaccine design.

Streptococcus pneumoniae has a highly adaptable genome due at least in part to its natural transformability and its ability to recombine with other pneumococci and related species. The emergence of antibiotic resistant clones of S. pneumoniae presents an opportunity to investigate how the genome has altered, spread and diversified within a defined time frame. We postulated that the genomes of epidemic, resistant isolates of S. pneumoniae would carry evidence of the genetic mechanisms that have shaped their evolution. We investigated this using eight to fifteen isolates from each of three S. pneumoniae clones; two multiply resistant clones Spain 9V-3 and Taiwan 19F-14 and one clone that has not acquired multiple resistance, England 14-9. Genome diversity in each of the three clones was investigated using pulsed field gel electrophoresis and multilocus sequence typing. Polymorphisms identified as a result of changes in the size of restriction fragments were found to be caused mainly by genomic rearrangements rather than restriction site mutations. Several deletion/insertion events in addition to one large inversion were identified. A number of polymorphisms correlated with previously known variable regions. Database analysis of multilocus sequence data from all three clones showed that recombination leads to sequence divergence more frequently than de novo mutation, but was significantly less common in England 14-9. The lower frequency of recombination events in England14-9 was in line with a transformation deficiency observed in vitro, and may explain the rare occurrence of penicillin resistance in this clone. Analysis of competence and recombination gene sequences available from databases revealed a potential cause of transformation deficiency: a four amino acid deletion in CelA, involved in DNA uptake and transport. Recombination can act as a DNA repair mechanism, but the significantly low occurrence in England14-9 suggests other mechanisms act to repair severe damage. Although S. pneumoniae does not have a typical SOS response it does possess DNA polymerase IV, encoded by dinB, which is predicted to be involved in error-prone DNA replication and repair of double strand breaks. DinB knockout mutants were created to investigate the effect in isogenic backgrounds. DinB mutants presented a lower frequency of spontaneous rifampicin resistance mutations than wild type 3 isolates. DinB mutants were more sensitive to killing by three different DNA damaging agents as well as by hydrogen peroxide. Isolates of the natural dinB mutant clone Spain 9V-3 were also shown to be more sensitive to DNA damaging agents than clones England 14-9 and Taiwan 19F-14. It is concluded that genetic differences between the three clones investigated do influence their patterns of evolution, and may account for differences in their antibiotic resistance profiles. Furthermore DNA polymerase IV does function as an error prone repair polymerase capable of protecting pneumococci from DNA damage despite the lack of a coordinated SOS response in pneumococci, and the absence of the gene in one successful multi-resistant clone.

Citrobacter rodentium is a gut pathogen, which infects the distal colon of mice. It has many similarities to human Enteropathogenic and Enterohemorrhagic E.coli in terms of mechanisms of pathogenicity and methods of transmission. Like many other gram negative bacteria, C. rodentium has developed a complex and highly specialised protein secretion system, known as type three (T3SS), to deliver bacterial proteins into eukaryotic cells. By injecting effector proteins into host cell cytoplasm, the pathogens are able to modulate host cellular functions to facilitate their own survival and replication. There is growing evidence that Attaching Effacing (AE) pathogens can inject effector proteins into gut epithelial cells, which dampen pro-inflammatory responses. There is also evidence that EPEC, Yersinia and Shigella can inject effectors into immune cells and also modulate their function. The objective of this work was to visualise and identify the host cells targeted for type III secretion by C. rodentium, and consequently determine the effect on host immune responses. The method chosen to detect cells targeted for effector protein delivery was the β-lactamase reporter system, where cells loaded with the fluorogenic substrate CCF2-AM emit a green FRET signal upon excitation by UV light, but emit a blue signal when cleaved by β-lactamase. By creating reporter strain of C.rodentium expressing fusion proteins between NleD effector and β-lactamase, I was able to show that C.rodentium is capable of injecting NleD in a wide variety of murine cell lines including Swiss 3T3 fibroblasts, J774 macrophages, CMT93 epithelial cells and BW715 T cells in a dose and time dependent manner in vitro. In addition, I found that C.rodentium has the ability to inject proteins into the cytoplasm of immune cells isolated from mouse lymphoid tissues including the spleen, mesenteric lymph nodes and Peyer's patches. Detailed analysis of the types of cells injected with effectors in vitro showed that NleD- injected cells represented B cells, dendritic cells and T cells. After inoculation of mice with the reporter strain of CitropACYCnleD, the plasmid encoded reporter fusion remained stable throughout infection and was able to inject cells in vitro after passage through the mouse gut. Unfortunately under the conditions described in this study, we were unable to visualise any gut cells targeted for protein delivery by C. rodentium in vivo, thus highlighting the complex nature of the host pathogen relationships in the gut. Although there is a need to develop better strategies to visualise effector translocation in vivo, our study has demonstrated, for the first time, the ability of C. rodentium to target immune cells for effector injection in vitro.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The general problem of alteration in viral susceptibility by the irradiation of monolayers of tissue cells in culture was examined in this study; specifically an increased susceptibility of HeLa (an established cell line which was derived from an epidermaid carcinoma of the cervix) to destruction by herpes simplex virus (the virus commonly associated with cold sores or fever blisters). The experimental procedures included the study of the radiosensitivity of the cell line, survival curve analyses expressed as the efficiency of plating, that is the per cent of viable cells capable of forming colonies visible to the unaided eye within twelve days, and finally infectivity studies. Tissue culture has proved to be a very useful tool in the study of radiation effects on tissues of higher animals since the effects of radiation can apparently be explained on the cellular level. Many types of cells have been studied but in all cases the most striking characteristic in irradiated populations is the increased cell size. Ionizing radiation effects both the reproductive and synthesizing capacity of cells with the former being the more sensitive. Some irradiated cells never divide while others divide several times before reproduction stops. After the cells stop dividing, they continue to grow in size forming giants because synthesis of the cellular constituents continues. Giant cells resulting from x-irradiation are more readily destroyed by the action of viruses than are non-irradiated cells. PUCK & MARCUS reported that NDV when plated on a mixture of giant and normal cells, destroyed more of the giants than normal cells. An enhancement of cell susceptibility following irradiation was also demonstrated for two enteroviruses by HSIUNG. The increased susceptibility of x-ray-induced giant cells to CPE of virus and the earlier release of virus by such cells was also demonstrated by LEVINE in studies with the Leon strain of type 3 polio. Many tissue culture-virus systems have been used to demonstrate alterations in susceptibility induced by x-irradiation, but few investigators have used a HeLa-HSV system to study this altered susceptibility using low levels of irradiation (50 roentgens to 500 roentgens) [TRUNCATED] / 2031-01-01

Bakgrund: Multiresistenta bakterier, däribland MRSA, är idag ett globalt samhällsproblem. Infektioner förorsakade av MRSA skapar ett onödigt lidande för patienter med utdragen vårdtid som i värsta fall kan resultera i ökad dödlighet. Enligt Smittskyddsinstitutet (2010) drabbades 1479 patienter förra året i Sverige. Med få verksamma antibiotika måste andra åtgärder tillämpas, så som basala hygienrutiner, screening, isoleringsvård och utökad städning av sjukhusmiljön. Det är dock viktigt att utvärdera åtgärdernas effekter för att kunna utföra smittskyddsarbete på bästa möjliga sätt. Syfte: Syftet var att undersöka effekterna av olika åtgärder i smittskyddsarbetet mot MRSA. Metod: Litteraturstudie med kvantitativ ansats baserad på tio vetenskapliga original artiklar. Analysen gjordes enligt Forsberg och Wengströms (2008) riktlinjer för meta-analys. Resultat: Studien visar att MRSA förekommer i kliniska miljöer samt förutom hos patienten även hos vårdpersonal. Förebyggande åtgärder som bland annat noggrann städning, basala hygienrutiner och screening hade varierande effekt och reducerade MRSA- förekomsten bäst i kombination. I vissa fall kunde brister i vårdpersonalens följsamhet (compliance) av hygienrutiner ses. Slutsats: För att reducera MRSA- förekomst och spridning är det viktigt att implementera de åtgärder som finns idag och som visat sig har effekt. För att genomföra detta krävs det att vårdpersonalens följsamhet blir bättre.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that for years was considered to belong of the past, but tuberculosis is back causing over 2 million deaths per year. The infection can be dormant for decades and an active immune response can prevent the infection from progressing into active disease. However, the HIV/AIDS epidemic has caused an alarming rise in tuberculosis cases. The main infectious route for Mtb is through the airways into the lungs, where they encounter alveolar macrophages. Mtb are phagocytosed by these macrophages, but instead of being killing within the phagosome, Mtb modulates the cell to become a host in which the bacteria thrive. The lack of capacity to eradicate the infection stimulate cells of the immune system to gather around infected macrophages and form a granuloma that walls off the infection. Within this granuloma, Mtb can wait silently and later progress into active disease. However, only a fraction of exposed individuals develop disease, indicating that initial eradication of Mtb infections is possible. Such immediate response must be directed by the innate immunity comprised of phagocytes such as neutrophils (PMNs) and non-activated macrophages. Upon Mtb infection, macrophages become anergic and PMNs enter apoptosis. PMNs have a short lifespan and are cleared by neighbouring phagocytes, a mechanism described to resolve the inflammation and modulate tissue regeneration. We found that Mtb-induced apoptosis in PMNs was not dependent on phagocytosis of the bacteria, indicating that Mtb have the capacity to induce apoptosis in multiple PMNs. Complement-mediated phagocytosis induce survival signals such as Akt in PMNs, but despite this, complement-opsonized Mtb was able to override the anti-apoptotic activation in the cells. Since phagocytes clear apoptotic cells, we investigated how clearance of Mtb-induced apoptotic PMNs affected macrophages. We found that Mtb-induced apoptotic PMNs inflicted pro-inflammatory activation of the macrophages that cleared them. In addition, this activation was mediated by Hsp72 released from the Mtb-induced apoptotic PMNs. Furthermore, apoptotic PMNs can work in synergy with phagocytosed Mtb to activate macrophages and enhance intracellular killing of Mtb. Since dendritic cells are important for the regulation of immunity, we investigated whether Mtb-induced apoptotic PMNs affected the inflammatory response and maturation of dendritic cells. We found that Mtb-induced apoptotic PMNs trigger dendritic cells to enter a mature state able to activate naïve T-cell proliferation. We propose that infected apoptotic PMNs is a potent activator of the inflammatory response during infections. Taken together, PMNs not only kill their share of pathogens but also modulate other immune cells, thereby forming a link between the early innate and the adaptive immune response during microbial challenge with Mtb.