Synthetic biology approaches for engineering diverse bacterial species

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, June 2016.Cataloged from PDF version of thesis. "May 2016."Includes bibliographical references (pages 113-134).When engineers control gene expression, cells can be re-programmed to create living therapeutics or materials by initiating expression of biosynthetic pathways in response to specific signals. In this thesis, two new genetic tools were developed to aid the construction of genetic circuits and facilitate their delivery to bacteria isolated from diverse environments. First, antisense transcription was explored as a new tool for tuning gene expression in Escherichia coli. Antisense transcription was found to reliably repress gene expression and was applied tune simple genetic circuits. Second, an integrative conjugative element from Bacillus subtilis, ICEBsJ, was engineered to deliver exogenous DNA to diverse strains of undomesticated Gram-positive bacteria. Engineered ICEBsI conjugation was demonstrated in twenty different bacterial strains, spanning sixteen species and five genera. To demonstrate ICE's utility in creating new probiotics, the element was used to deliver functional nitrogen fixation pathways (nif clusters) to bacteria isolated from agricultural soils. Collectively, the tools presented here in provide a platform for programing bacteria from diverse environments for advanced applications.by Jennifer Ann Noelani Brophy.Ph. D

Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival

tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm[superscript 5]U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm5U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm[superscript 5]U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.United States. National Institutes of Health (grant CA055042)United States. National Institutes of Health (grant ES002109)United States. National Institutes of Health (grant ES01701)National Institutes of Health (U.S.). Intramural Research ProgramWestaway Research FundNational Center for Research Resources (U.S.) (grant S10-RR023783

Attachment Predicts College Students’ Knowledge, Attitudes, and Skills for Working with Infants, Toddlers, and Families

Research Findings:Adults’ attitudes about attachment relationships are central to how they perceive and respond to children. However, little is known about how attachment styles are related to teachers’ attitudes toward and interactions with infants and toddlers. From a survey of 207 students taking early childhood (EC) courses at 4 U.S. universities, we report relations among students’ attachment styles and their (a) career goals, (b) attitudes about caring for and educating infants and young children, and (c) interaction skills for responding in developmentally supportive ways. Overall, attachment security was positively associated with career goals focused on working with younger children, knowledge about infant/toddler development, attitudes that acknowledge the importance of adult support in children’s development, and developmentally supportive interaction skills. Students who scored high on attachment fearfulness minimized the importance of adults in children’s lives, minimized the importance of the early years for later learning, and endorsed strict and controlling forms of child guidance. Practice or Policy: A conceptual mediation model linking a path from attachment to caregiving skill through knowledge and attitudes is articulated. We propose a person-centered pedagogy for infant/toddler professional preparation that provides opportunities for reflection on one’s own attachment and its effects on work with young childre

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Antisense transcription as a tool to tune gene expression

A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip‐based oligo synthesis was applied to build a large library of 5,668 terminator–promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.United States. Office of Naval Research. Multidisciplinary University Research Initiative (4500000552)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)National Science Foundation (U.S.). Graduate Research Fellowshi

Attachment Predicts College Students’ Knowledge, Attitudes, and Skills for Working with Infants, Toddlers, and Families

Research Findings:Adults’ attitudes about attachment relationships are central to how they perceive and respond to children. However, little is known about how attachment styles are related to teachers’ attitudes toward and interactions with infants and toddlers. From a survey of 207 students taking early childhood (EC) courses at 4 U.S. universities, we report relations among students’ attachment styles and their (a) career goals, (b) attitudes about caring for and educating infants and young children, and (c) interaction skills for responding in developmentally supportive ways. Overall, attachment security was positively associated with career goals focused on working with younger children, knowledge about infant/toddler development, attitudes that acknowledge the importance of adult support in children’s development, and developmentally supportive interaction skills. Students who scored high on attachment fearfulness minimized the importance of adults in children’s lives, minimized the importance of the early years for later learning, and endorsed strict and controlling forms of child guidance. Practice or Policy: A conceptual mediation model linking a path from attachment to caregiving skill through knowledge and attitudes is articulated. We propose a person-centered pedagogy for infant/toddler professional preparation that provides opportunities for reflection on one’s own attachment and its effects on work with young childre

Maternal history of adverse experiences and posttraumatic stress disorder symptoms impact toddlers’ early socioemotional wellbeing: The benefits of infant mental health-home visiting

Background: The present study examined the efficacy of the Michigan Model of Infant Mental Health-Home Visiting (IMH-HV) infant mental health treatment to promote the socioemotional wellbeing of infants and young children. Science illuminates the role of parental “co-regulation” of infant emotion as a pathway to young children’s capacity for self-regulation. The synchrony of parent–infant interaction begins to shape the infant’s own nascent regulatory capacities. Parents with a history of childhood adversity, such as maltreatment or witnessing family violence, and who struggle with symptoms of post-traumatic stress may have greater challenges in co-regulating their infant, thus increasing the risk of their children exhibiting social and emotional problems such as anxiety, aggression, and depression. Early intervention that targets the infant–parent relationship may help buffer the effect of parental risk on child outcomes. Methods: Participants were 58 mother–infant/toddler dyads enrolled in a longitudinal randomized control trial testing the efficacy of the relationship-based IMH-HV treatment model. Families were eligible based on child age (\u3c24 months at enrollment) and endorsement of at least two of four socio-demographic factors commonly endorsed in community mental health settings: elevated depression symptoms, three or more Adverse Childhood Experiences (ACEs) parenting stress, and/or child behavior or development concerns. This study included dyads whose children were born at the time of study enrollment and completed 12-month post-baseline follow-up visits. Parents reported on their own history of ACEs and current posttraumatic stress disorder (PTSD) symptoms, as well as their toddler’s socioemotional development (e.g., empathy, prosocial skills, aggression, anxiety, prolonged tantrums). Results: Maternal ACEs predicted more toddler emotional problems through their effect on maternal PTSD symptoms. Parents who received IMH-HV treatment reported more positive toddler socioemotional wellbeing at follow-up relative to the control condition. The most positive socioemotional outcomes were for toddlers of mothers with low to moderate PTSD symptoms who received IMH-HV treatment. Conclusion: Results indicate the efficacy of IMH-HV services in promoting more optimal child socioemotional wellbeing even when mothers reported mild to moderate PTSD symptoms. Results also highlight the need to assess parental trauma when infants and young children present with socioemotional difficulties

Attachment Predicts College Students\u27 Knowledge, Attitudes, and Skills for Working with Infants, Toddlers, and Families

Research Findings: Adults’ attitudes about attachment relationships are central to how they perceive and respond to children. However, little is known about how attachment styles are related to teachers’ attitudes toward and interactions with infants and toddlers. From a survey of 207 students taking early childhood (EC) courses at 4 U.S. universities, we report relations among students’ attachment styles and their (a) career goals, (b) attitudes about caring for and educating infants and young children, and (c) interaction skills for responding in developmentally supportive ways. Overall, attachment security was positively associated with career goals focused on working with younger children, knowledge about infant/toddler development, attitudes that acknowledge the importance of adult support in children’s development, and developmentally supportive interaction skills. Students who scored high on attachment fearfulness minimized the importance of adults in children’s lives, minimized the importance of the early years for later learning, and endorsed strict and controlling forms of child guidance. Practice or Policy: A conceptual mediation model linking a path from attachment to caregiving skill through knowledge and attitudes is articulated. We propose a person-centered pedagogy for infant/toddler professional preparation that provides opportunities for reflection on one’s own attachment and its effects on work with young children. © 2015 Taylor & Franci

Relationships heal: Reducing harsh parenting and child abuse potential with relationship-based parent-infant home visiting

Background: Childhood maltreatment and harsh parenting can have lasting effects on young children\u27s development, and home visiting interventions provide critical opportunities to mitigate this risk. Objective: This study aimed to examine associations between participation in the Michigan Model of Infant Mental Health Home Visiting (IMH-HV) and both harsh parenting and child abuse potential. Participants and Setting: Participants were 76 mothers and their infants/toddlers (M age = 9.9 months at baseline) receiving IMH-HV with Community Mental Health Service Provider agencies in Michigan. Methods: Data were collected at baseline (shortly after initiation of services), and 3, 6, 9, and 12 months post baseline assessment. Mothers provided demographic information and completed several questionnaire measures including the Brief Child Abuse Potential Inventory (BCAP). Harsh parenting was rated by a trained research assistant using subscales from the Home Observation of Measurement of the Environment (HOME). Treatment dosage was collected from IMH-HV clinicians. Results: Mothers with higher numbers of IMH-HV visits showed lower levels of observed harsh parenting over the three time points in the study (estimate = 0.050, p = 0.002) and higher cumulative societal factors associated with oppression (SFAO) predicted higher harsh parenting over the three time points. A higher number of IMH-HV visits also predicted lower child abuse potential across the time points of the study (estimate = −0.78, p = 0.043), controlling for SFAO. Conclusions: Participation in the Michigan Model of IMH-HV, delivered in the community, is associated with reductions in both harsh parenting and child abuse potential