An interpretative phenomenological analysis of adults’ accounts of the lived experience of parental death in adolescence

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the award of Professional Doctorate in Counselling Psychology.This qualitative study aimed to hear the subjective lived experiences of adults who experienced parental death during adolescence in order to gain insight into how their experience has impacted them from adolescence to adulthood. The findings from this research endeavour to contribute to a theoretical understanding of this experience and highlight the clinical implications for Counselling Psychologists working with individuals who have experienced parental death during adolescence. An Interpretative Phenomenological Analysis was the methodological design used to facilitate an understanding of how participants make sense of their experience. Seven participants were recruited via a purposive snowball sampling method. Participants spoke about their experiences of parental death from adolescence to adulthood in 45-50-minute semi-structured telephone interviews which were then transcribed and analysed. The Findings illuminated the following four superordinate themes, Managing Emotions, Interpersonal Changes, Complex Grief and Positive Changes. Participants appeared to have an impaired ability to manage and regulate their emotions relying instead on maladaptive coping and defence mechanisms to attenuate their emotions. This could be attributed to their difficulties with grief expression, lack of support in both aiding their grief and helping the development of these regulatory skills. Emotional regulatory difficulties could also be symptomatic of their unique developmental period. Participants appear to exhibit an array of symptoms pertaining to the diagnosis of Persistent Complex Bereavement Disorder (DSM-5, 2013) and Prolonged Grief Disorder (ICD-11r, 2018). Participants reported experiencing vast interpersonal changes and experienced subsequent attachment difficulties which were influenced by a range of developmental, psychological and psychosocial factors and stressors post parental death. Although indicators of growth were also apparent, the findings support the potential association of parental death in adolescence to ongoing developmental, psychological and psychosocial effects, from adolescence to adulthood. Implications for Counselling Psychologists’ clinical practice, training and consultancy have been addressed. How the humanistic and psychodynamic counselling framework addresses the needs of the individual parentally bereaved in adolescence are illuminated. Furthermore, a range of directive and non-directive therapeutic interventions and clinical suggestions have been recommended as Counselling Psychologists work in an integrative manner in line with the nuanced need of the individual client. Early intervention inclusive of a comprehensive complex grief assessment and a developmentally informed formulation is suggested. Psychological therapy to aid the development of emotional regulation skills is also suggested. Parent-child/Family therapy and a range of treatment and preventative initiatives and interventions across the education, health and community sector is recommended with both the bereaved individual and existing family

Mechanism of Cytochrome c Biogenesis by the Integral Membrane System II Synthetase CcsBA

Cytochromes c are electron transport proteins that contain a heme moiety covalently ligated to the protein at a conserved CXXCH motif. Organisms employ one of three biogenesis systems (system I, II, or III) to facilitate transmembrane delivery, reduction, and ligation of the apoprotein and heme. System II, employed by Gram positive bacteria, cyanobacteria, some proteobacteria, and chloroplasts, consists of four proteins: CcsB, CcsA, DsbD, and CcsX. Only CcsB and CcsA are absolutely required for function and represent the minimal system II synthetase. Naturally occurring fusions of the CcsB and CcsA proteins (CcsBA) from different bacteria were investigated for robustness of cytochrome c assembly in recombinant Escherichia coli and purification of CcsBA was optimized. The recombinant Helicobacter hepaticus CcsBA yielded the highest levels of diheme cytochrome c4. CcsBA can synthesize diverse cytochromes c, including those naturally assembled by systems I (monoheme cytochrome c2) and III (human cytochrome c). E. coli disulfide bond formation (Dsb) mutants and chemical reducing agents were employed to address the redox requirements for function of recombinant CcsBA. Under aerobic conditions DsbC and DsbD are required for function while under anaerobic conditions only DsbD is required. CcsBA contains a conserved motif called the WWD domain and is a member of the heme handling protein (HHP) superfamily. Detergent solubilized GST-tagged CcsBA purifies with heme trapped in the protein. Two conserved histidines in transmembrane domains (TMDs) are required for heme binding and mutants can be complemented for function by addition of the histidine side chain analogue imidazole to growth media. Results suggest the presence of a well defined heme binding site within a channel comprised of TMD3 and TMD8 where histidines in these TMDs function as axial ligands. To further refine the model of heme binding and trafficking by CcsBA, chemical complementation by imidazole analogues, low temperature spectral features, and binding of alternative porphyrins were investigated. The results and models presented in this thesis provide new insight into the mechanisms of system II heme trafficking and synthetase activity and will guide future investigations of the CcsBA protein and efforts to reconstitute cytochrome c biogenesis in vitro

Topology and Function of CcmD in Cytochrome c Maturation▿

The system I cytochrome c biogenesis pathway requires CcmD, a small polypeptide of 69 residues in Escherichia coli. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain

Heme Concentration Dependence and Metalloporphyrin Inhibition of the System I and II Cytochrome c Assembly Pathways

Studies have indicated that specific heme delivery to apocytochrome c is a critical feature of the cytochrome c biogenesis pathways called system I and II. To determine directly the heme requirements of each system, including whether other metal porphyrins can be incorporated into cytochromes c, we engineered Escherichia coli so that the natural system I (ccmABCDEFGH) was deleted and exogenous porphyrins were the sole source of porphyrins (ΔhemA). The engineered E. coli strains that produced recombinant system I (from E. coli) or system II (from Helicobacter) facilitated studies of the heme concentration dependence of each system. Using this exogenous porphyrin approach, it was shown that in system I the levels of heme used are at least fivefold lower than the levels used in system II, providing an important advantage for system I. Neither system could assemble holocytochromes c with other metal porphyrins, suggesting that the attachment mechanism is specific for Fe protoporphyrin. Surprisingly, Zn and Sn protoporphyrins are potent inhibitors of the pathways, and exogenous heme competes with this inhibition. We propose that the targets are the heme binding proteins in the pathways (CcmC, CcmE, and CcmF for system I and CcsA for system II)

Cytochrome c Biogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

Summary: Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments

Nitric Oxide Disrupts Zinc Homeostasis in Salmonella enterica Serovar Typhimurium

Nitric oxide (NO·) produced by mammalian cells exerts antimicrobial actions that result primarily from the modification of protein thiols (S-nitrosylation) and metal centers. A comprehensive approach was used to identify novel targets of NO· in Salmonella enterica serovar Typhimurium (S. Typhimurium). Newly identified targets include zinc metalloproteins required for DNA replication and repair (DnaG, PriA, and TopA), protein synthesis (AlaS and RpmE), and various metabolic activities (ClpX, GloB, MetE, PepA, and QueC). The cytotoxic actions of free zinc are mitigated by the ZntA and ZitB zinc efflux transporters, which are required for S. Typhimurium resistance to zinc overload and nitrosative stress in vitro. Zinc efflux also ameliorates NO·-dependent zinc mobilization following internalization by activated macrophages and is required for virulence in NO·-producing mice, demonstrating that host-derived NO· causes zinc stress in intracellular bacteria.Nitric oxide (NO·) is produced by macrophages in response to inflammatory stimuli and restricts the growth of intracellular bacteria. Mechanisms of NO·-dependent antimicrobial actions are incompletely understood. Here, we show that zinc metalloproteins are important targets of NO· in Salmonella, including the DNA replication proteins DnaG and PriA, which were hypothesized to be NO· targets in earlier studies. Like iron, zinc is a cofactor for several essential proteins but is toxic at elevated concentrations. This study demonstrates that NO· mobilizes free zinc in Salmonella and that specific efflux transporters ameliorate the cytotoxic effects of free zinc during infection