Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Construindo novos túneis : subterfúgios das engenheiras para deslocar as fronteiras da divisão sexual da ciência e da tecnologia

O domínio do conhecimento científico é historicamente masculino. Mesmo com a crescente inserção das mulheres nesse campo, pode-se dizer que a lógica de fazer ciência ainda está pautada em valores masculinizados. Nosso trabalho pretende mapear a percepção das engenheiras sobre suas escolhas pelas ciências exatas e por um campo profissional tradicionalmente masculino, a engenharia. A nossa hipótese de trabalho inicial afirmava que as escolhas das engenheiras durante sua trajetória acadêmica e profissional são dificultadas pela forte tendência à perpetuação da divisão sexual da educação superior, da ciência e do trabalho. O objetivo principal da pesquisa foi desvendar os argumentos que levam à naturalização da separação entre cursos de homens e cursos de mulheres, áreas de trabalho femininas e masculinas ainda que dentro da mesma área do saber ou do campo profissional. Para tanto, foi realizado estudo qualitativo com entrevistas semiestruturadas à sete engenheiras de diferentes áreas de uma empresa estatal, sociedade anônima de economia mista, com sede no Distrito Federal, atuante no serviço público de energia elétrica. Observou-se a perpetuação da divisão sexual horizontal e vertical da ciência, tecnologia e do trabalho, que se reinventa nas trajetórias traçadas pelas engenheiras, formando guetos “permitidos” às mulheres, ainda que dentro da própria engenharia. _________________________________________________________________________________________________ ABSTRACT

Poorer subjective sleep quality is related to higher fantasy-induced sexual arousal in women of reproductive age

Lack of sleep enhances erections and lubrication the next day. This raises the possibility that poorer subjective sleep quality is related to sexual arousal. To test this hypothesis, sexual arousal was elicited in 70 Portuguese women of reproductive age by means of fantasy. The level of salivary testosterone before and shortly after fantasy was determined by luminescence immunoassays. Participants completed the Pittsburgh Sleep Quality Index (PSQI), reported their sexual arousal before and during fantasy, and how anxious they were after the fantasy. The hypothesis was confirmed. Anxiety did not explain the association, but testosterone response (poststimulus minus baseline) had a slight explanatory effect.

Avaliação do impacto do modelo biográfico em educação sexual na erotofília/erotofobia e no duplo padrão sexual

O impacto do modelo biográfico em educação sexual no constructo da erotofília/erotofobia e do duplo padrão sexual é avaliado, a partir das hipóteses de que este modelo contribui para a promoção da erotofília e atenuação do duplo padrão sexual. Os resultados não permitiram confirmar qualquer uma das hipóteses. Os indivíduos avaliados, independentemente da sua experiência no modelo biográfico, são mais erotofóbicos do que erotofílicos, e o duplo padrão sexual, de acordo com as pontuações médias e os resultados da análise de discurso, só se faz sentir com maior expressividade e significado na perceção social do duplo padrão sexual referente à liberdade sexual dos homens, com ou sem experiência no modelo biográfico, e das mulheres com experiência no modelo biográfico. Em todas as outras dimensões avaliadas do duplo padrão sexual predominou o padrão singular e o padrão sexual invertido (liberdade sexual relativa ao número de parceiros e expressões e vocábulos utilizados para caracterizar homens e mulheres com múltiplos parceiros).

A multi-hypothesis topological SLAM approach for loop closing on edge-ordered graphs

We present a method for topological SLAM that specifically targets loop closing for edge-ordered graphs. Instead of using a heuristic approach to accept or reject loop closing, we propose a probabilistically grounded multi-hypothesis technique that relies on the incremental construction of a map/state hypothesis tree. Loop closing is introduced automatically within the tree expansion, and likely hypotheses are chosen based on their posterior probability after a sequence of sensor measurements. Careful pruning of the hypothesis tree keeps the growing number of hypotheses under control and a recursive formulation reduces storage and computational costs. Experiments are used to validate the approach.

Multi-victim sexual assault : a case study in rural Australia

Objective: There is a paucity of information regarding cases of multi-victim sexual assault of children. The reported incidence suggests that these cases are rare. The aim of this paper is to provide practitioners with information about effective intervention strategies arising out of the direct experience of managing a case of multi-victim sexual assault in an Australian rural community. --------- Method: A descriptive, case-report methodology summarizing the investigation and intervention in a case of multi-victim sexual assault is reported. A community based intervention arising out of the disclosures of 21 male children is described. The intervention occurred at an individual, group, and community level using a coordinated multi-disciplinary team and natural helping networks. ---------- Results: The coordination of police and welfare services increased the communication flow to victims, their families, and the community. The case also demonstrated the utility in regularly briefing political and bureaucratic authorities as well as local officials about emergent issues. Coordinating political and bureaucratic responses was essential in obtaining ongoing support and sufficient researching to enable the effective delivery of services. ---------- Conclusions: Interventions were focused at an individual, group, and community level using a coordinated multi-disciplinary team and natural helping networks. This provided a choice of services which were sensitive to the case setting. Recommendations are offered for practitioners who are confronted with similar events. While this paper describes an approach for intervening in a case of multi-victim sexual assault, further empirical research is needed to enable service deliverers to efficaciously target interventions which offer choice to victims and their families.

Inhibiting outrage: tactics and counter-tactics in legal cases of sexual harassment

Sexual harassment can be conceptualised as an interaction between harassers and targets. Utilising 23 detailed legal transcripts, this study explored evidence of a range of perpetrator tactics and target counter-tactics. These tactics can be readily fitted into the backfire framework, which proposes that powerful perpetrators of perceived unjust acts are likely to cover up the actions, devalue the target, reinterpret the events, use official channels to give an appearance of justice, and intimidate or bribe people involved. Targets can respond using counter-tactics of exposure, validation, reframing, mobilisation of support, and resistance. The findings have implications for raising awareness of harassing tactics and recommendations for effective informal responses in organisations.