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The presence of ubiquitylated protein aggregates in neurons and surrounding cells is considered one of the hallmarks of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). Since the identification of SOD1 as the first causative gene in 1993, extensive research has been carried out to investigate the role Cu/Zn superoxide dismutase-1 (SOD1) aggregation plays in ALS pathogenesis. Recently, it has been reported that SOD1 inclusions could propagate in a prion-like manner, by seeding the aggregation of soluble functional proteins and transmitting aggregation to neighbouring cells. HSJ1 (DnaJB2) is a chaperone that can reduce protein aggregation in several neurodegenerative disease models; such as, Huntington’s disease and Parkinson’s disease. HSJ1a overexpression has also been shown to improve motor neuron survival in an animal model of SOD1-ALS. In this study, I tested the hypothesis that HSJ1 could alter SOD1-aggregation and seeding in vitro and in cell models. I developed an in vitro system with purified proteins to explore the tendency of SOD1 wild type (SOD1WT) and mutants (SOD1MT) to aggregate and seed further aggregation. The results showed that SOD1WT is resistant to seeding unless the protein is destabilized and partially unfolded. Purified HSJ1 could reduce SOD1 aggregation. I then developed transient and inducible cell models to investigate the effects of HSJ1 on SOD1 aggregate formation and expansion. In cells, HSJ1 interacted preferentially with SOD1MT, and could reduce SOD1 inclusion formation, and disassembled pre-existing SOD1 inclusions. Using an inducible stable cell line expressing HSJ1a, unfolded protein response (UPR) markers were modestly reduced after ER stress, suggesting HSJ1a expression could potentially reduce disease-related intracellular stress. Collectively, these findings shed light on HSJ1 as a potential candidate targeting misfolded and aggregated SOD1 for future investigation.

Amorphous solid dispersions are a promising way to improve the dissolution rate and bioavailability of hydrophobic drugs. However, they have a tendency to crystallise to the stable crystalline form during processing or storage, leading to changed physicochemical properties. The study of the stability for amorphous solid dispersions is important. Here, the thermodynamic stability of griseofulvin and indomethacin in polymers were studied using differential scanning calorimetry (DSC), and the effect of moisture on the kinetic stability of indomethacin or griseofulvin solid dispersions with PVP was studied using dynamic vapour sorption – near infrared (DVS - NIR). DSC could estimate the solubility of griseofulvin in HPMCAS accurately but not for the other models. DVS-NIR showed that the water uptake by the solid dispersions of indomethacin and PVP was lower than the values predicted from the sum of the water isotherm for individual components. The reason for this reduction was due to the hydrogen bonds formed between IMC and PVP occupied the water binding sites on PVP. The moisture sorption reduction in the amorphous solid dispersions can stabilise the solid dispersions. A reduced water uptake was also observed for the solid dispersion of 70% GSF and PVP compared to the predicted value. As no hydrogen bonds formed between GSF and PVP, the reason for this reduction was that the water binding sites on PVP were sterically hindered by GSF through the formation of the solid dispersion. A weight loss was absent in the DVS curve for the solid dispersion while crystallisation happened. This was due to the expelled water from amorphous GSF was sorbed by PVP. DVS-NIR offers a great promise in understanding the mechanism of stabilisation of solid dispersions, and therefore may be useful to predict the stability of new API dispersions.

Background: Family carers of people with dementia report high levels of anxiety and depression. More emotion-focused and less dysfunctional coping appear protective against symptoms in observational studies, but no randomised controlled trial (RCT) has investigated emotion-focused coping as a mechanism of effective therapy. Method: We recruited 260 family carers of people with dementia (referred to services in past year) into a pragmatic RCT of 8-sessions manualised, individual-based coping skills intervention versus treatment-as-usual (TAU). Blinded raters measured carers’ psychological morbidity (Hospital Anxiety and Depression Scale, HADS-T) and coping (Brief COPE: emotion-focused, problem-focused, dysfunctional subscales) at 4 and 8 months. My hypothesis that increased emotion-focused coping mediated treatment effects in reducing symptoms was tested using regression. As baseline symptoms moderated treatment effects on coping, post-hoc subgroup efficacy analyses were performed in carers with different baseline morbidity levels. Finally moderated mediation was tested using regression models. Results: Emotion-focused coping did not mediate treatment effects in reducing psychological symptoms in the whole sample. It appeared to mediate such effects only in psychological morbidity cases (baseline HADS-T 16+). Increased emotion-focused coping over 4 months predicted reduced symptoms at 8 months regardless of treatment status (b = -0.24, p = 0.005). Intervention had no overall effects on coping, but more severe cases (HADS-T 20+) increased emotion-focused coping (b = 4.57 [95% CI: 1.83, 7.30]), and maintained dysfunctional coping while TAU decreased (b = 0.14 [95% CI: 0.02, 0.26]). Non-cases (HADS-T <8) in TAU increased dysfunctional coping versus intervention (b = -0.09 [95% CI: -0.17, -0.003], log). Conclusions: Emotion-focused coping appeared to mediate treatment effects on psychological morbidity only in carers with high baseline symptoms. The most distressed increased helpful coping strategies and improved; the least distressed maintained low use of unhelpful strategies and remained well. Carers found different ways to benefit from standardised therapy.

Background: Resting-state functional magnetic resonance imaging (fMRI) studies have demonstrated that basal ganglia functional connectivity is altered in Parkinson’s disease (PD) as compared to healthy controls. However, such functional connectivity alterations have not been related to the dopaminergic deficits that occurs in PD over time. Objectives: To examine whether functional connectivity impairments are correlated with dopaminergic deficits across basal ganglia subdivisions in patients with PD both cross-sectionally and longitudinally. Methods: We assessed resting-state functional connectivity of basal ganglia subdivisions and dopamine transporter density using 11C-PE2I PET in thirty-four PD patients at baseline. Of these, twenty PD patients were rescanned after 19.9 ± 3.8 months. A seed-based approach was used to analyze resting-state fMRI data. 11CPE2I binding potential (BPND) was calculated for each participant. PD patients were assessed for disease severity. Results: At baseline, PD patients with greater dopaminergic deficits, as measured with 11C-PE2I PET, showed larger decreases in posterior putamen functional connectivity with the midbrain and pallidum. Reduced functional connectivity of the posterior putamen with the thalamus, midbrain, supplementary motor area and sensorimotor cortex over time were significantly associated with changes in DAT density over the same period. Furthermore, increased motor disability was associated with lower intraregional functional connectivity of the posterior putamen. Conclusions: Our findings suggest that basal ganglia functional connectivity is related to integrity of dopaminergic system in patients with PD. Application of resting-state fMRI in a large cohort and longitudinal scanning may be a powerful tool for assessing underlying PD pathology and its progression.

Ovarian cancer is a gynaecological malignancy associated with the highest level of death. Its resistance against anti-cancer drugs in recent time has exacerbated the problem. Hence, the search for novel drugs from plants used in traditional medicine is imperative. This study investigated the in vitro anti-ovarian cancer activities of Justicia insularis T. Anderson and mechanisms of action of its bioactive constituents, two plant-derived diterpenoids (andrographolide and triptolide) and four sesquiterpene lactones (dehydroleucodine, alantolactone, parthenolide and costunolide). The bioactive compounds from J. insularis were isolated and characterised using bioassay-guided fractionation, mass spectrometry and nuclear magnetic resonance spectroscopy. The antiproliferative activity of the sesqui- and diterpenoids was evaluated against human ovarian cancer cell lines (CIS-A2780, OVCAR- 8 and OVCAR-4) using Sulforhodamine B and trypan blue exclusion assays. Pro-apoptotic activity was evaluated using enzymatic assays and flow cytometry. Global DNA methylation and DNA methyltransferases (DNMTs) activity were measured using ELISA assay. Gene-specific CpG methylation and mRNA expression of panel of tumour suppressor genes were quantified using pyrosequencing and RT-qPCR respectively. The two major bioactive compounds in J. insularis were identified as diterpenoids, 16α/β-hydroxy-cleroda- 3,13(14) dien-15,16-olide and 16-oxocleroda-3,13(14) dien-15-oic acid. Sesqui- and diterpenoids showed strong growth inhibitory activity, decreased cell viability and induced apoptosis via intrinsic and extrinsic pathways. They possessed DNMT inhibitory activity, upregulated the expression of notable tumour suppressor genes. Furthermore, sesquiterpene lactones showed significant global and gene specific CpG methylation activity. In conclusion, sesqui- and diterpenoids possess strong anti-ovarian cancer activity and bear the potential to be developed as drug candidates for cancer treatment. Therefore, in vivo studies of their anti-ovarian cancer activity would be essential in the future.

Ovarian cancer is the second leading cause of death among women in the gynaecological category of cancers. The current post surgery treatment which involves the use of platinum-based therapy with its attendant adverse drug-resistance often results in the return of the cancer. This research work, explored the role of natural products as the major source of new drugs by the evaluation of the anti-ovarian cancer activities of three selected medicinal plants and the semi-synthesis of analogues of an anti-cancer agent; thymoquinone from Nigella sativa. Using a bioassay-guided approach, an investigation of the cell growth inhibition of the extracts/fractions of these plants on four human ovarian cancer cell lines, A2780, OVCAR 4, OVCAR 8, and CIS-A2780 showed that Acalypha wilkesiana, Margaritaria discoidea and Rutidea parviflora had promising anti-ovarian cancer activities. This is the first report of the anti-cancer activities of M. discoidea and R. parviflora. The bioactive compounds of the plants were isolated by HPLC, identified by mass spectrometry/NMR spectroscopy and investigated for cytotoxicity. Gallic acid, (IC50 range of 6.2±0.3 – 26.9±4.1 μM) was the active compound in A. wilkesiana. The significantly bioactive compounds of M. discoidea were securinine, (IC50 range of 2.7±0.7 – 8.7±0.1 μM), betulinic acid, (IC50 of 16.0±1.9 μM) and gallic acid. While palmatine, (IC50 range of 5.5±0.9 – 7.9±0.5 μM) was the major active compound in R. parviflora. Twenty-one thymoquinone analogues including eleven semi-synthetic ones were evaluated for cytotoxicity. A synthetic 2-dimethylamino-5-methyl-1,4-benzoquinone demonstrated optimum activity (IC50 range of 4.7±0.5 – 11.2±1.9 μM) and superior aqueous solubility. Palmatine, securinine and 2-dimethylamino-5-methyl-1,4-benzoquinone showed induction of apoptosis via increased caspase 3/7 activity. Palmatine demonstrated PAPR cleavage by western blot analysis. 2-dimethylamino-5-methyl-1,4-benzoquinone showed synergy with carboplatin and paclitaxel. These studies have provided scientific evidences for the potential treatment of ovarian cancer with these traditional medicinal plants and hit compounds for future optimization towards clinical trials.