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http://theses.ncl.ac.uk/jspui/handle/10443/5256
2024-03-29T07:53:59ZAlternate channel therapy for cystic fibrosis lung disease
http://theses.ncl.ac.uk/jspui/handle/10443/6098
Title: Alternate channel therapy for cystic fibrosis lung disease
Authors: Delpiano, Livia
Abstract: Cystic Fibrosis (CF) is the most common, severe, autosomal recessive disease in the Caucasian
population, and is caused by mutations in the cystic fibrosis transmembrane conductance
regulator (CFTR) gene. A triple-combination drug therapy that restores CFTR function is now
available for around 85-90% of the CF population. However, the remaining 10-15% carry rare
and poorly characterized mutations that cannot benefit from this treatment. An alternative
approach for this group is to use a CFTR-independent therapy, exploiting alternative ion
channels and transporters (AICTs), to restore anion and fluid secretion. The main aim of this
work was to identify drug candidates that target AICTs, and to determine if increasing the
activity of the specific AICTs, TMEM16A, SLC26A9 and SLC26A4, could restore the airways
environment in CF cells.
1400 FDA-approved drugs were screened on nasal airway organoids derived from CF patients
with different mutations for their ability to boost fluid secretion. From the screen, the most
promising 13 ‘hit’ compounds, that induced fluid secretion, were further evaluated on fully
differentiated monolayers from three CF donors with rare CF mutations. Electrophysiological
and ASL pH assays, combined with CRISPR-Cas gene knock-out of selective AICTs, showed that
SLC26A9 was not active under the conditions tested, and none of the FDA compounds
modulated TMEM16A activity, even when overexpressed under inflammatory conditions.
However, under inflammatory conditions, TMEM16A regulated ASL pH after sustained calcium
stimuli. Furthermore, SLC26A4 also regulated ASL pH in response to cAMP agonists and was
the target of 2 FDA compounds. However, significant donor variability was observed in these
functional measurements.
Overall, this study clearly demonstrates that patients-derived airway organoids are a suitable
in vitro model to screen compound libraries, which encourages a personalised approach to
identify new therapies for orphan mutations. The results also support the use of AICTs as an
alternative therapy for CF.
Description: PhD Thesis2023-01-01T00:00:00ZMitigation of radiation-induced premature frailty and cognitive decline by ablation of senescent cells
http://theses.ncl.ac.uk/jspui/handle/10443/6092
Title: Mitigation of radiation-induced premature frailty and cognitive decline by ablation of senescent cells
Authors: Weigand, Bettina Melanie
Abstract: A fundamental characteristic of radiotherapy is induction of cellular senescence and
presentation of a senescence associated secretory phenotype (SASP). Of clinical
importance, the initial accumulation of senescent cells halts cancer progression. However,
post-radiotherapy, the accumulation of senescent cells (SCs) negatively impacts clinical
outcomes. This is because SCs with a pro-inflammatory and pro-apoptotic SASP target
surrounding healthy cells. This bystander effect is alleviated when senescent cells are
destroyed using senolytic, or genetical drugs.
SCs have been associated with a wide range of age-related diseases, premature frailty,
reduced health and lifespan. Frailty is a geriatric syndrome that reduces the host’s
resilience to stressors and is associated with decline in physical and cognitive function,
susceptibility to disease, prolonged hospitalisation, increased vulnerability to adverse drug
effects, morbidity and early death.
There are currently no treatments for these radiation-induced adverse effects and, as a
result, life expectancy and quality of life is reduced for many cancer survivors. No previous
studies have focused on investigating gender specific impact of senolytic drugs or
genetical intervention after radiation-induced frailty in mice.
This study investigates whether SC removal can ameliorate the clinical impact of
radiotherapy in mice and whether these effects are gender-specific. Additionally, this
study considers the impact of single and multiple dose treatment regimens with senolytic
or pharmacogenetic drugs and the ability to alleviate premature radiation-induced frailty,
physical and cognitive decline and impact on lifespan in male and female mice after 5-7
month old mice received fractionated whole body irradiation.
The results demonstrate that female mice are more sensitive to radiation than male mice,
displaying greater levels of premature frailty, cognitive decline and expression of SC
markers as well as reduced lifespan as compared to irradiated males. Irradiated male and
female mice treated with senolytic drugs or pharmacogenetic interventions were also
examined. Late genetic clearance with AP20187 rescued premature frailty, physical and
cognitive decline and, in irradiated female mice, extended lifespan without altering
prevalence of pathologies. Strikingly, a single dose of dasatinib and quercetin treatment
one month post-irradiation prevented premature frailty and physical dysfunction, extended
median lifespan and reduced number of pathologies in irradiated female mice.
These results indicate intervention- and gender-specific elimination of SCs may rescue
cognitive decline and radiation-induced frailty and, thus, promote health and extend
lifespan. This is consistent with current views that interventions that reduce SC levels
present a potential opportunity to counteract the major side effects associated with
radiotherapy and, indeed, a single dose of treatment is sufficient to confer lasting benefits
in both genders.
Description: PhD Thesis2023-01-01T00:00:00ZThe analysis of candidate analgesics utilising a model of nociception derived from human induced pluripotent stem cells
http://theses.ncl.ac.uk/jspui/handle/10443/6091
Title: The analysis of candidate analgesics utilising a model of nociception derived from human induced pluripotent stem cells
Authors: Thornton, Jack Ronald
Abstract: The sensation of pain is an adaptive mechanism vital for the wellbeing of an organism. However,
in the case of chronic pain, this essential mechanism is corrupted, resulting in catastrophic
suffering for the individual and devastating socioeconomic burdens for the community. Cur rent methods used to treat pain are largely ineffective, associated with deleterious unintended
consequences, and diminishing beneficial outcomes. Despite the obvious need and urgency
for improved therapeutics, novel drug development has been severely impacted by the poor
recapitulation of animal model findings to human diseases, and the difficulties associated with
acquiring human primary neuronal tissue. These shortcomings highlight the necessity for a new
stratagem to address the pain problem.
This study demonstrated the efficient differentiation of human induced pluripotent stem cells
(HiPSCs) into cultures possessing key biomarkers associated with the nociceptor phenotype.
HiPSC-derived neurons were capable of generating action potentials in response to noxious
stimulation measurable by both patch clamp and microelectrode array. Furthermore, HiPSC nociceptors derived from inherited erythromelalgia patients were shown to be electrically active
in the absence of stimulation, indicating functional continuity with genetic pain disorders. Drug
screening of 295 compounds through the application of spontaneously active HiPSC-derived
nociceptors resulted in the selection of twelve candidates able to inhibit spike activity by greater
than 40 %. Compound inhibition of HiPSC-derived nociceptor spiking was shown to be
reversible and demonstrated less than 5 % cytotoxicity over 24 hours.
Identification of dose-response relationships for candidate compounds allowed for the cal culation of IC50s, which further indicated efficacy and nontoxicity. Investigation into the
transcriptomic and electrophysiological profiles of selected compounds alluded to promising
mechanisms of action for targeted analgesia. The scale, reproducibility, and effectiveness of this
drug screen demonstrate a high throughput approach to human neuronal disease modelling which
may enable the discovery of new analgesics for the treatment of both general and neuropathic
pain
Description: PhD Thesis2023-01-01T00:00:00ZAnalysis of DNA methylation patterns in hepatocellular carcinoma to identify novel therapeutic targets and biomarkers that predict response to therapy
http://theses.ncl.ac.uk/jspui/handle/10443/6089
Title: Analysis of DNA methylation patterns in hepatocellular carcinoma to identify novel therapeutic targets and biomarkers that predict response to therapy
Authors: Permtermsin, Chalermsin
Abstract: Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting
for over 90% of cases. The treatment outcome for HCC patients is very poor and over 70% of
patients present with disease that is incurable by current therapies. Furthermore, current therapies
for HCC patients often have low efficacy and significant toxicities. Thus, there is a critical need
for the development of novel therapeutic approaches and the optimisation of currently used
therapies to improve the outcomes for HCC patients. We developed a novel bioinformatics
pipeline, which integrates genome-wide DNA methylation and gene expression data, to identify
genes required for the survival of cancer cells but not normal cells. Targeting these genes may
induce "synthetic lethality" (SL), specifically killing cancer cells with little or no impact on healthy
ones. Based on global DNA methylation patterns, five potential HCC subgroups were identified.
Subgroup 2 exhibited the most unique methylation profile and was utilised for SL gene analysis.
This identified two candidate SL genes, T-lymphoma invasion and metastasis 1 (TIAM1) and
lactate dehydrogenase B (LDHB). Available HCC cell lines were characterised for their global
methylation patterns and expression of TIAM1 and LDHB. Analysis releveled SNU182 and
PLC/PRF-5 as potential HCC subgroup 2 representative cell lines (positive expression and
hypomethylation of TIAM1 and LDHB) and HepG2 and Huh-7 (negative expression and
hypermethylation of TIAM1 and LDHB) as non-subgroup 2 cell lines. TIAM1 belongs to a family
of guanine nucleotide exchange factors (GEFs) known to activate RAC1 signalling and plays a role
in cancer cell growth, adhesion, and invasion. The HCC cell lines as well as additional non-HCC
liver cell lines positive for TIAM1 and LDHB SK-Hep1 (cholangiocarcinoma) and HHL5
(immortalised hepatocytes) were used to investigate the functional relevance of TIAM1 utilising
siRNA silencing and a specific TIAM1/Rac1 signalling inhibitor (NSC23766). siRNA targeting
TIAM1 inhibited cell proliferation in TIAM1 positive (subgroup 2) HCC cell lines but had no effect
on TIAM1 negative cell lines and cell proliferation was also suppressed at significantly lower
NSC23766 concentrations in the TIAM1 positive compared with the TIAM1 negative HCC lines.
Thus, confirming TIAM1 as a potential SL gene for HCC subgroup 2. LDHB is a metabolic gene
that encodes the B subunit of the lactate dehydrogenase enzyme, which catalyses the
interconversion of pyruvate and lactate. Since the HCC subgroup 2 representative cell lines exhibit
subgroup specific expression of LDHB, we tested their sensitivity to metabolic inhibitors (2-DG
and metformin) and GNE-140 (LDH inhibitor). The result showed that the sensitivity of 2-DG and
metformin was HCC subgroup 2 independent. In addition, the sensitivity to the LDH inhibitor
GNE-140 did not correlate with LDHB expression status. Radiotherapy is a common treatment
choice for cancer patients. The development of radioresistance is common in numerous cancer
types including HCC, but metabolic interventions have shown promise as radiosensitisers.
Hence, we evaluated radiation synergy with metabolic inhibitors (2-DG and metformin) and its
potential correlation with the LDHB status of the cells. No radiosensitisation effect for either of
the inhibitor individually or in combination was observed in any of the HCC cell lines tested.
However, potential synergy between 2-DG and metformin was observed in some cell lines
independent of their LDHB status. Overall, the study provides evidence towards exploiting TIAM1
as a potential therapeutic target for a subgroup of HCC. In addition, a combination of 2-DG and
metformin should be explored further as a potential treatment strategy for HCC.
Description: PhD Thesis2023-01-01T00:00:00Z