Neuroblastoma (NB) is a very heterogeneous tumour derived from undifferentiated cells of the neural crest. It is the most common extracranial solid tumour in children, characterised by a large variability of the clinical outcome. The clinically aggressive form of the disease, the high-risk neuroblastoma, affects about 50% of patients, which, unfortunately, despite the intensity of therapies, have a survival rate of less than 50%. Although the introduction of two new therapeutic solutions based on the use of 13-cis-retinoic acid and the immunotherapy with a chimeric monoclonal antibody against the GD2 ganglioside, 20% of patients affected by the high-risk disease are still entirely refractory to treatments, and 60% will relapse. In this panorama, identifying new strategies specific for critical NB targets is endowed with great potential to improve the survival rate and long-term quality of life and reduce the elevated toxicity of current treatments. LIN28B is an RNA binding protein extensively overexpressed in NB. Its exogenous expression in mouse sympatho-adrenergic lineage is able to reproduce the human disease, underlying the importance of LIN28B in NB pathogenesis. LIN28B prevents the maturation of let-7 miRNA family members, an important group of tumour suppressors that induce differentiation and, at the same time, decrease cell proliferation. We hypothesised that interfering with the LIN28B/let-7 miRNA interaction could lead to an increase in let-7 miRNA levels and, consequently, to a decrease in cell proliferation and an induction of cell differentiation, ultimately reducing NB aggressiveness. First, we created NB cell lines with stable LIN28B down-regulation, and we observed an expected increase in let-7 miRNA levels. We then verified if the rise in the let-7 miRNAs could induce the cells' differentiation by analysing a panel of stemness and differentiation markers such as SOX2, SOX9 and β-III-tubulin, detecting a decrease in the stemness markers and an increase in the differentiation-related markers. Following a high-throughput screening, performed and validated with two orthogonal biochemical techniques, the Alpha screen and the REMSA, we identified molecule A as the molecule with the best inhibitory activity on the LIN28B/let-7 miRNA interaction. After the biochemical validation, we proceeded to assess molecule A activity in vitro on NB cell lines. Molecule A resulted to be very unstable in cell culture conditions, therefore we decided to include the molecule in PLGA-PEG nanoparticles to preserve its stability in solution and improve its activity. Upon encapsulation, we observed a substantial increase in molecule A effects, leading to a strong increment in mature let-7 miRNAs and a consequent inhibition of cellular growth. Finally, we tested if the let-7 miRNAs increment caused by molecule A treatment was sufficient to induce NB cells differentiation, as observed with stable LIN28B-downregulation. We detected an increase in differentiation marker levels suggesting that the treatment with Molecule A nanoparticles is able to lead to the induction of neuronal differentiation processes in NB cells. Although these last results need to be confirmed with further experiments, they clearly show that the LIN28B/let-7 miRNA axis represents a good therapeutic target and that molecule A and/or other molecules able to interfere with this interaction deserve further preclinical and clinical evaluation.

Diverse clinical presentation in neurodevelopmental disorders (NDDs) leads to difficulty in matching individuals with effective treatments. Autism spectrum disorders (ASD) and attention deficit hyperactivity disorder (ADHD) are the two most prevalent neurodevelopmental disorders (NDDs), characterized by deficits in communication, social interactions, and behaviours. There is high within-diagnosis heterogeneity and striking overlap between diagnoses. The literature suggests that current diagnostic criteria do not align well with behaviour metrics. Therefore, identifying novel biomarkers underlying behaviour in NDDs may provide a reliable way to group individuals with similar behavioural phenotypes. This thesis examines how gut-immune biology is linked to clinical heterogeneity in children with NDDs. The first study used unsupervised machine learning to cluster typically developing (TD), ADHD, and ASD participants by their behaviour metrics in a diagnosis-agnostic approach. The results produced a six-cluster solution, five of which were a mix of all diagnostic categories. Further, gastrointestinal (GI) symptoms were mapped to the clusters, revealing a link between constipation, social communication deficits and restrictive-repetitive behaviours. The second study used hierarchical clustering to group TD and NDD participants based on a profile of gut and inflammatory markers. Participants clustered into two biotypes, both containing TD and NDD participants. Additionally, using regression analysis, novel markers were linked to anxiety. The third study evaluated the multisite biospecimen collection protocol of the Province of Ontario Neurodevelopmental Disorders (POND) Network. The final study used biospecimens collected from the POND network to phenotype peripheral blood mononuclear cells in TD and NDD participants. In NDD groups, monocyte and B cell activation markers were differentially expressed compared to TD. Overall, this thesis demonstrates that gut-immune mechanisms contribute to clinical heterogeneity in a subset of people and contribute to the search for biomarkers in NDDs. / Thesis / Doctor of Philosophy (PhD) / Autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) are diagnosed based on behavioural symptoms. However, symptoms can vary a lot from person to person, and some symptoms are shared between ASD and ADHD. Understanding the biological reasons for symptom differences between people can help pinpoint treatments which work best for an individual. This thesis looks at the role of the gut and immune system in ASD and ADHD. Blood samples and behaviour questionnaires were collected to study how immune cells, inflammation, and intestinal permeability shape behaviour symptoms. The results show that diagnosis is not the most accurate way to group people. Anxiety symptoms were different when people were grouped based on their inflammation levels. Also, specific immune cells appear to work differently in people with ASD and ADHD. These findings clarify some of the biology that affects behavioural symptoms in ASD and ADHD.

Bakgrund: Demens drabbar en stor del av den äldre befolkningen och antalet insjuknade väntas stadigt stiga. Det finns i dagsläget ingen sjukdomsbotande behandling och etiologin är oklar. Hypotalamus-hypofys-binjurebark (HPA)-axeln är ett neuroendokrint system som aktiveras vid exponering för endo- respektive exogena stressorer. Aktivering leder till frisättning av glukokortikoider från binjurebarken. Cirkulerande glukokortikoider (kortisol) påverkar kognitiva funktioner och verkar ha effekt på plasticiteten vid hippocampus, som är en region viktig för minnesfunktioner. Förhöjda kortisolhalter har korrelerats med snabbare kognitiv nedgång och pågående Alzheimers sjukdom. HPA-axeldysfunktion och förhöjda kortisolhalter har föreslagits vara en möjlig bidragande orsak till kognitiv funktionsnedsättning och demens. Syfte: Undersöka om HPA-axeldysfunktion med förhöjda kortisolhalter bidrar till demensutveckling. Metod: Systematisk litteratursökning med på förhand implementerade inklusions- respektive exklusionskriterier. Sökningar utfördes på databaserna Pubmed och Web of Science med sökorden ”dementia”, ”Alzheimer’s”, ”longitudinal”, ”cortisol”. Resultat: Av sex granskade artiklar kunde fem korrelera förhöjda kortisolhalter med senare nedsatt kognitiv förmåga eller demens. Resultaten varierade beroende på vilka effektvariabler som användes i samband med analys. Slutsats: Inkluderade studier uppvisade viss ökad förekomst av HPA-axeldysfunktion och förhöjda kortisolhalter i samband med senare demens eller kognitiv funktionsnedsättning. Det går dock inte att utesluta att förhöjda kortisolhalter orsakas av patofysiologiska förändringar i samband med det begynnande sjukdomsförloppet. / Background: Dementia affects a large part of the elderly population, and the incidence is expected to keep rising. However, there is no available disease-curing treatment nor is the etiology yet fully understood. The hypothalamus-pituitary-adrenal (HPA) axis is a neuroendocrine system that is activated under exposure to endo- and/or exogenous stressors. Activation leads to the release of glucocorticoids from the adrenal cortex. Circulating glucocorticoids (cortisol) affects cognitive functions including hippocampal plasticity. Elevated cortisol levels have been associated with ongoing Alzheimer’s disease and a more rapid cognitive decline. Thus, HPA axis dysfunction with elevated cortisol levels have been suggested as a possible contributor to cognitive impairment and dementia. Objective: To investigate if HPA axis dysfunction and elevated cortisol levels are a contributing cause of dementia. Method: Systematic literature search with pre-implemented inclusion and exclusion criteria. Searches were carried out on Pubmed and Web of Science using the words "dementia", "Alzheimer's", "longitudinal", "cortisol". Results: Associations between elevated cortisol levels and later cognitive decline or dementia was made in five out of six reviewed studies. The results varied depending on the effect variables used in connection with the analysis. Conclusion: Reviewed studies showed some degree of associations between high cortisol levels and later cognitive decline or dementia. It is however impossible to rule out the possibility of elevated cortisol levels as part of the initial disease progression, rather than a cause of disease.

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