The following chapters provide additional proof of the enduring vitality of theatre about science. They portray a number of diverse initiatives exploring different scientific subjects, with social intervention facets, addressing pressing contemporary issues such as ecological (un)sustainability, mental health prevalence, and the ethical implications of technological advances. They also reveal a progressive integration of theatre companies and practitioners in scientific projects.

Cellular senescence drives aging and age-related dysfunction across multiple tissues, including the brain. Through a high-content, senescent cell-based phenotypic screen of a small panel of natural products, we identified tomatidine, an aglycone of tomatine found in tomatoes, as a previously unrecognized senotherapeutic agent. In senescent human brain microvascular endothelial cells and fibroblasts, tomatidine selectively suppressed SASP expression without affecting p16Ink4a or p21Cip1 levels consistent with a senomorphic effect. In aged mice, tomatidine reduced frailty and improved motor coordination and cognitive performance. These functional benefits were accompanied by reduced senescence markers (p16 Ink4a, p21 Cip1, and telomere-associated DNA damage foci) in liver, skin, and hippocampal neurons, along with decreased neuroinflammation and microglial activation. Tomatidine also diminished brain endothelial cell senescence while enhancing tight junction protein expression, suggesting preserved blood-brain barrier integrity. Together, these findings identify tomatidine as a promising senescence-targeting compound with beneficial effects in aged mice and support its further evaluation in mechanistic and translational studies.

Chronic Insomnia is a prevalent sleep disorder that remains difficult to diagnose due to subjective symptoms and heterogeneous presentations. The most severe form, insomnia with short sleep duration (ISSD), is defined by a total sleep time of less than six hours on polysomnography. However, objective assessments are rarely recommended in diagnostic guidelines, highlighting the need for alternative biomarkers. Disruptions in the circadian clock system may contribute to chronic insomnia, though the extent of these effects remains unclear. In this study, we investigate sleep and circadian rhythm-related alterations in chronic insomnia and its subtypes, ISSD and insomnia with normal sleep duration (INSD), by assessing plasma cortisol, wrist and axillary temperature, and clock gene expression in peripheral blood mononuclear cells (PBMCs). Additionally, we use machine learning to identify the most relevant clock genes for detecting insomnia and classifying its subtypes. Chronic insomnia patients exhibited reduced body temperature rhythms, elevated cortisol levels during wake before sleep, and significant alterations in clock gene expression, including in BMAL1, PER1-2, REV-ERBα, and REV-ERBβ, compared to controls. Most alterations were more significant in the ISSD group. Moreover, associations between clock gene expression, sleep-related parameters and Insomnia Severity Index (ISI) scores were identified. Using machine learning, we identified three genes as sensitive biomarkers distinguishing chronic insomnia from controls and differentiating between ISSD and INSD subtypes. Our findings suggest that circadian markers and machine learning could improve understanding of chronic insomnia and aid biomarker discovery for diagnosis.

Circadian rhythms are endogenous oscillations of approximately 24 h that regulate a wide range of cellular and physiological processes, including gene expression, metabolism, and behavior. These rhythms arise from interconnected transcriptional-translational feedback loops that respond to temporal and environmental cues. Because circadian regulation is highly dynamic, even minor experimental variations can influence phase, amplitude, and rhythmicity, making standardized experimental workflows essential for generating reliable and reproducible results. The goal of the present protocol is to provide a practical and reproducible workflow for synchronizing cultured cells, performing time-course sampling, and analyzing circadian clock gene expression under standard laboratory conditions. The protocol describes serum shock-based synchronization, staggered sample collection over 24-72 h to avoid overnight sampling, ribonucleic acid extraction, complementary deoxyribonucleic acid synthesis, quantitative real-time polymerase chain reaction, and circadian rhythm analysis using appropriate statistical approaches. The workflow also highlights critical experimental considerations, including synchronization conditions, sample quality assessment, reference gene selection, and data analysis, to improve reproducibility across experiments. This method provides an accessible approach for investigating molecular circadian mechanisms and evaluating rhythmic gene expression in cultured cells, facilitating studies of circadian regulation in physiological and disease-related experimental models.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by a CAG over-repetition in the ATXN3 gene, resulting in a toxic gain-of-function in the ataxin-3 protein. Despite all the advances, its molecular mechanisms remain unclear, and no disease-modifying treatments are available. Aging is the major risk factor for neurodegenerative diseases, including Alzheimer’s and Huntington’s. Nuclear membrane proteins (lamins) and related processing proteins like ZMPSTE24 are altered not only in aging but also in neurodegeneration. To explore aging’s role in MJD, we examined age-related markers in human and animal MJD models. Reduced levels of lamins B, C and ZMPSTE24 were observed, along with nuclear shape abnormalities - hallmark of aging. Additionally, overexpressing progerin (mutant lamin A that causes premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS)) in a relevant brain area of a lentiviral MJD mouse model, aggravated MJD-related neuropathology. These findings suggest that aging mechanisms may contribute to MJD progression, offering potential targets for therapy.

The ketogenic diet (KD) is increasingly recognized as a promising therapeutic strategy for neurodegenerative disorders because of its multifaceted impacts on key pathophysiological mechanisms. This review explores the molecular pathways through which KD may protect against neurodegeneration, including the use of ketone bodies as alternative energy substrates, reduction of oxidative stress and inflammation, modulation of autophagy and protein aggregation, and impact on the gut microbiome. The potential benefits of KD are explored across neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and multiple sclerosis, based on both preclinical and clinical evidence that supports its feasibility. However, challenges in long-term safety, patient adherence, and clinical practicality limit its widespread adoption. This review underscores the potential of KD for treating neurodegeneration on the basis of current scientific evidence while highlighting the need for further research to optimize its application and address existing gaps.

The hypothalamus is a central regulator of circadian rhythms, metabolism, and endocrine function, integrating internal and external cues to maintain physiological homeostasis. Aging impairs hypothalamic function, leading to metabolic changes, sleep disturbances, and a higher risk of age-related disease. Laminopathies - rare genetic disorders marked by premature aging - exhibit profound neuroendocrine and circadian rhythm dysfunction, offering insights into mechanisms of hypothalamic aging. The complex interplay between the hypothalamus, circadian rhythms, and systemic aging highlights the critical role of neuroendocrine crosstalk in the regulation of health span and life span. This review summarizes emerging molecular and physiological insights into hypothalamic aging and circadian misalignment, and highlights potential interventions, such as chronotherapy and caloric restriction, that may alleviate hypothalamic alterations and promote healthy aging.

Machado-Joseph disease (MJD) is caused by an abnormal CAG repeat expansion in the ATXN3 gene, leading to the expression of a mutant ataxin-3 (mutATXN3) protein. Patients with MJD exhibit a wide range of clinical symptoms, including motor incoordination. Emerging evidence highlights circadian rhythm disruptions as early indicators and potential risk factors for the progression of neurodegenerative conditions. Circadian rhythms are regulated by internal clocks, with the suprachiasmatic nucleus (SCN) acting as the master pacemaker to synchronize timing across the body's behavioural and physiological functions. While sleep disturbances have been observed in MJD, the role of clock regulation in its pathophysiology remains largely unexplored in spinocerebellar ataxias. This study aimed to investigate circadian rhythms, characterize associated disruptions and uncover the mechanisms underlying clock dysregulation in patients and preclinical models of MJD. Circadian activity in MJD patients was assessed over 2 weeks using actigraphy, while in a YAC-MJD transgenic mouse model, circadian rhythms were examined through: (i) wheel-running experiments; (ii) telemetry-based monitoring of core body temperature; (iii) immunohistochemical analysis of the neuropeptides arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) in the SCN and paraventricular nucleus (PVN); and (iv) quantitative real-time PCR evaluation of clock gene expression in the cerebellum. The impact of mutATXN3 on clock mechanisms was further investigated using Bmal1/Per2-luciferase reporters. MJD patients exhibited a progressive decline in robustness of behavioural rhythms, demonstrated by negative correlations between the circadian function index, rest-activity fragmentation and sleep efficiency with MJD clinical scales. YAC-MJD mice exhibited reduced activity levels and increased behavioural fragmentation, and they required three additional days to re-entrain after a jet lag protocol compared to controls. Disrupted core body temperature rhythms were observed, including a phase advance and elevated temperature (∼1°C) at the onset of the active period. Furthermore, transgenic mice showed reduced levels of VIP and AVP in the SCN and PVN and decreased clock gene expression in the cerebellum. Lastly, we found new mechanistic evidence that wild-type ATXN3 activates the promoters of Bmal1 and Per2, whereas mutATXN3 loses the capacity to drive Per2 upon polyglutamine expansion. Overall, our findings indicate that central clock dysfunction in MJD is associated with impaired clock gene expression and disruptions in activity and temperature rhythms. This study provides the first robust evidence of circadian rhythm dysregulation and underlying mechanisms in MJD, paving the way for identifying new biomarkers and developing novel circadian-based interventions to tackle MJD and possibly other spinocerebellar ataxias.

Rhythmicity is a central feature of behavioral and physiological processes, including sleep, immune responses, and metabolism. Research on brain control of these processes has largely focused on neurons, with less known about the role of clock genes in glial cells. In this study, we addressed the function of glial clocks by targeting the expression of key clock genes in glia of Drosophila melanogaster. Loss of the period (per) gene in glia increases sleep following aseptic injury and loss of either per or timeless (tim) significantly reduces locomotor activity in light:dark cycles and in constant dark, but other than this, the major effect of clock gene loss in glia is on metabolic function. We demonstrate that disruption of either tim or per in glia affects glycogen stores and reduces metabolic rate. Disruption of either tim or per in glia also affects rhythms of feeding and overall food consumption. Notably, these effects of clock disruption are mediated by distinct glial subtypes, especially cortex glia. We propose that the major role of glial clocks is in the control of energy homeostasis and metabolic rhythms, which likely also accounts for effects on locomotor activity. These findings link metabolism and behavior via circadian regulation in glia.

Melatonin, N-acetyl-5-methoxytryptamine, is a tryptophan-derived hormone mostly produced in the pineal gland, despite being synthesized locally at several tissues and organs. This production is rhythmically controlled by complex clock gene networks in the master pacemaker located in the suprachiasmatic nucleus of the hypothalamus. Melatonin is usually secreted only during the dark phase of the day and is essential to synchronize circadian rhythms and neuroendocrine physiological processes. Its main clinical use is associated with the treatment of jet lag and other circadian rhythm sleep disorders, with a growing number of other promising therapeutic applications due to the diverse physiological roles of melatonin. In this review, we explore melatonin and its receptors and provide an updated overview on research concerning the role of melatonin, either as an endogenous molecule or as a drug, in: sleep-wake cycle regulation; circadian rhythms; inflammatory processes that may compromise cardiovascular, respiratory, gastrointestinal, renal, and reproductive system functions; and neurodegenerative disorders such as Alzheimer's and Parkinson's disease. The most recent and promising research findings concerning melatonin synthetic analogs such as agomelatine and ramelteon are highlighted, pointing toward new compounds with promising pharmacological activity while emphasizing their structural differences and advantages when compared to melatonin.

The hypothalamus has been recognized as a regulator of whole-body aging. Neuropeptide Y (NPY), highly abundant in the central nervous system and produced by the hypothalamus, enhances autophagy in this brain region and mediates autophagy triggered by caloric restriction, suggesting a potential role as a caloric restriction mimetic and an aging regulator. Considering that hypothalamic NPY levels decline during aging, we investigated if reestablishment of NPY levels mitigate aging phenotype, using a mouse model of premature aging – Zmpste24−/− mouse. The results show that reestablishing hypothalamic NPY levels delayed aging-associated features, including lipodystrophy, alopecia, and memory. Moreover, these results suggest that strategies that promote maintenance of hypothalamic NPY levels might be relevant to counteract aging progression and age-related deteriorations.

Compromised cellular resilience in bipolar disorder (BD) has been associated with structural brain changes and cognitive deficits caused by perturbation of redox status, endoplasmic reticulum (ER) stress and innate immunity. These crucial cellular events are regulated by the ER‑mitochondria close contacts at mitochondria‑associated membranes (MAM) through Ca2+ transfer and lipids exchange between these organelles. The present study aimed to investigate the structural and functional alterations in MAM during BD early stages using patient‑ and control‑derived cellular models, namely dermal fibroblasts. Morphological alterations in close ER‑mitochondria contacts at MAM occur in BD cells and correlate with functional changes, as shown by lipid droplets accumulation. The MAM dysfunction in BD cells parallels changes in Ca2+ homeostasis, namely inhibition of store‑operated Ca2+ entry (SOCE), ER Ca2+ depletion and attenuation of ER‑mitochondria Ca2+ transfer, as well as enhanced ER and oxidative stress and NOD‑like receptor family pyrin domain‑containing 3 (NLRP3) inflammasome activation leading to sterile inflammation. The absence of inflammasome activation upon lipopolysaccharide exposure supports the compromised ability of BD cells (fibroblasts as well as monocytes) to deal with stressful conditions. In conclusion, MAM disruption is highlighted as a potential pathophysiological mechanism driving impaired cellular resilience in BD. Skin fibroblasts are a particularly attractive cellular model for studying mental illnesses, such as BD, due to the shared developmental origin of epidermal and neural tissues. The ectodermal origins of the skin‑brain axis have been proposed as a novel route for understanding brain development, neurodevelopmental conditions and behavior modulation.

Obstructive sleep apnoea (OSA) is a major public health concern, strongly linked to cardiovascular disease and cancer. Extracellular vesicles (EVs) have emerged as key mediators in intercellular communication, oxidative stress and inflammation, carrying molecules that can influence OSA pathophysiology. However, their role in OSA pathophysiology remains underexplored. This systematic review consolidates current research on EVs in OSA, focusing on their cargo, surface proteins and impact on oxidative stress, inflammation, cancer progression and cardiovascular dysfunction. Registered in the International Prospective Register of Systematic Reviews (ID CRD 42024537136), it explores the intricate links between OSA and EVs to uncover disease mechanisms and identify potential biomarkers. The search was conducted in PubMed/Medline and Web of Science databases to identify studies exploring OSA and EVs in clinical studies, animal studies and in vitro studies. Among the 600 unique studies screened, 27 met the inclusion criteria. These studies demonstrated that OSA-derived EVs influence key biological processes, such as endothelial dysfunction, inflammation and tumour cell proliferation. Transcriptomic and proteomic analyses revealed dysregulation of specific microRNAs and proteins in EVs from OSA patients in comparison with controls. Notably, EVs studies in clinical, animal and in vitro settings were shown to enhance cancer cell migration and endothelial dysfunction, underscoring their potential as biomarkers for OSA-related comorbidities. EVs hold great promise as minimally invasive, cost-effective biomarkers for understanding OSA mechanisms, diagnosis and prognosis. However, stricter characterisation and comprehensive profiling of their dynamics and cargo are essential to standardise methodologies and clarify their role in the disease.

Infertility affects millions of couples globally, with male factors contributing up to 50 % of cases. Despite the existing Assisted Reproduction Technologies (ART), decreased sperm quality and male infertility persist. Evidence suggests that sleep is crucial for normal reproductive function, and the noticeable increase in infertility rates aligns with the growing prevalence of sleep deprivation, suggesting a connection between both conditions. This literature review explores the possible links between sleep disturbances, with a special emphasis on insomnia, the most prevalent sleep disorder, and male reproductive health. It delves into the latest findings regarding factors such as sperm quality, hormone levels, and overall reproductive function from studies in mammals, both rodents and humans. Through a concise synthesis of these findings, we unveil potential mechanisms and provide crucial insights for clinicians and researchers in the field of reproductive health.