Developmental origins of polycystic ovary syndrome : role of early adverse life events on adult health

The research focus of this thesis is polycystic ovary syndrome (PCOS), the most common endocrine disorder among women of reproductive age, associated with reproductive, cardio-metabolic, and mental health complications. Despite the high prevalence, little is known about the etiology of the syndrome. The scope of the current thesis was to investigate aspects of the developmental origins of the syndrome, with focus on the impact of adverse environmental factors during pregnancy on PCOS-associated features in the offspring. To reach these aims we employed three mouse model of PCOS and data from a Swedish national register-based cohort study. The thesis is divided into two parts. Part 1 investigates the effects of maternal androgen excess and maternal obesity on the mental health of adult male and female offspring. Study I demonstrated that maternal androgen exposure increases anxiety-like behavior in the first generation of female mouse offspring, but not male offspring, while maternal obesity affects male offspring behavior, but not female offspring. This sexually dimorphic response to the two prominent environmental stimuli was further supported by sex-specific changes in gene expression within the amygdala and hypothalamus. Study II provided evidence that daughters of women with PCOS are at increased risk to be diagnosed with anxiety disorders, but not the sons, using a Swedish national register-based cohort study. It further showed that maternal androgen exposure can lead to transgenerational transmission of anxiety-like behavior in the third generation of female mice, but not the male offspring, without major impact of maternal obesity. Finally, the male germline (first generation of male offspring with no change in behavior) did also transgenerationally transmit an anxiety-like behavior to the third generation of male offspring. Part 2 investigates the effects of an adverse maternal and/or postnatal environment on the cardiovascular and metabolic health of females, using mouse models of PCOS. Study III revealed that maternal androgen excess leads to cardiac hypertrophy in adult female offspring, accompanied by gene expression changes in the left ventricle, without the presence of metabolic dysfunction. It was further shown that this adverse cardiac phenotype is the result of an early cardiac reprogramming. In addition, cardiovascular assessment of a mouse model of PCOS with continuous exposure to androgens from prepuberty to adulthood, demonstrated cardiac hypertrophy and increased vasocontractile responses, while simultaneous administration of the antiandrogen flutamide could only partially prevent the observed phenotype. Finally, in study IV, developmental, reproductive and metabolic complications were revealed in a transgenic mouse model of PCOS that overexpresses ovarian nerve growth factor. Ovarian excess of nerve growth factor led to developmental defects in the female fetus, including growth restriction, reduction of germ cell number and delayed primary oocyte maturation. In addition, the adult transgenic female mice displayed disrupted estrous cyclicity and ovarian expression changes of steroidogenic genes and epigenetic markers. Lastly, these mice developed metabolic complications, as shown by impaired glucose metabolism, aberrant adipose tissue function, and liver steatosis

An FTIR Microspectroscopy Ratiometric Approach for Monitoring X-ray Irradiation Effects on SH-SY5Y Human Neuroblastoma Cells

The ability of Fourier transform infrared (FTIR) spectroscopy in analyzing cells at a molecular level was exploited for investigating the biochemical changes induced in protein, nucleic acid, lipid, and carbohydrate content of cells after irradiation by graded X-ray doses. Infrared spectra from in vitro SH-SY5Y neuroblastoma cells following exposure to X-rays (0, 2, 4, 6, 8, 10 Gy) were analyzed using a ratiometric approach by evaluating the ratios between the absorbance of significant peaks. The spectroscopic investigation was performed on cells fixed immediately (t0 cells) and 24 h (t24 cells) after irradiation to study both the initial radiation-induced damage and the effect of the ensuing cellular repair processes. The analysis of infrared spectra allowed us to detect changes in proteins, lipids, and nucleic acids attributable to X-ray exposure. The ratiometric analysis was able to quantify changes for the protein, lipid, and DNA components and to suggest the occurrence of apoptosis processes. The ratiometric study of Amide I band indicated also that the secondary structure of proteins was significantly modified. The comparison between the results from t0 and t24 cells indicated the occurrence of cellular recovery processes. The adopted approach can provide a very direct way to monitor changes for specific cellular components and can represent a valuable tool for developing innovative strategies to monitor cancer radiotherapy outcome

NEPTUNE (Nuclear process-driven Enhancement of Proton Therapy UNravEled)

Protontherapy is an important radiation modality that has been used to treat cancer for over 60 years. In the last 10 years, clinical proton therapy has been rapidly growing with more than 80 facilities worldwide [1]. The interest in proton therapy stems from the physical properties of protons allowing for a much improved dose shaping around the target and greater healthy tissue sparing. One shortcoming of protontherapy is its inability to treat radioresistant cancers, being protons radiobiologically almost as effective as photons. Heavier particles, such as 12C ions, can overcome radioresistance but they present radiobiological and economic issues that hamper their widespread adoption. Therefore, many strategies have been designed to increase the biological effectiveness of proton beams. Examples are chemical radiosensitizing agents or, more recently, metallic nanoparticles. The goal of this project is to investigate the use of nuclear reactions triggered by protons generating short-range high- LET alpha particles inside the tumours, thereby allowing a highly localized DNA-damaging action. Specifically, we intend to consolidate and explain the promising results recently published in [2], where a significant enhancement of biological effectiveness was achieved by the p-11B reaction. Clinically relevant binary approaches were first proposed with Boron Neutron Capture Therapy (BNCT), which exploits thermal neutron capture in 10B, suitably accumulated into tumour before irradiation. The radiosensitising effects due to the presence of 10B will be compared to those elicited by p-11B, using the same carrier and relating the observed effects with intracellular 11B and 10B distribution as well as modelled particle action and measured dose deposition at the micro/nanometer scale. Moreover, the p-19F reaction, which also generates secondary particles potentially leading to local enhancement of proton effectiveness, will be investigated. The in-vivo imaging of 11B and 19F carriers will be studied, in particular by optimizing 19F-based magnetic resonance

Micro Sensing of pH Levels in Biological Samples by Graphene-Based Raman Spectroscopy

Graphene provides a unique way for sensing local pH level of substances on micrometric scale, with important implications for the monitoring of cellular metabolic activities where protonic excretion could occur. Doping modifications of graphene, induced by the contact of the graphene with different pH solutions were investigated by micro-Raman spectroscopy in order to develop a pH biosensor. To test the developed biosensor with real biological systems, the pH values of cell culture media in different conditions were evaluated

Sleep Respiratory Disorders in Children and Adolescents with Cystic Fibrosis and Primary Ciliary Dyskinesia

Cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are genetic respiratory diseases featured by chronic upper and lower airway inflammation and infection, mainly due to impaired mucociliary clearance due to genetic mutations. Sleep is crucial to healthy children’s normal physical and psychological development and has an important value in chronic respiratory diseases. Impaired sleep quality, such as sleep deprivation or insufficient sleep during the night, and sleep respiratory disorders (SRDs) are common in 5% to 30% of the general population. Sleep disruption leads to attention deficits, daytime sleepiness, fatigue and mood disorders and correlates to a worsened quality of life. Furthermore, sleep respiratory disorders (SRSs) are under-recognized comorbidities in CF and PCD patients. SRSs include a spectrum of symptoms ranging from primary snoring through upper airway resistance to obstructive sleep apnea (OSA), nocturnal hypoventilation and hypoxemia occurring in people with moderate to severe lung disease and damaging the disease-related outcomes and quality of life. Effective screening during sleep with polysomnography is very important for the timely initiation of efficacious treatments and to prevent worsened respiratory, metabolic and cardiovascular outcomes. However, the impact of SRDs on health and quality of life is still underinvestigated.</p

Cystic fibrosis transmembrane conductance regulator (CFTR): beyond cystic fibrosis

Abstract Background The cystic fibrosis transmembrane conductance regulator (CFTR) gene has been traditionally linked to cystic fibrosis (CF) inheritance in an autosomal recessive manner. Advances in molecular biology and genetics have expanded our understanding of the CFTR gene and its encoding products expressed in different tissues. Aim The study's aim consists of reviewing the different pathological CF phenotypes using the existing literature. We know that alterations of the CFTR protein's structure may result in different pathological phenotypes. Methods Open sources such as PubMed and Science Direct databases have been used for this review. We focused our selection on articles published within the last 15 years. Critical terms related to the CFTR protein have been used: "CFTR AND cancer," "CFTR AND celiac disease," "CFTR AND pancreatitis," "children," "adults," "genotype," "phenotype," "correlation," "mutation," "CFTR," "diseases," "disorders," and "no cystic fibrosis." Results We analyzed 1,115 abstracts in total. Moreover, only 189 were suitable for the topic. We focused on the role of CFTR in cancer, gastrointestinal disorders, respiratory diseases, reproductive system, and systemic hypertension. Conclusions Mutations in CFTR gene are often associated with CF. In this review, we highlighted the broad spectrum of alterations reported for this gene, which may be involved in the pathogenesis of other diseases. The importance of these new insights in the role of CFTR relies on the possibility of considering this protein/gene as a novel therapeutic target for CF- and CFTR-related diseases

Nasal Nitric Oxide and Nasal Cytology as Predictive Markers of Short-Term Sublingual Allergen-Specific Immunotherapy Efficacy in Children with Allergic Rhinitis

Background Few studies have been conducted on the short-term response to sublingual immunotherapy (SLIT). Objective The purpose of our experimental trial was to evaluate if two markers such as nasal nitric oxide (nNO) and nasal cytology could be useful to identify a precocious clinical efficacy of SLIT treatment. Methods We enrolled 34 children aged 6 to 14 years old with diagnosis of allergic rhinitis (AR) and documented sensitization towards house dust mites. We started allergoid-monomeric tablets immunotherapy along with any conventional therapy for AR and we evaluated at baseline (T0), after one (T1), two (T2), three (T3), and six months (T6) the effects of the treatment through the study of: i) a visual analogue scale (VAS 1-10); ii) measurement of nNO; iii) measurement of FeNO; iv) nasal cytology; v) spirometry; and vi) evaluation of any conventional therapy. Results We observed an improvement in symptoms evaluated by global VAS (T0 vs. T6: 47.13 vs. 17.57; p &lt; .05) and a statistically significant reduction of nNO (1035.2 +/- 956.08 vs. 139.2 +/- 59.01; p &lt; .05). In this case, significance was reached when the patients completed the 6 months of treatment. Cytological evaluation revealed significant reduction in nasal eosinophils (T0 vs. T6: 87% vs. 16%; p &lt; .01). Moreover, at T0, 56% of patients had also neutrophils that were reduced up to the 8% at T6 (p &lt; .05). Conclusions Our data confirm the effectiveness of SLIT treatment from a clinical perspective and identifies two biomarkers, such as nNO and nasal cytology, as predictive of treatment efficacy in the short term

Evaluation of Proton-Induced Biomolecular Changes in MCF-10A Breast Cells by Means of FT-IR Microspectroscopy

Radiotherapy (RT) with accelerated beams of charged particles (protons and carbon ions), also known as hadrontherapy, is a treatment modality that is increasingly being adopted thanks to the several benefits that it grants compared to conventional radiotherapy (CRT) treatments performed by means of high-energy photons/electrons. Hence, information about the biomolecular effects in exposed cells caused by such particles is needed to better realize the underlying radiobiological mechanisms and to improve this therapeutic strategy. To this end, Fourier transform infrared microspectroscopy (-FT-IR) can be usefully employed, in addition to long-established radiobiological techniques, since it is currently considered a helpful tool for examining radiation-induced cellular changes. In the present study, MCF-10A breast cells were chosen to evaluate the effects of proton exposure using -FT-IR. They were exposed to different proton doses and fixed at various times after exposure to evaluate direct effects due to proton exposure and the kinetics of DNA damage repair. Irradiated and control cells were examined in transflection mode using low-e substrates that have been recently demonstrated to offer a fast and direct way to examine proton-exposed cells. The acquired spectra were analyzed using a deconvolution procedure and a ratiometric approach, both of which showed the different contributions of DNA, protein, lipid, and carbohydrate cell components. These changes were particularly significant for cells fixed 48 and 72 h after exposure. Lipid changes were related to variations in membrane fluidity, and evidence of DNA damage was highlighted. The analysis of the Amide III band also indicated changes that could be related to different enzyme contributions in DNA repair

FT-IR Transflection Micro-Spectroscopy Study on Normal Human Breast Cells after Exposure to a Proton Beam

Fourier transform infrared micro-spectroscopy (mu-FT-IR) is nowadays considered a valuable tool for investigating the changes occurring in human cells after exposure to ionizing radiation. Recently, considerable attention has been devoted to the use of this optical technique in the study of cells exposed to proton beams, that are being increasingly adopted in cancer therapy. Different experimental configurations are used for proton irradiation and subsequent spectra acquisition. To facilitate the use of mu-FT-IR, it may be useful to investigate new experimental approaches capable of speeding up and simplifying the irradiation and measurements phases. Here, we propose the use of low-e-substrates slides for cell culture, allowing the irradiation and spectra acquisition in transflection mode in a fast and direct way. In recent years, there has been a wide debate about the validity of these supports, but many researchers agree that the artifacts due to the presence of the electromagnetic standing wave effects are negligible in many practical cases. We investigated human normal breast cells (MCF-10 cell line) fixed immediately after the irradiation with graded proton radiation doses (0, 0.5, 2, and 4 Gy). The spectra obtained in transflection geometry showed characteristics very similar to those present in the spectra acquired in transmission geometry and confirm the validity of the chosen approach. The analysis of spectra indicates the occurrence of significant changes in DNA and lipids components of cells. Modifications in protein secondary structure are also evidenced

An FTIR Spectroscopy Investigation on Different Methods of Lipid Extraction from HepG2 Cells

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