1,145 research outputs found

    From cancer to forensics: the immunohistochemical characterization of TP63, TP53, and MDM2 proteins tissue expression in skin basal cell and squamous cell carcinomas

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    Skin cancer is one of the most occurring cancers in the world and its occurrence is rising. Many factors contribute to the development of skin cancers such as UV light exposure, chronic inflammation, and genetic susceptibility. Cancers such as basal cell carcinomas and squamous cell carcinomas arise from cells within the epidermis. TP53 and TP63, members of the p53 protein family, play a major role in skin tumorigenesis and progression. While p53 is the most mutated gene in human cancers, p63 is seldom mutated in cancers. The functions of p53, “guardian of the genome”, have been well documented and studied. Nonetheless, many studies show that p63 is overexpressed in tumors. Other studies show that p63 is lost during tumorigenesis and cancer progression. Consequently, whether the p63 gene is a tumor suppressing gene or an oncogene remains a matter of the p63 protein’s isoforms present in these tumors and their interactions with p53 and MDM2. This challenge has two facets. First, the multiple spliced isoforms of p53 and p63 which have different functions in skin development as well as skin cancers. Second, the Mouse Double Minute 2 (MDM2) protein, a negative regulator of p53, which has been shown to have different proteomic affinities by which it binds these different p53 and p63 isoforms. The findings of this project are geared toward characterizing and semi-quantifying the histopathological expression of p63, p53 and MDM2 in cutaneous basal cell carcinomas as well as squamous cell carcinomas, while addressing the aggregation propensities between these proteins. The project also aims to summarize the forensic use of p53 and p63 as potential biomarkers for the estimation of age in antemortem and postmortem wounds and lesions. We have used advanced softwares such as ImageJ from the NIH and QuPath to extract important data to better characterize the layer-by-layer expression of p53, p63, and MDM2 in specific areas such as the epidermis, the dermal layer, stroma, and tumor nests. We used multiple skin tumor biopsies and excisions from three different skin cancer patients. The molecular simulations using the PASTA 2.0, AGGRESCAN, and FELLS webservers is to compare and contrast the aggregation propensities of p53 and p63 and what locations of their protein sequences are hot spots for aggregation. Results showed that p63 has the most stain intensity and was strictly nuclear in all three cases (p-value of 0.0007). The consistent expression of p63 in all three cases indicates the important role of p63 in the tumorigenesis of skin epithelial cells and dysplasia. MDM2 has been shown to have a strong stain intensity both nuclear and cytoplasmic in all three cases but was of no significance in differentiating our cases (p-value of 0.718). p53 was partially absent in all three cases and had a weak stain intensity when present (p-value of 0.086). Data from the FELLS, PASTA 2.0, and AGGRESCAN showed that p53 has the highest number of residues susceptible to aggregation at 27.48% (108 residues), whereas p63 and MDM2 have aggregation percentages of 7.94% (54 residues) and 3.67% (18 residues) respectively. Clinically, studies show that p63, particularly the ∆Np63 isoform, has an essential role in epithelial wound repair. Forensically, studies show that p53’s expression increased in wounds with an interval of post-infliction of three to 77 days. This makes p53 a potential candidate for wound age estimation in ante-mortem wounds with longer survival time after the injury. Research also shows that these later conclusions cannot be applied to postmortem wounds and that further research is needed to evaluate the potential use of p53 in a postmortem setting

    AFM-STED correlative nanoscopy provides a new view on the formation process of misfolded protein aggregates

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    The main part of my PhD work focused on the application of an advanced integrated technique, based on the coupling of an atomic force microscope (AFM) and a stimulated emission depletion (STED) microscope in the study of amyloid fibrils formation. This coupled system allows the acquisition of super-resolution fluorescence images, perfectly overlapped with AFM topography. Exploiting the extended capability offered by this technique, I highlighted some important features on the mechanisms followed by the labeled and unlabeled proteins through their aggregation pathway. The results demonstrates that labeled molecules are involved only in selected pathways of aggregation, among the multiple that are present in the aggregation reaction. In a second part of my work, I investigated the process of interaction between Alpha-synuclein (\u3b1-Syn), the pathological peptide associated to the Parkinson\u2019s disease, and model lipid membranes. The aim of this study was to identify molecular mechanisms that are indicated as the base of neurodegeneration, not only in Parkinson\u2019s disease, but also in a large class of disorders, indicated as protein misfolding diseases

    Factors Influencing Huntingtin Aggregation at Surfaces: Implications for Huntington’s Disease

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    Huntington’s Disease (HD) is a genetic, neurodegenerative disease characterized by an abnormal polyglutamine (polyQ) expansion in the first exon of the huntingtin protein (htt). The polyQ domain facilitates aggregation and initiates the formation of a diverse collection of aggregate species, including fibrils, oligomers and annular aggregates. The first 17 amino acids of htt (Nt17) directly flank the polyQ domain and is a key factor in htt’s association to membranous structures. In addition to Nt17 being an amphipathic αhelix, it also promotes aggregation through self-association and contains numerous posttranslational modifications (PTMs) that can modulate toxicity and subcellular localization. For in depth understanding of these mechanisms, particularly in the presence of lipid membrane surfaces, the PTM phosphorylation and macromolecular crowders found in subcellular environments were explored. Through the application of phosphomimetic mutations of htt to a variety of lipid systems, lipid-specific impacts of electrostatic interactions involved in htt/lipid interactions were elucidated. Cytosolic conditions mimicked through the addition of macromolecular crowders and htt were evaluated at both solid/liquid and membrane/liquid interfaces, with each crowder having a distinct effect on htt aggregation. The results presented here aid in the understanding of the multi-faceted nature of htt aggregation in the presence of cellular and subcellular surfaces

    Development of an amyloid protein-based composite biomaterial for coating applications

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    Streptomyces bacteria are highly versatile micro-organisms, which have been recognised as potent biochemical, soil and biomaterials engineers. In particular, functional non-pathogenic amyloid proteins can be formed from the expression of ÎČ-sheet proteins. These ÎČ-sheet proteins known as chaplins, which aggregate to form a fibrillar morphology, has been shown to protect against desiccation in hydrophobic environments. These robust Chaplin proteins have served here as a source of inspiration for materials development, based on the chaplins’ ability to modulate the properties of its own surface and that of its natural environment.This study has developed more economical and environmentally friendlier methods for chaplin protein production by replacing the existing TES buffer for a potassium bicarbonate buffer and by modifying the downstream processing to assist in the removal of trifluoroacetic acid. Combinations of different media and buffers were tested for alternative fermentations that support Streptomyces morphological differentiation in liquid media, in which a potassium bicarbonate buffer system proved as efficient as well as more economical when compared to conventional fermentations with expensive organic buffer systems that support differentiation. Downstream processing of amyloid proteins was furthermore improved by adopting synthetic peptide procedures resulting in an environmentally friendlier amyloid purification method. This modified medium was also demonstrated within a bioreactor at 1.7 L scale which further enhances the economic benefit which could be implemented for production of other secondary metabolites.The resulting chaplin proteins were then applied with ÎČ-glucans to form a biocomposite for different industrial applications. Material properties and anti-corrosion were determined by goniometry and high-resolution imaging, and by qualitative and quantitative electrochemistry. Our protein-based corrosion resistant nano-coating has great potential for the manufacturing, defence and other industries, including healthcare and biomaterials manufacturing

    From Research to Diagnostic Application of Raman Spectroscopy in Neurosciences: Past and Perspectives

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    In recent years, Raman spectroscopy has been more and more frequently applied to address research questions in neuroscience. As a non-destructive technique based on inelastic scattering of photons, it can be used for a wide spectrum of applications including neurooncological tumor diagnostics or analysis of misfolded protein aggregates involved in neurodegenerative diseases. Progress in the technical development of this method allows for an increasingly detailed analysis of biological samples and may therefore open new fields of applications. The goal of our review is to provide an introduction into Raman scattering, its practical usage and also commonly associated pitfalls. Furthermore, intraoperative assessment of tumor recurrence using Raman based histology images as well as the search for non-invasive ways of diagnosis in neurodegenerative diseases are discussed. Some of the applications mentioned here may serve as a basis and possibly set the course for a future use of the technique in clinical practice. Covering a broad range of content, this overview can serve not only as a quick and accessible reference tool but also provide more in-depth information on a specific subtopic of interest

    Cardiac Amyloidosis and Endomyocardial Biopsy: Correlation of Extent and pattern of Deposition with Amyloid Immunophenotype by immunogold in a single institution

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    Background: Amyloidosis is an infiltrative systemic disease characterized by the extracellular deposition of fibrillar proteins in different organs leading to tissue damage and dysfunction. Cardiac involvement by amyloidosis is common. The identification of the type of amyloid deposits is critical to the correct diagnosis and treatment. Several methods are currently used to characterize amyloid deposits. Aim of the study: The aims of the present study were to (1) investigate the accuracy and reliability of light microscopy compared to immune-electron microscopy (IEM) study in diagnosis and characterization of cardiac amyloidosis on fixed paraffin-embedded tissues; (2) to study at light microscopy the association in endomyocardial biopsies (EMB) between the amyloid burden and its patterns of deposition (perimisial, interstitial nodular-like, perivascular) and the type of amyloid characterized by means of IEM; (3) to evaluate the diagnostic value of abdominal fat pad excisional biopsies (FPEB) vs endomyocardial biopsy (EMB) when both are performed, (4) and to find clinico-pathological correlations in cases of cardiac amyloidosis. Methods: Consecutive EMBs, of patients with clinical evidence of hypertrophic-restrictive cardiomyopathy, suspected to be due to amyloidosis, submitted to our Pathology Unit over the last 5 years, were re-examined. Cases with FPEB besides EMB were also reviewed. All EMBs underwent to H&E, Congo Red and Thioflavin stains. IEM, performed with gold-labelled antibodies against light chains (AL, kappa light chains, lambda light chains), and transthyretin (TTR), was used to identify specific amyloid fibrils. Forty cases of the Hospital of Padua were re-examined to find clinical-pathological correlations. Results: In the time interval 2017-2022, a series of 171 EMBs were collected (118 males, 69%; average age at diagnosis 66.6±11.5 years). A subgroup of 36 patients (21%) underwent also FPEB; in 27 (75%) of them, EMB was positive, 21 of which negative at FPEB. In 45 cases (26%) EMB amyloid deposits were not identified by means of H&E, Congo Red, and Thioflavin stains nor by IEM. In the remaining 126 EMB cases (74%), IEM was diagnostic, revealing even small amounts of amyloid fibrils, positive for AL in 82/126 (65%) and TTR in 44/126 (35%). Both the interstitium and the vessels were affected, with different burden based on the type of amyloid. Cardiac structural damage and dysfunction is more relevant in ATTR amyloidosis. Conclusion: IEM represents a sensitive, reliable, and low-cost method for amyloid typing. It can be used to test the reliability of light microscopy. EMB with IEM amyloid typing is crucial to establish diagnosis, prognosis, and appropriate treatment. Localized amyloidosis can only be diagnosed by biopsy of the affected organ or tissue. Choosing the correct tissue/organ to biopsy is essential to avoid false negatives and delays to diagnosis. The choice of a surrogate tissue should be discouraged.Background: Amyloidosis is an infiltrative systemic disease characterized by the extracellular deposition of fibrillar proteins in different organs leading to tissue damage and dysfunction. Cardiac involvement by amyloidosis is common. The identification of the type of amyloid deposits is critical to the correct diagnosis and treatment. Several methods are currently used to characterize amyloid deposits. Aim of the study: The aims of the present study were to (1) investigate the accuracy and reliability of light microscopy compared to immune-electron microscopy (IEM) study in diagnosis and characterization of cardiac amyloidosis on fixed paraffin-embedded tissues; (2) to study at light microscopy the association in endomyocardial biopsies (EMB) between the amyloid burden and its patterns of deposition (perimisial, interstitial nodular-like, perivascular) and the type of amyloid characterized by means of IEM; (3) to evaluate the diagnostic value of abdominal fat pad excisional biopsies (FPEB) vs endomyocardial biopsy (EMB) when both are performed, (4) and to find clinico-pathological correlations in cases of cardiac amyloidosis. Methods: Consecutive EMBs, of patients with clinical evidence of hypertrophic-restrictive cardiomyopathy, suspected to be due to amyloidosis, submitted to our Pathology Unit over the last 5 years, were re-examined. Cases with FPEB besides EMB were also reviewed. All EMBs underwent to H&E, Congo Red and Thioflavin stains. IEM, performed with gold-labelled antibodies against light chains (AL, kappa light chains, lambda light chains), and transthyretin (TTR), was used to identify specific amyloid fibrils. Forty cases of the Hospital of Padua were re-examined to find clinical-pathological correlations. Results: In the time interval 2017-2022, a series of 171 EMBs were collected (118 males, 69%; average age at diagnosis 66.6±11.5 years). A subgroup of 36 patients (21%) underwent also FPEB; in 27 (75%) of them, EMB was positive, 21 of which negative at FPEB. In 45 cases (26%) EMB amyloid deposits were not identified by means of H&E, Congo Red, and Thioflavin stains nor by IEM. In the remaining 126 EMB cases (74%), IEM was diagnostic, revealing even small amounts of amyloid fibrils, positive for AL in 82/126 (65%) and TTR in 44/126 (35%). Both the interstitium and the vessels were affected, with different burden based on the type of amyloid. Cardiac structural damage and dysfunction is more relevant in ATTR amyloidosis. Conclusion: IEM represents a sensitive, reliable, and low-cost method for amyloid typing. It can be used to test the reliability of light microscopy. EMB with IEM amyloid typing is crucial to establish diagnosis, prognosis, and appropriate treatment. Localized amyloidosis can only be diagnosed by biopsy of the affected organ or tissue. Choosing the correct tissue/organ to biopsy is essential to avoid false negatives and delays to diagnosis. The choice of a surrogate tissue should be discouraged

    Novel insights in cardiac imaging to evaluate disease progression and mechanisms in cardiac amyloidosis

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    Introduction: Systemic amyloidosis is characterised by the extracellular deposition of amyloid fibrils that misfold and aggregate causing disease when accumulation is sufficient to disrupt the structure and integrity of affected organs. Cardiac involvement causes a restrictive cardiomyopathy and determines prognosis. / Aims: My aims were 1) To assess the echocardiographic phenotype of patients with transthyretin (ATTR) cardiomyopathy and evaluate determinants of prognosis at baseline 2) To describe the echocardiographic progression in patients with ATTR cardiomyopathy over serial assessment 3) To assess the role of cardiac magnetic resonance (CMR)-derived extracellular volume (ECV) mapping to detect and estimate extra-cardiac amyloid load against Serum Amyloid P (SAP) scintigraphy 4) To assess the presence and mechanisms of myocardial ischaemia using stress perfusion CMR. / Results and Conclusions: Results demonstrate that 1) At baseline echocardiographic assessment in ATTR cardiomyopathy, stroke volume index, right atrial area index, longitudinal strain, E/e’ and severe aortic stenosis were independently associated with mortality. Compared to other genotypes, patients with V122I-associated ATTR cardiomyopathy had the most severe degree of dysfunction. 2) Worsening in the degree of mitral and tricuspid regurgitation at 12 and-24 months was associated with worse prognosis by echocardiography, and patients with V122-associated ATTR cardiomyopathy had more rapid progression when compared to other genotypes. 3) CMR derived ECV mapping shows high diagnostic performance to detect splenic and hepatic amyloid deposits with good correlation between ECV and amyloid load assessed by SAP scintigraphy. 4) Myocardial blood flow assessed by stress perfusion CMR is severely reduced in patients with cardiac amyloidosis and histological analysis of endomyocardial tissue shows evidence of hypoxia, vascular infiltration and capillary rarefaction. In conclusion, these results demonstrate that imaging in cardiac amyloidosis has an important role in optimising diagnosis, tracking individual changes over time and characterising amyloid organ involvement

    Unraveling the mechanisms of alpha-synuclein aggregation and toxicity

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    Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s disease and affects about 1% of the population over 65 years old. This disorder can be both sporadic and familial and some genetic forms are due to mutations in SNCA gene, encoding for the protein alpha-synuclein (aS). PD pathological hallmarks are the prominent death of the dopaminergic neurons in the substantia nigra pars compacta and the presence of proteins and lipid inclusions, termed Lewy’s body (LBs), in the surviving neurons in parkinsonian brains. The main constituent of LBs is an aggregated fibrillar beta-sheet rich form of aS. aS aggregation process was widely studied in the past years: the protein is unfolded in its native state, but in pathological conditions it tends to aggregate forming oligomeric species. These oligomers constitute a heterogeneous and transient ensemble and rapidly convert into amyloid fibrils when they reach a critical concentration. Amyloid fibrils then deposit in LBs along with several other proteins and lipids. aS aggregation was mainly studied in vitro, but recently more efforts were put into the study of this process in cell and animal models, to identify not only aS aggregation intermediates, but also the associated toxic mechanism(s) that lead to neurons cell death in PD. In this thesis two main issues were faced: the study of aS aggregation in cells using unconventional methods and the characterization of the effects of the family of chaperone-like proteins 14-3-3, on aS aggregation. In the first part, two cellular models for the study of aS aggregation were set and characterized: the first one is obtained just overexpressing aS and allowed the characterization of an ensemble of heterogeneous oligomeric species (about 6±4 monomers per oligomer) using a new fluorescence microscopy method termed Number and Brightness analysis. These oligomeric species induced autophagic lysosomal pathway activation and mitochondrial fragmentation in this model. The second cellular model provides a method to study aS fibrils and larger aggregates in a physiological environment: aS was overexpressed in cells and aggregation was triggered by introducing in cell cytoplasm recombinant aS fibrils fragments, termed seeds. In both cases aS overexpression and aggregation cause cellular death, in good agreement with what was previously published by others groups. The characterization of aS aggregation in cells went further looking at the variation in cellular metabolism, possibly induced by mitochondrial damage. These changes were quantified measuring NADH fluorescence properties in the two models with respect to the control. These results showed that in cells presenting aS oligomer or aggregates, NADH fluorescence lifetime and emission spectra change, suggesting that these measurements may be used to detect aS aggregates in live cells and in vivo using a non-invasive dye-free method. The second part of the thesis concerns the ability of 14-3-3 chaperone-like proteins of interacting with aS and of interfering with aS aggregation process rescuing the induced toxicity in cells. Among the seven 14-3-3 isoforms, 14-3-3 eta can re-route aS amyloidogenic process in vitro, leading to the formation of curved objects rather than aS fibrils. These curved objects have diameters and curvatures that depend on 14-3-3 eta amount in the aggregation assays; moreover, 14-3-3 eta molecules were found in these aggregates, suggesting the formation of a stable complex between the two proteins. When aS amount is too large or seeds are used to trigger the aggregation process in vitro, 14-3-3 eta is not able any more to affect aS aggregation and is sequestered into aS fibrils. In cell models, 14-3-3 eta overexpression leads to a rescue when aS was only overexpressed, but not when aggregation in cell cytoplasm was triggered by seeds. Overexpressed 14-3-3 eta was found to interact with overexpressed aS using image correlation spectroscopy methods (cross raster image correlation spectroscopy and cross Number and Brightness analysis), mainly at plasma membrane. Moreover, 14-3-3 eta is sequestered into aggregates when aS aggregation is triggered by seeds, highlighting another possible toxic mechanism due to aS aggregation. All the results obtained in cells are in good agreement with the in vitro results previously reported, further suggesting that 14-3-3 proteins and eta isoform in particular are interesting in aS aggregation frame and may be used to interfere in the process to rescue its toxic effects
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