Phenotypic characterization of the epithelial and myoepithelial components in canine and feline mammary tumours

In veterinary medicine, the ability to classify mammary tumours based on the molecular profile and also determine whether the immunophenotype of the regional lymph node and/or systemic metastases is equal to that of the primary tumor may be predictive on the estimation of the effectiveness of various cancer treatments that can be scheduled. Therefore, aims, developed as projects, of the past three years have been (1) to define the molecular phenotype of feline mammary carcinomas and their lymph node metastases according to a previous modified algorithm and to demonstrate the concordance or discordance of the molecular profile between the primary tumour and lymph node metastasis, (2) to analyze, in female dogs, the relationship between the primary mammary tumor and its lymph node metastasis based on immunohistochemical molecular characterization in order to develop the most specific prognostic-predictive models and targeted therapeutic options, and (3) to evaluate the molecular trend of cancer from its primary location to systemic metastases in three cats and two dogs with mammary tumors. The studies on mammary tumours, particularly in dogs, have drawn gradually increasing attention not exclusively to the epithelial component, but also to the myoepithelial cells. The lack of complete information on a valid panel of markers for the identification of these cells in the normal and neoplastic mammary gland and lack of investigation of immunohistochemical changes from an epithelial to a mesenchymal phenotype, was the aim of a parallel research. While investigating mammary tumours, it was noticed that only few studies had focused on the expression of CD117. Therefore, it was decided to further deepen the knowledge in order to characterize the immunohistochemical staining of CD117 in normal and neoplastic mammary tissue of the dog, and to correlate CD117 immunohistochemical results with mammary histotype, histological stage (invasiveness), Ki67 index and patient survival time.

Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor gene

During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity.

The role of interleuchin 6 (IL-6) in the promotion of an aggressive and stem cell-like phenotype in human breast cancer cells and in stem/progenitor cells expanded in vitro as mammospheres

High serum levels of Interleukin-6 (IL-6) correlate with poor outcome in breast cancer patients. However no data are available on the relationship between IL-6 and stem/progenitor cells which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in mammospheres (MS), multi-cellular structures enriched in stem/progenitor cells of the mammary gland, and also in MCF-7 breast cancer cells. We show that MS from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. We find that IL-6 mRNA is detectable only in basal-like breast carcinoma tissues, an aggressive variant showing stem cell features. Our results reveal that IL-6 triggers a Notch-3-dependent up-regulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and MCF-7 derived spheroids. Moreover, IL-6 induces a Notch-3-dependent up-regulation of the carbonic anhydrase IX gene, which promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, an autocrine IL-6 loop relies upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3 expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

Anticancer drug screening from images of zebrafish embryogenesis

During the previous 10 years, global R&D expenditure in the pharmaceuticals and biotechnology sector has steadily increased, without a corresponding increase in output of new medicines. To address this situation, the biopharmaceutical industry's greatest need is to predict the failures at the earliest possible stage of the drug development process. A major key to reducing failures in drug screenings is the development and use of preclinical models that are more predictive of efficacy and safety in clinical trials. Further, relevant animal models are needed to allow a wider testing of novel hypotheses. Key to this is the developing, refining, and validating of complex animal models that directly link therapeutic targets to the phenotype of disease, allowing earlier prediction of human response to medicines and identification of safety biomarkers. Morehover, well-designed animal studies are essential to bridge the gap between test in cell cultures and people. Zebrafish is emerging, complementary to other models, as a powerful system for cancer studies and drugs discovery. We aim to investigate this research area designing a new preclinical cancer model based on the in vivo imaging of zebrafish embryogenesis. Technological advances in imaging have made it feasible to acquire nondestructive in vivo images of fluorescently labeled structures, such as cell nuclei and membranes, throughout early Zebrafishsh embryogenesis. This In vivo image-based investigation provides measurements for a large number of features at cellular level and events including nuclei movements, cells counting, and mitosis detection, thereby enabling the estimation of more significant parameters such as proliferation rate, highly relevant for investigating anticancer drug effects. In this work, we designed a standardized procedure for accessing drug activity at the cellular level in live zebrafish embryos. The procedure includes methodologies and tools that combine imaging and fully automated measurements of embryonic cell proliferation rate. We achieved proliferation rate estimation through the automatic classification and density measurement of epithelial enveloping layer and deep layer cells. Automatic embryonic cells classification provides the bases to measure the variability of relevant parameters, such as cell density, in different classes of cells and is finalized to the estimation of efficacy and selectivity of anticancer drugs. Through these methodologies we were able to evaluate and to measure in vivo the therapeutic potential and overall toxicity of Dbait and Irinotecan anticancer molecules. Results achieved on these anticancer molecules are presented and discussed; furthermore, extensive accuracy measurements are provided to investigate the robustness of the proposed procedure. Altogether, these observations indicate that zebrafish embryo can be a useful and cost-effective alternative to some mammalian models for the preclinical test of anticancer drugs and it might also provides, in the near future, opportunities to accelerate the process of drug discovery.

mRNAs translation and tumorigenesis

Translational control has a direct impact on cancer development and progression. Quantitative and qualitative changes of cap-dependent translation initiation contribute to neoplastic transformation and progression. However, the idea that “alternative” mechanisms of translation initiation, such as IRES-dependent translation, can be involved in the tumorigenesis is emerging. Because the relevance of this kind of translation initiation in cancer progression is not so well clarified, the purpose of my work was to study the impact of IRES-dependent mRNA translation on tumourigenesis and cancer progression with particular regard for breast cancer. The data obtained clarify the function of cap-independent translation in cancer. Particularly they suggested that the deregulation of IRES-dependent translation can be considered a sort of pro-oncogenic stimulus characterized by the inhibition of the expression of some proteins that block cell growth and proliferation and by the over expression of other proteins that contributed to cell survival. In addition, under stress condition, such as a hypoxia, in immortalized epithelial cell lines, changes in cap-independent translation are associated with an induction of expression of stem cell markers, and with the selection of a sub group of cells that have an increased ability to self-renewing and therefore in the acquisition of a more aggressive phenotype.

Down Syndrome: Neuropsychological phenotype and mitochondrial DNA

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth.

Phenotype and genotype characterization of Monilinia spp. isolates and preformed antifungal compounds in peach peel fruit at different developmental stages

The brown rot fungi belong to a group of fungal pathogens that causes considerable damage to cultivated fruits trees, particularly stone fruits and apples in the temperate regions of the World and during the postharvest with an important economic impact. In particular in Italy, it is important to monitor the Monilinia population to control economic losses associated to the peach and nectarine market. This motivates the research steps presented in this dissertation on Monilinia Italian isolates. The Monilinia species collected from stone fruits have been identified using molecular analysis based on specific primers. The relevant role of M. fructicola was confirmed and, for the first time, it was found also on apple fruits. To avoid the development of resistant strains and implement valid treatment strategies, the understanding of the fruit natural resistance during different developmental stages and the assessment of the Monilinia sensitivity/resistance to fungicides are required. The relationship between the inhibition spots and the phenolic compounds in peach fruit peel was highlighted in this research. Three methods were used to assess isolate resistance/sensitivity, the amended medium, the Spiral Gradient Endpoint Method (SGD) and the Alamar Blue method. The PCR was used to find possible mutation points in the b-tubulin gene that is responsible for fungicide resistance. Interestingly, no mutation points were observed in resistant M. laxa isolates, suggesting that the resistance could be stimulated by environmental factors. This lead to the study of the effect of the temperature on the resistance and the preliminary results of in vitro tests showed that maximum inhibition was observed at 30°C.

Environmental influence on the phenotype: Morphological variation in human dentition

This work is about the role that environment plays in the production of evolutionary significant variations. It starts with an historical introduction about the concept of variation and the role of environment in its production. Then, I show how a lack of attention to these topics may lead to serious mistakes in data interpretation. A statistical re-analysis of published data on the effects of malnutrition on dental eruption, shows that what has been interpreted as an increase in the mean value, is actually linked to increase of variability. In Chapter 3 I present the topic of development as a link between variability and environmental influence, giving a review of the possible mechanisms by which development influences evolutionary dynamics. Chapter 4 is the core chapter of the thesis; I investigated the role of environment in the development of dental morphology. I used dental hypoplasia as a marker of stress, characterizing two groups. Comparing the morphology of upper molars in the two groups, three major results came out: (i) there is a significant effect of environmental stressors on the overall morphology of upper molars; (ii) the developmental response increases morphological variability of the stressed population; (iii) increase of variability is directional: stressed individuals have increased cusps dimensions and number. I also hypothesized the molecular mechanisms that could be responsible of the observed effects. In Chapter 5, I present future perspectives for developing this research. The direction of dental development response is the same direction of the trend in mammalian dental evolution. Since malnutrition triggers the developmental response, and this particular kind of stressor must have been very common in our class evolutionary history, I propose the possibility that environmental stress actively influenced mammals evolution. Moreover, I discuss the possibility of reconsidering the role of natural selection in the evolution of dental morphology.

Role of Magnesium and its mitochondrial transporter MRS2 in the modulation of drug-induced apoptosis leading to multidrug resistance phenotype

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique.