Phenotypic and molecular characterization of ethylene biosynthesis in apples

Ethylene plays an important role in apple fruit development. Its biosynthesis is catalyzed by two enzymes ACS and ACO. The first is considered to catalyzes the rate-limiting step of ethylene production and in apple two different alleles (MdACS1-1 and MdACS1-2) of this gene have been identified. The presence in the promoter region of MdACS1-2 allele of a SINE insertion is considered to be responsible for a low transcription level and a pronounced reduction in ethylene production in apple cultivar homozygous for this allele. However, the specific expression of each MdACS1 allele has never been reported as well as any in vivo analysis of its 5’-flanking region. With the present study we addressed these issues by developing a set of qPCR allele specific primers for MdACS1 and by a functional characterization of the MdACS1 promoters by transient expression analysis. qPCR analysis on different apple tissues and stages of development demonstrated that MdACS1-2 allele is never express and that MdACS1-1 allele is ripening-related and expresses predominantly but not exclusively in apple fruit. To test MdACS1 promoter in fruit the only protocol available in literature for transient transformation of apple fruit was evaluated and optimized. Twenty chimeric promoter::reporter constructs were generated and analyzed by Agrobacterium-transient transformation. The in vivo analysis allowed to identify an enhancer-like region of 261 bp in MdACS1 promoter and a region of 57 bp in MdACS1-2 responsible, also if not alone, in the inactivation of the MdACS1-2 allele. Through the assessment of ethylene production in a segregating progeny derived from the cross between Fuji and Mondial Gala (homozygous for MdACS1-2 allele) we demonstrated that at least two other genes may be involved in apple ethylene production. An hypothesis that could explain the difference between Fuji and Mondial Gala have been proposed.

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.

Effect of host genotype and prion strain on the phenotypic heterogeneity of Creutzfeldt-Jakob disease: the peripheral nervous system involvement and the spectrum of the genetic form

The first study was designed to assess whether the involvement of the peripheral nervous system (PNS) belongs to the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease (sCJD). To this aim, we reviewed medical records of 117 sCJDVV2, 65 sCJDMV2K, and 121 sCJDMM(V)1 subjects for symptoms/signs and neurophysiological data. We looked for the presence of PrPSc in postmortem PNS samples from 14 subjects by western blotting and real-time quaking-induced conversion (RT-QuIC) assay. Seventy-five (41.2%) VV2-MV2K patients, but only 11 (9.1%) MM(V)1, had symptoms/signs suggestive of PNS involvement and neuropathy was documented in half of the VV2-MV2K patients tested. RT-QuIC was positive in all PNS samples, whereas western blotting detected PrPSc in the sciatic nerve in only one VV2 and one MV2K. These results support the conclusion that peripheral neuropathy, likely related to PrPSc deposition, belongs to the phenotypic spectrum of sCJDMV2K and VV2, the two variants linked to the V2 strain. The second study aimed to characterize the genetic/molecular determinants of phenotypic variability in genetic CJD (gCJD). To this purpose, we compared 157 cases of gCJD to 300 of sCJD. We analyzed: demographic aspects, neurological symptoms/signs, histopathologic features and biochemical characteristics of PrPSc. The results strongly indicated that the clinicopathological phenotypes of gCJD largely overlap with those of sCJD and that the genotype at codon 129 in cis with the mutation (i.e. haplotype) contributes more than the latter to the disease phenotype. Some mutations, however, cause phenotypic variations including haplotype-specific patterns of PrPSc deposition such as the “dense” synaptic pattern (E200K-129M), the intraneuronal dots (E200K-129V), and the linear stripes perpendicular to the surface in the molecular layer of cerebellum (OPRIs-129M). Overall, these results suggest that in gCJD PRNP mutations do not cause the emergence of novel prion strains, but rather confer increased susceptibility to the disease in conjunction with “minor” clinicopathological variations.

Phenotypic and genotypic characterisation of antimicrobial resistance in Escherichia coli indicator of animal, food and human origin.

This thesis presents AMR phenotypic evaluation and whole genome sequencing analysis of 288 Escherichia coli strains isolated from different sources (livestock, companion animal, wildlife, food and human) in Italy. Our data reflects general resistance trends in Europe, reporting tetracycline, ampicillin, sulfisoxazole and aminoglycosides resistance as the most common phenotypic AMR profile among livestock, pets, wildlife and humans. Identification of human and animal (livestock and companion animal) AMR profiles in niches with a rare (fishery, mollusc) or absent (vegetable, wild animal, wild boar) direct exposure to antimicrobials, suggests widespread environmental pollution with ARGs conferring resistance to these antimicrobials. Phenotypic resistance to highest priority critically important antimicrobials was mainly observed in food-producing animals and related food such as rabbit, poultry, beef and swine. Discrepancies between AMR phenotypic pattern and genetic profile were observed. In particular, phenotypic aminoglycoside, cephalosporin, meropenem, colistin resistance and ESBL profile did not have a genetic explanation in different cases. This data could suggest the diffusion of new genetic variants of ARGs, associated to these antimicrobial classes. Generally, our collection shows a virulence profile typical of extraintestinal pathogenic Escherichia coli (ExPEC) pathotype. Different pandemic and emerging ExPEC lineages were identified, in particular in poultry meat (ST10; ST23; ST69, ST117; ST131). Rabbit was suggested as a source of ST20-ST40 potential hybrid pathogens. Wildlife carried a high average number (10) of VAGs (mostly associated to ExPEC pathotype) and different predominant ExPEC lineages (ST23, ST117, ST648), suggesting its possible involvement in maintenance and diffusion of virulence determinants. In conclusion, our study provides important knowledge related to the phenotypic/genetic AMR and virulence profiles circulating in E. coli in Italy. The role of different niches in AMR dynamics has been discussed. In particular, food-producing animals are worthy of continued investigation as a source of potential zoonotic pathogens, meanwhile wildlife might contribute to VAGs spread.

Stayability in Italian Simmental dual-purpose cows: phenotypic and genetic associations

In recent years, dairy farmers have observed a substantial decrease in cows’ survival, with a direct negative consequence on the profitability. Shorter lifespan raises questions about animal welfare and farming conditions at which cows are exposed to. However, the length of productive life depends also on voluntary culling due to low productivity and, in dual-purpose breed, to low price of carcasses (meat). The general aim of the thesis was to investigate the genetic and phenotypic relationship of functional longevity with morphological features like muscularity and body condition score (BCS) and productive traits within Italian Simmental dual-purpose dairy cattle raised in Emilia-Romagna herds.

Phenotypic and molecular investigation of circulating tumor cells (CTCs) isolated from breast cancer patients at single cell resolution

Despite the paramount advances in cancer research, breast cancer (BC) still ranks one of the leading causes of cancer-related death worldwide. Thanks to the screening campaign started in developed countries, BC is often diagnosed at early stages (non-metastatic BC, nmBC), but disease relapse occurrence even after decades and at distant sites is not an uncommon phenomenon. Conversely, metastatic BC (mBC) is considered an incurable disease. The major perpetrators of tumor spread to secondary organs are circulating tumor cells (CTCs), a rare population of cells detectable in the peripheral blood of oncologic patients. In this study, CTCs from patients diagnosed with luminal nmBC and mBC (hormone receptor positive, Human Epidermal Growth Factor Receptor 2 (HER2) negative) were characterized at both phenotypic and molecular levels. To better understand the molecular mechanisms underlying their biology and their metastatic potential, next-generation sequencing (NGS) analyses were performed at single-cell resolution to assess copy number aberrations (CNAs), single nucleotide variants (SNVs) and gene expression profiling. The findings of this study arise hints in CTC detection, and pave the way to new application in CTC research.