Miracle-dz/UniFAR-FTUs huggingface dataset:
The Human Pancreatic Islet Methylome and Its Role in Type 2 Diabetes

Islet dysfunction is central to the development and progression of type 2 diabetes (T2D). Epigenetic modifications are essential for establishing and maintaining cell identity and function in normal circumstances. Exposure to adverse environmental factors may alter the epigenome, and result in changes of gene expression and the resulting phenotype. The aim of this thesis was to analyze DNA methylation levels of specific genes, as well as genome-wide DNA methylation, in order to determine whether epigenetic dysregulation of pancreatic islets contributes to islet dysfunction in subjects with T2D. We also assessed the relationship between genetic variation and DNA methylation. We further examined the potential use of DNA methylation in blood DNA to predict future T2D. At the specific gene level, we found that DNA methylation of INS and PDX-1 was increased in pancreatic islets from subjects with T2D (Studies I and II). Conversely, their mRNA expression, insulin content and glucose-stimulated insulin secretion (GSIS) were decreased in the same islets. We next analyzed genome-wide DNA methylation in human pancreatic islets from both T2D and non-diabetic donors (Study III). Nearly 1,500 CpG sites (853 genes) were differentially methylated in T2D islets, with the majority showing decreased DNA methylation. 102 genes showed both altered DNA methylation and mRNA expression in T2D islets, including CDKN1A, PDE7B, SEPT9 and EXOC3L2. Our functional experiments provided further evidence that altering the expression of these genes, by modeling the situation in T2D, results in impaired insulin and glucagon secretion in cell line models. Furthermore, we showed that nearly half of the single nucleotide polymorphisms (SNPs) associated with T2D are CpG-SNPs, which can introduce or remove a CpG site (Study IV). Accordingly, we found that the degree of DNA methylation at CpG-SNP sites varied between individuals with different genotypes, and that some of the CpG-SNPs were associated with differential gene expression, alternative splicing and hormonal secretion. In Study V, we showed that altered DNA methylation at two CpG sites in the ABCG1 and PHOSPHO1 genes in blood from non-diabetic individuals was associated with a higher risk of future T2D. Subsequently, we found that CpG sites annotated to these genes were differentially methylated in T2D target tissues. Taken together, our findings suggest that epigenetic dysregulation of pancreatic islets play a role in islet dysfunction in subjects with T2D, and can be influenced by genetic variation and the environment.
ANALYSIS OF THE INTERACTION BETWEEN TOMATO TORRADO VIRUS PROTEINS USING THE YEAST TWO-HYBRID SYSTEM

Ten years ago for the first time the new picorna-like virus species - Tomato torrado virus (ToTV) - was found and described on tomato plants. The isolates of this pathogen were reported in Europe, America, and Oceania including Australia. Because of its unique biological and molecular features, ToTV was classified to the new genus Torradovirus, in the Secoviridae family. In Poland, three isolates: Wal'03, Kra, and Ros ToTV were identified on greenhouse tomato cultivars. At present, the biology and the genome structure of this virus are characterised. But there is no data extending beyond the bioinformatics analyses about the function of viral proteins, polyproteins, and non-coding sequences, as well as possible interactions between viral, host and vector factors that may be important for the infection process, encapsidation, transport in plants, and transmission. In this study, we have undertaken a search for the possible protein-protein interaction of ToTV encoded proteins using the yeast two-hybrid (Y2H) system. The viral genome fragments covering full sequences for nine known proteins of ToTV were amplified using specific primers with characteristic recom- bination sites. This process enabled the construction of basic entry clones for each protein that further facilitated manipulations with prepared constructs using Gateway technology. Two-hybrid assays were performed in the yeast strain and tested interactions of ToTV proteins were analysed in several combinations using auxotrophy markers. Our analyses did not reveal the presence of interactions between ToTV domains. Surprisingly, no interactions were found in the case of various CP subunits as well as between CP subunits and 3A protein, that in some virus families are known to play a role in viral life cycle. This role includes virion assembly or cell-to-cell transport. The lack of interactions may be a result of the limitation of this experimental system, or suggest that these proteins may interact indirectly, or require the presence of genomic RNAs or some host factors.
A PRELIMINARY EARLY -SEASON FLOWER -VISITATION WEB FOR THE KIRINDY FOREST , MADAGASCAR

Tropical dry deciduous forest is an endangered ecosystem whose plant-pollinator relationships are little known. We characterised a portion of the web of interactions between flowering plants and flower visitors in the Kirindy Forest of the Menabe region of west-central Madagascar. Taking a plant-centered approach, we observed individuals of the 5 most abundant native plant species that were coming into flower at the end of the annual dry season, and recorded all identifiable flower-visitors. Taking a visitor-centered approach, we walked a network of established trails and listened for distinctive calls of a common flower-visiting bird, noting the plant species visited. The former approach revealed connections among the early-flowering species via birds and insects, whereas the latter confirmed these connections and added an additional plant species. Flowers of the 6 plant species were visited on average by 5.5 animal species, while 10 visitor species for which we had reasonable samples frequented on average the flowers of 3.3 plant species. These qualitative results resemble those reported from other temperate and tropical webs, in that interactions appeared to be relatively generalised by pollinator species and body plan (e.g., birds vs. bees). Also in agreement, the visitation web was significantly nested, with more-specialised species tending to interact with mutualistic partners that were themselves more generalised. In addition to documenting previously-unreported interactions, therefore, this preliminary web conforms to more widespread patterns emerging for pollination systems at the community level.
The use of seedling bark by voles sustained by high proteinic content of food

Meadow voles (M. pennsylvanicus) are known to select herbaceous plants with high protein contents and low phenolic values. However, they eat the bark of coniferous and deciduous seedlings with low dosages of protein and high phenolic contents. We tested the hypothesis that voles can use bark of red oak seedlings (Q. rubra) when their regular diet contains high proteinic components (> 8% dry matter). Three groups of 10 non reproducing female meadow voles were maintained on 15%, 8%, and 4% protein diets supplemented with red oak seedlings during two weeks. Variations in body mass, total food intake, ingestion of bark and the assigned diets, chemical constituents of fecal matter, protein digestibility, and phenolic recovery rates in fecal matter were compared between the experimental groups. Animals of all groups used extensively the bark of red oak seedlings. Ingestion of bark tissues explained 46% and 20% of the body mass variation of voles maintained on the 15% and 4% protein diets, respectively. Fecal matter yielded significantly higher contents of phenolics and total nonstructural carbohydrates and lower dosages of protein for voles maintained on the 4% proteinic diet. The opposite was true for animals maintained on the 15% proteinic diet. Protein digestibility was similar for every treatment which indicates that voles maintained on a 4% proteinic diet can keep an excellent proteinic balance if they have access to seedlings with high proteinic values in bark. Phenolic recovery rates from fecal matter varied between 12 and 23% for the three vole categories but were not statistically different. These results suggest that low proteinic content of food does not regulate the use of bark by voles. Bark tissues during short periods can be added to the vole diet without any apparent costs.
Stability of fungal immunomodulatory protein, FIP-gts and FIP-fve, in IFN-γ production

Abstract Fungal immunomodulatory proteins (FIPs) are a group of novel proteins, purified from medicinal fungi or edible mushrooms that possess immunomodulatory properties. FIP-gts and FIP-fve have been isolated and purified from Ganoderma tsugae and Flammunlina velutipes, respectively. The evaluation of FIP immunomodulatory activity was based on their ability to stimulate human peripheral blood lymphocytes to release interferon-gamma (IFN-γ). We found that FIP-gts exhibited better immunomodulatory activity than FIP-fve. Activities were both greatly reduced with duration of heating. For digestibility, FIP-fve was more resistant than FIP-gts to digestive enzymes in simulated gastric fluid and simulated intestinal fluid. IFN-γ production is only detectable in dimers of FIP-gts as opposed to polymer of FIP-fve. These results suggest that FIP-gts and FIP-fve have activities that are stable and have a strong potential of being applied to food or pharmaceutical products for commercial development.
The interface between peripheral axons, Schwann cells and biosynthetic nerve guides

Schwann cells are essential for axonal regrowth following injury, and Schwann cell transplantation has been proposed to encourage peripheral nerve regeneration. However there is the need to develop a methodology to enable investigation and characterisation of Schwann cells following transplantation, Initially, an identifiable and pure population of cultured Schwann cells was obtained, and transduction of lacZ genetic label was carried out. Transduced Schwann cells properties and lacZ expression were preserved for 6 month of continuous culture, demonstrating the stability of the genetically modified cells. Suspension matrix and a conduit are required for Schwann cell transplantation, and the suitability of alginate hydrogel and PHB filaments was confirmed by in vitro tests to support Schwann cell proliferation and neurite sprouting in neuron-glial co-cultures.Constructs of PHB conduits containing Schwann cells in alginate were tested in vivo, and an optimal number of cells required to enhance axonal regeneration was determined. Staining of transduced cells with X-gal histochemistry confirmed the viability of transplanted Schwann cells within the conduit, as they could be identified for up to 6 weeks post-operatively clearly associated with regenerating axons. Quantitative immunohistochemistry was used to characterise Schwann cells and axonal regrowth into the composite conduit, and it showed that transplantation of Schwann cells improved the rate and quantity of axonal regeneration. In conclusion, genetically modified Schwann cells retain their ability to enhance nerve regeneration and bioengineered nerve conduits might provide the ideal environment for optimal regeneration following peripheral nerve injury.
Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze-induced lesions: expansion-induced lysis (EIL) between −2 and −4 °C and loss of osmotic responsiveness (LOR) between −5 and −12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at −5 and −6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at −5 and −6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low-temperature-induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.
Radiation epigenetically modulates tumor cells and alters activation and function of effector T cells

Radiation is a common therapeutic modality for cancer however it fails to control advanced and malignant disease. As a result, novel approaches that aim to stimulate immune attack of tumors are currently being investigated and approved by the US FDA. It is clear, however, that no single agent will be responsible for achieving long-term control and treatment of cancer and that combination therapies will be required. Previous studies indicate that colorectal cancer cells that survive radiation up-regulate surface expression of cytotoxic T lymphocyte (CTL) relevant proteins including death receptors, cell adhesion molecules and tumor-associated antigens (TAA). The aim of this dissertation was to investigate the effect of non-cytolytic doses of ionizing radiation (IR) on co-stimulatory molecule expression on tumor cells and evaluate the impact of their modulation on effector T-cell biology and tumoricidal activity. Here, several human tumor cell lines were exposed to various doses of radiation (0-10Gy) and TAA-specific T-cell tumoricidal activity and expression of effector CTL co-stimulatory molecules were evaluated. I found OX40L and 41BBL to be the most consistently upregulated proteins post-IR by flow cytometry. Furthermore, I saw enhanced survival and activation of human CD8+ T-cells exposed to irradiated tumor cells. Importantly, enhanced killing of irradiated tumor cells by TAA-specific CTLs in cytotoxicity assays was reported. Blocking OX40L and 41BBL reversed radiation-enhanced T-cell killing. My data also indicate that expression of 41BBL and OX40L can be epigenetically regulated, as inhibition of histone deacetylases (HDAC) and of DNA methyltransferases (DNMT) resulted in increased OX40L and 41BBL mRNA and protein expression. Furthermore, chromatin immunoprecipitation experiments revealed increased histone H3 acetylation specifically at the 41BBL promoter following irradiation. Last, I began exploring the ability of IR to reverse immune suppression by evaluating the impact of radiation on regulatory T cells (TREGS) that can suppress the function of effector CTLs. I found that RT could reduce TREG numbers likely by altering their phenotype. Overall, this dissertation demonstrates that radiation can be used to make human tumors more immunogenic through epigenetic modulation of genes stimulatory to effector T-cells and that it may also reverse immune suppression by phenotypically altering TREG cells. INDEX WORDS: radiation therapy, cancer immunotherapy, ionizing irradiation, immunogenic modulation, CTLs, epigenetic, effector costimulation, and regulatory T cells. RADIATION EPIGENTICALLY MODULATES TUMOR CELLS AND ALTERS ACTIVATION AND FUNCTION OF EFFECTOR T CELLS
7 Auditory Systems in Insects

Evolution has endowed insects with an extraordinary capacity for miniaturization. Virtually all aspects of insect biology convey the sense of successfully uniting form and function in exquisitely small, diverse, and sophisticated motor, sensory, and metabolic systems (Grimaldi and Engel 2005). The ability of insects to fly in an efficient and controlled manner well illustrates how, through evolution by natural selection, they adapted to solve what we consider serious problems of engineering. Insects are little marvels of “evolutionary engineering. ” The seemingly boundless ingenuity and creativity of the process of evolutionary adaptation are also reflected in the sensory systems of insects. As we try to make clear in this chapter, hearing in insects is a sophisticated process; understanding its fundamental mechanisms and trying to understand its evolution present many challenges but are likely to be very rewarding. Perhaps because insects are so small, their ears have generally been considered to be simple compared to those of vertebrates. Anatomically, they may be simpler, but their capacity for sound reception and processing turns out to be remarkably elaborate (for reviews, see Fullard and Yack 1993; Hoy 1998; Robert and Gopfert 2002; Robert 2005; Hedwig 2006; Gopfert and Robert 2007). The ears of insects can be as sensitive and acute as their vertebrate counterparts (Webster et al. 1992; Hoy 1998). Indeed, in some cases their feats of detection surpass the capabilities of vertebrates (Robert and Gopfert 2002). For example, the ultrafast ears of the parasitoid fly Ormia ochracea can distinguish time differences in the arrival.
InVivoSplicing ofthe,3Tropomyosin Pre-mRNA:A Role forBranchPoint andDonorSiteCompetition

Thechicken atropomyosin genecontains twosetsofalternatively spliced, mutually exclusive exonswhose utilization isdevelopmentally regulated. Exons6Aand6Bareusedinnonmuscle cells (orundifferentiated muscle cells) andskeletal muscle cells, respectively. A complex arrangement ofcis-acting sequenceelements is involved inalternative splicing regulation. We haveperformed an extensive mutational analysis on the sequencespanning theregion fromexon6Atotheconstitutive exon7.A large numberofmutantminigenes havebeentested intransfection assaysofcultured myogenic cells, andthesplicing products havebeenanalyzed bycDNApolymerase chain reaction. We demonstrate thatinundifferentiated myoblasts, exon6Bisskipped asaresult ofanegative control onitsselection, while exon6Aisspliced asadefault choice. We provide evidence thatthefocal point ofsucha regulation islocalized intheintron upstream ofexon 6Bandprobably involves theblockage ofitsassociated branchpoint. Indifferentiated myotubes, incontrast, bothexonsareaccessible tothesplicing machinery. We showthatthepreferential choice ofexon6Binthis splicing environment depends on theexistence ofa competition between thetwoexons fortheflanking constitutive splice sites. We demonstrate thatboththedonors andthebranchpoints ofthetwoexonsareinvolved inthiscompetition. Split genesineukaryotes require aprecise assembly ofthe coding information prior toexpression. Thestepofthe maturation pathway inwhichthenoncoding interspersed regions (introns) areexcised fromthefull geneRNA copy (primary transcript) inorder toyield thefinal maturemessageiscalled splicing. Thedefinitions ofcoding andnoncodingsegments oftheprimary transcript maysometimes vary andleadtothestochastic orcontext-dependent production
Lactation Curve and Milk Flow

The buffalo Mediterranean Italian breed was very much studied for lactation curve. Data on lactation curves after the first, the second and the third calving are reported. In all the 3 lactations we have similar curves, with the production peak on the 40 th day, in the second and third lactations, while in the first lactation it is shown on the 50 th day and with a longer duration. Lactation curves were different for the first and later parities with lactations of different duration also in Nili-Ravi buffaloes. Milk production and milk flow profiles are important parameters to be recorded and evaluated. In fact they give various advices on milking management. The milk flow curve is typical for each animal and must be recorded singly. Buffaloes are characterized by longer teat and teat canal, and particularly by stronger muscular resistance of teat wall, than in cow: it is necessary a high vacuum level for opening the teat canal and beginning milk ejection. A recent trial is reported, where the manual stimulation at different times provoked the increasing of released oxytocin and of the intra-mammary pressure and finally the increasing of cisternal teat largeness and the reduction of teat canal length. Buffalo is characterized by smaller cisternal fraction than in cattle. The cisternal milk fraction is immediately available while the alveolar fraction needs an oxytocin stimulation to release. In Mediterranean Italian buffaloes it has been observed a higher cisternal fraction of total milk than in Murrah breed. In buffalo management, the milking room and the milking machine are really a critical point and the characteristics of milking vacuum and milking pulsations are strictly connected with milk flow observations. Different curves obtained in recent trials during the milking with normal milking machine and with Automatic Milking System are reported and discussed. A recent study on Mediterranean Italian buffaloes from 104 different herds showed that 88.86% of milk is ejected in 4.24 min (main milking time), as 8.49% is ejected in 1.98 min (milk let-down) corresponding to the cisternal milk fraction. Only 2.65% is the milk ejected in the stripping phase. These results suggest toapply a premilking udder stimulation to reduce milk let-down time increasing the average milk flow and reducing total milking time. These results suggest to the detachment of milking cluster to reduce the blind phase with the following advantages: a) reducing the total milking time and consequently worker’s time, b) improving the farmer’s income and the milk quality thought the mastitis incidence decreasing.
COMPARATIVE MORPHOLOGY, CROSSABILITY, AND TAXONOMY WITHIN THE CALOGLOSSA CONTINUA (DELESSERIACEAE, RHODOPHYTA) COMPLEX FROM THE WESTERN PACIFIC

Systematic studies were undertaken on the Caloglossa continua (Okamura) King et Puttock complex from Japan, Singapore, and Australia, based on morphology and reproductive compatibility. Specimens from Japan had two to six cell rows derived from a nodal axial cell, at the margin opposite the branch, whereas those from Australia possessed only a single cell row. Specimens from Singapore formed one to four cell rows per nodal axial cell and always contained at least one single cell row on any one thallus. These differences were maintained in cultured materials over a range of temperatures or salinities. Type material of C. continua had the same morphology as the Japanese specimens in this study. Carpospores discharged from the Japanese isolate germinated at 10°C, whereas those from Singapore and Australia died at this temperature. In hybridization experiments, the Japanese entity was completely nonfertile with both the Singaporean and Australian isolates. Many pseudocystocarps were produced in the crossing between the male from Australia and the female from Singapore, although the reciprocal combination did not produce any such structures. On the basis of the discontinuous morphology coupled with the complete reproductive isolation, the entities from Singapore and Australia are described here as C. monosticha sp. nov. The entities with multiple cell rows likely expanded their geographic range from tropical regions, where the majority of Caloglossa species are now distributed, to high‐latitude regions, and such an expansion would be associated with acquiring low‐temperature tolerance.