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Landscape ecology and genetics of the wolf in ItalyMilanesi, Pietro <1982> 08 May 2014 (has links)
This PhD Thesis includes five main parts on diverse topics. The first two parts deal with the trophic ecology of wolves in Italy consequently to a recent increase of wild ungulates abundance. Data on wolf diet across time highlighted how wild ungulates are important food resource for wolves in Italy. Increasing wolf population, increasing numbers of wild ungulates and decreasing livestock consume are mitigating wolf-man conflicts in Italy in the near future.
In the third part, non-invasive genetic sampling techniques were used to obtain genotypes and genders of about 400 wolves. Thus, wolf packs were genetically reconstructed using diverse population genetic and parentage software. Combining the results on pack structure and genetic relatedness with sampling locations, home ranges of wolf packs and dispersal patterns were identified. These results, particularly important for the conservation management of wolves in Italy, illustrated detailed information that can be retrieved from genetic identification of individuals.
In the fourth part, wolf locations were combined with environmental information obtained as GIS-layers. Modern species distribution models (niche models) were applied to infer potential wolf distribution and predation risk. From the resulting distribution maps, information pastures with the highest risk of depredation were derived. This is particularly relevant as it allows identifying those areas under danger of carnivore attack on livestock.
Finally, in the fifth part, habitat suitability models were combined with landscape genetic analysis. On one side landscape genetic analyses on the Italian wolves provided new information on the dynamics and connectivity of the population and, on the other side, a profound analysis of the effects that habitat suitability methods had on the parameterization of landscape genetic analyses was carried out to contributed significantly to landscape genetic theory.
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Epigenetic role of N-Myc in NeuroblastomaMilazzo, Giorgio <1985> 09 April 2015 (has links)
Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas.
N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1.
Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin.
Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.
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Roles of Ecdysone signaling in cell survival and epithelium morphogenesis during Drosophila melanogaster developmentRomani, Patrizia <1982> 05 May 2011 (has links)
In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act.
During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.
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Structural and functional analysis of centromeric chromatinZoli, Monica <1982> 05 May 2011 (has links)
Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres.
We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes.
Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever.
Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.
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Studio del controllo trascrizionale del gene VM32E in Drosophila: analisi genetica delle vie di segnalazione coinvolteBernardi, Fabio <1977> 02 April 2007 (has links)
No description available.
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Wing shape evolution: a role for cell competition in shaping the proximal distal axis of Drosophila wingZiosi, Marcello <1978> 13 May 2008 (has links)
No description available.
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Analisi funzionale dei recettori per le neurotrofine p75NTR e Trka in neuroblastomaPapa, Antonella <1978> 03 April 2008 (has links)
The biological complexity of NGF action is achieved by binding two distinct Neurotrophin
receptors, TrkA and p75NTR. While several reports have provided lines of evidence on the
interaction between TrkA and p75NTR at the plasma membrane, much fewer data are
available on the consequence of such an interaction in terms of intracellular signaling. In
this study, we have focused on how p75NTR may affect TrkA downstream signaling with
respect to neuronal differentiation. Here, we have shown that cooperation between p75NTR
and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a
sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite
outgrowth is concomitant with a selective enhancement of the AP-1 activity and the
transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be
involved in the differentiation process. Collectively, our results unveil a functional link
between the specific expression profile of neurotrophin receptors in neuronal cells and the
NGF-mediated regulation of the differentiation process possibly through a persistent ERKs
activation and the selective control of the AP-1 activity. In our studies we discuss the
functional role of the neurotrophin receptor p75NTR and TrkA in a ligand-dependent signal
transduction.
It is known that p75NTR is also involved in the mediation of cell death ligand dependent.
Here we show for the first time that the membrane receptor p75NTR, upon binding to b-
Amyloid (Ab) peptide, is able to transduce a cytotoxic signal through a mechanism very
similar to the one adopted by Tumor Necrosis Factor Receptor 1 (TNFR1), when activated
by TNFa. We define that in neuroblastoma cell line Ab cytotoxicity signals through a
pathway depending on p75NTR death domain (DD), mostly through some specific
conserved residues. We identified that TRADD is the first interactor recruiting to the
membrane and activates JNK and NF-kB transcription factors. Since Ab is defined as the
most important aetiologic element associated with the Alzheimer’s Disease (AD),
characterization of the mechanism involved in the mediation of the neurodegeneration can
suggest also new therapeutic approaches.
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Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activationPasini, Luigi <1978> 03 April 2008 (has links)
Transcription is controlled by promoter-selective transcriptional factors (TFs),
which bind to cis-regulatory enhancers elements, termed hormone response
elements (HREs), in a specific subset of genes. Regulation by these factors
involves either the recruitment of coactivators or corepressors and direct
interaction with the basal transcriptional machinery (1). Hormone-activated
nuclear receptors (NRs) are well characterized transcriptional factors (2) that
bind to the promoters of their target genes and recruit primary and secondary
coactivator proteins which possess many enzymatic activities required for
gene expression (1,3,4).
In the present study, using single-cell high-resolution fluorescent microscopy
and high throughput microscopy (HTM) coupled to computational imaging
analysis, we investigated transcriptional regulation controlled by the estrogen
receptor alpha (ERalpha), in terms of large scale chromatin remodeling and
interaction with the associated coactivator SRC-3 (Steroid Receptor
Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a
steroid-dependent transcriptional factor (16) that belongs to the NRs
superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2),
regulates transcription of distinct target genes involved in development,
puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the
nucleus and undergoes a rapid (within minutes) intranuclear redistribution
following the addition of either agonist or antagonist (17,18,19).
We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated
reporter gene array (PRL-array) containing multicopy hormone
response-binding elements for ERalpha that are derived from the physiological
enhancer/promoter region of the prolactin gene. Following GFP-ER
transfection of PRL-HeLa cells, we were able to observe in situ ligand
dependent (i) recruitment to the array of the receptor and associated
coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout
of the reporter gene. Addition of E2 causes a visible opening
(decondensation) of the PRL-array, colocalization of RNA Polymerase II, and
transcriptional readout of the reporter gene, detected by mRNA FISH. On the
contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI),
a dramatic condensation of the PRL-array was observed, displacement of
RNA Polymerase II, and complete decreasing in the transcriptional FISH
signal.
All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid
Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160
family member and a known oncogenic protein (4,34). SRC-3 is regulated by a
variety of posttranslational modifications, including methylation,
phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These
events have been shown to be important for its interaction with other
coactivator proteins and NRs and for its oncogenic potential (37,39). A number
of extracellular signaling molecules, like steroid hormones, growth factors and
cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by
a wide range of kinases, including extracellular-regulated kinase 1 and 2
(ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs)
(41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein,
whose cellular localization is regulated by phosphorylation and interaction with
ERalpha. Using a combination of high throughput and fluorescence microscopy,
we show that both chemical inhibition (with U0126) and siRNA downregulation
of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in
SRC-3 localization, whereas stimulation by EGF signaling enhances its
nuclear localization by inducing phosphorylation at T24, S857, and S860, known
partecipants in the regulation of SRC-3 activity (39). Accordingly, the
cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further
supports these results. In the presence of ERalpha, U0126 also dramatically
reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the
nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell
lysates; localization of SRC-3 at the ER-targeted prolactin promoter array
(PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also
function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of
estrogen receptor.
While a wealth of studies have revealed the molecular functions of NRs and
coregulators, there is a paucity of data on how these functions are
spatiotemporally organized in the cellular context. Technically and
conceptually, our findings have a new impact upon evaluating gene
transcriptional control and mechanisms of action of gene regulators.
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Insulin and TOR pathways regulate cellular and organismal growth through the myc oncogene in DrosophilaParisi, Federica <1981> 21 April 2009 (has links)
A large body of literature documents in both mice and Drosophila the
involvement of Insulin pathway in growth regulation, probably due to its role
in glucose and lipid import, nutrient storage, and translation of RNAs
implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover
several lines of evidence implicate this pathway as a causal factor in cancer
(Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008).
With regards to Myc, studies in cell culture have implied this family of
transcription factors as regulators of the cell cycle that are rapidly induced in
response to growth factors. Myc is a potent oncogene, rearranged and
overexpressed in a wide range of human tumors and necessary during
development. Its conditional knock-out in mice results in reduction of body
weight due to defect in cell proliferation (Trumpp et al. 2001).
Evidence from in vivo studies in Drosophila and mammals suggests a
critical function for myc in cell growth regulation (Iritani and Eisenman 1999;
Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al.
2001). This role is supported by our analysis of Myc target genes in
Drosophila, which include genes involved in RNA binding, processing,
ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al.,
2005, Hulf et al, 2005).
The fact that Insulin signaling and Myc have both been associated
with growth control suggests that they may interact with each other.
However, genetic evidence suggesting that Insulin signaling regulates Myc
in vivo is lacking.
In this work we were able to show, for the first time, a direct
modulation of dMyc in response to Insulin stimulation/silencing both in vitro
and in vivo. Our results suggest that dMyc up-regulation in response to
DILPs signaling occurs both at the mRNA and potein level. We believe
dMyc protein accumulation after Insulin signaling activation is conditioned to
AKT-dependent GSK3β/sgg inactivation. In fact, we were able to
demonstate that dMyc protein stabilization through phosphorylation is a
conserved feature between Drosophila and vertebrates and requires
multiple events. The final phosphorylation step, that results in a non-stable
form of dMyc protein, ready to be degraded by the proteasome, is
performed by GSK3β/sgg kinase (Sears, 2004). At the same time we
demonstrated that CKI family of protein kinase are required to prime dMyc
phosphorylation.
DILPs and TOR/Nutrient signalings are known to communicate at
several levels (Neufeld, 2003). For this reason we further investigated TOR
contribution to dMyc-dependent growth regulation. dMyc protein
accumulates in S2 cells after aminoacid stimulation, while its mRNA does
not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient
pathway regulates dMyc mostly post-transcriptionally.
In support to this hypothesis, we observed a TORC1-dependent
GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and
Nutrients on dMyc protein stability.
On the other hand, our data show that Rheb but not S6K, both
downstream of the TOR kinase, contributes to the dMyc-induced growth of
the eye tissue, suggesting that Rheb controls growth independently of S6K..
Moreover, Rheb seems to be able to regulate organ size during
development inducing cell death, a mechanism no longer occurring in
absence of dmyc. These observations suggest that Rheb might control
growth through a new pathway independent of TOR/S6K but still dependent
on dMyc.
In order to dissect the mechanism of dMyc regulation in response to
these events, we analyzed the relative contribution of Rheb, TOR and S6K
to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic
clones and we further confirmed an interplay between Rheb and Myc that
seems to be indipendent from TOR.
In this work we clarified the mechanisms that stabilize dMyc protein in
vitro and in vivo and we observed for the first time dMyc responsiveness to
DILPs and TOR. At the same time, we discovered a new branch of the
Nutrient pathway that appears to drive growth through dMyc but
indipendently from TOR.
We believe our work shed light on the mechanisms cells use to grow
or restrain growth in presence/absence of growth promoting cues and for
this reason it contributes to understand the physiology of growth control.
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Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor geneDuchi, Serena <1979> 21 April 2009 (has links)
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.
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