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Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umanoPurgato, Stefania <1978> 06 June 2008 (has links)
MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult
tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a
restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the
latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene
transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the
MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in
Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four
ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric
soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)].
ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS
and ARMS, but the MYCN amplification and expression levels shows a significant correlation and
are greater in ARMS, in which they are associated with adverse outcome.
We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28
and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent
inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was
followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN
expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated-
PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix
microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced
or repressed, with both genes previously described as targets of N-myc or Myc, and new genes
undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility,
metastasis, angiogenesis and muscle development). The changes in the expression of the most
relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the
MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS
xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic
tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity),
whereas treatment with the mutated-PNA had no effect.
Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS,
particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.
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Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factorsBedini, Andrea <1979> 06 June 2008 (has links)
The organization of the nervous and immune systems is characterized by obvious differences and
striking parallels. Both systems need to relay information across very short and very long
distances.
The nervous system communicates over both long and short ranges primarily by means of more
or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange
communication in the immune system occurs mainly via the ordered and guided migration
of immune cells and systemically acting soluble factors such as antibodies, cytokines, and
chemokines. Its short-range communication either is mediated by locally acting soluble factors or
transpires during direct cell–cell contact across specialized areas called “immunological synapses”
(Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented
by a complex array of factors that induce cell growth and differentiation: these factors in the
immune system are called cytokines; in the nervous system, they are called neurotrophic factors.
Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either
system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most
potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived
neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et
al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth
factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000).
These factors may allow the two systems to “cross-talk” and eventually may provide a molecular
explanation for the reports that inflammation after central nervous system (CNS) injury has
beneficial effects (Moalem et al., 1999).
In order to shed some more light on such a cross-talk, therefore, transcription factors modulating
mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated.
More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell
receptor induction of MOPr expression in T-lymphocytes.
Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the
G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated
exogenous opioids or endogenous opioid peptides, and contribute to the regulation of
several functions including pain transmission, respiration, cardiac and gastrointestinal functions,
and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer
and Hollt 2006).
Recently, induction of MOPr expression in different immune cells induced by cytokines has been
reported (Kraus et al., 2001; Kraus et al., 2003).
The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has
clusters of potential binding sites for different transcription factors (Law et al. 2004).
Several studies, primarily focused on the upstream region of the OPRM1 promoter, have
investigated transcriptional regulation of MOPr expression. Presently, however, it is still not
completely clear how positive and negative transcription regulators cooperatively coordinate cellor
tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its
expression.
IGF-I and its receptors are widely distributed throughout the nervous system during
development, and their involvement in neurogenesis has been extensively investigated
(Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such
neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al.,
2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor
which, after ligand binding-induced autophosphorylation, associates with specific adaptor
proteins and activates different second messengers (Bondy and Cheng 2004). These include:
phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di
Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al.
2000; Yadav et al. 2005).
REST plays a complex role in neuronal cells by differentially repressing target gene expression
(Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during
neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated
in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al.
2006). Thus, the REST concentration seems to influence its function and the expression of
neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al.
2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural
induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes
not yet required by the differentiation program, but its expression decreases after five days of
treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in
which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the
disappearance of REST (Di Toro et al. 2005).
The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the
repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional
repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth
factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates
OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A
series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell
models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels
of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated
OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with
constructs deficient in the REST DNA binding element, or when REST was down-regulated in
retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of
transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3
DNA element located in the promoter, increases OPRM1 transcription.
T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major
histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular
signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the
genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat
CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study
whether its signalling influence OPRM1 expression. Results were that TCR engagement
determined a significant induction of OPRM1 expression through the activation of transcription
factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been
expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation
and recycling, whereas morphine did not.
Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a
critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how
human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates
OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as
well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune
cells suggest an important role for MOPr as a molecular bridge between neurons and immune
cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous
system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli
and analgesic and neuroprotective effects.
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Marcatura di molecole biologiche a funzione antigenica per lo studio e la caratterizzazione di protocolli di vaccinoterapia in oncologia medicaAncarani, Valentina <1978> 06 June 2008 (has links)
Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured in
GM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are the
most potent APCs known. These DCs have many features of primary DCs, including the
expression of molecules that enhance antigen capture and selective receptors that guide
DCs to and from several sites in the body, where they elicit the T cell mediated immune
response.
For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC)
with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials of
different cancers.
However, the efficacy of DCs in the development of immunocompetence is critically
influenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the time
of exposure of the tumor antigens used for loading in the presentation phase.
The aim of the present study was to create instruments to acquire more information about
DC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbased
vaccine.
In particular, two different tumor antigen were studied: the monoclonal immunoglobulin
(IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanoma
tissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate the
kinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetric
and fluorescence microscopy analysis, respectively.
iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours after
loading. By the cytofluorimetric analysis it was observed that the monoclonal antibody was
completely captured after 2 hours from pulsing, and was decreased into mDC in 5 hours
after maturation stimulus.
To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, then
were monitored in the 2h to 22 hours interval time after loading.
Then, to reveal difference between increasing lysate concentration, iDC were loaded with
20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h from
pulsing.
By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8
hours loading was completed reaching a plateau phase. For 200 and 400μg the mean
fluorescence of cells increased until 13h from pulsing.
The lysate localization into iDC was evaluated with conventional and confocal
fluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensive
and diffuse fluorescence was observed within the cytoplasmic compartment. Moreover,
after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shaded
area with a typical polarized aspect. In addition, small fluorescent spots clearly appeared
with an increment in the number and fluorescence intensity.
The nature of these spot-like formations and cloudy area is now being investigated
detecting the colocalization of the fluorescence lysate and specific markers for lysosomes,
autophagosomes, endoplasmic reticulum and MHCII positive vesicles.
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Genomic and non genomic effects of elevated concentration of anabolic steroids in human neuronal cellsGuarino, Goffredo <1979> 06 June 2008 (has links)
Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter
the expression and function of neurotransmitter systems and contribute to neuronal cell death.
This effect can explain the behavioural changes, drug dependence and neuro degeneration
observed in steroid abuser.
Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent
downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma
cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide.
Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated
cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that
nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human
MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene
transcription. These results suggest that nandrolone may regulate MOPr expression through
post-transcriptional mechanisms requiring the AR.
Cito-toxicity assays demonstrated a time- and concentration dependent decrease of
cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is
independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was
observed in cells treated with Nandrolone 10-6M for 48h.
Collectively, these data support the existence of two cellular mechanisms that might
explain the neurological syndromes observed in steroids abuser.
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Design and implementation of bioinformatics tools for large scale genome annotationPierleoni, Andrea <1979> 06 June 2008 (has links)
The continuous increase of genome sequencing projects produced a huge amount of data in the
last 10 years: currently more than 600 prokaryotic and 80 eukaryotic genomes are fully sequenced
and publically available. However the sole sequencing process of a genome is able to determine just
raw nucleotide sequences. This is only the first step of the genome annotation process that will deal
with the issue of assigning biological information to each sequence. The annotation process is done at
each different level of the biological information processing mechanism, from DNA to protein, and
cannot be accomplished only by in vitro analysis procedures resulting extremely expensive and time
consuming when applied at a this large scale level. Thus, in silico methods need to be used to
accomplish the task.
The aim of this work was the implementation of predictive computational methods to allow a
fast, reliable, and automated annotation of genomes and proteins starting from aminoacidic sequences.
The first part of the work was focused on the implementation of a new machine learning based
method for the prediction of the subcellular localization of soluble eukaryotic proteins. The method is
called BaCelLo, and was developed in 2006. The main peculiarity of the method is to be independent
from biases present in the training dataset, which causes the over‐prediction of the most represented
examples in all the other available predictors developed so far. This important result was achieved by
a modification, made by myself, to the standard Support Vector Machine (SVM) algorithm with the
creation of the so called Balanced SVM. BaCelLo is able to predict the most important subcellular
localizations in eukaryotic cells and three, kingdom‐specific, predictors were implemented. In two
extensive comparisons, carried out in 2006 and 2008, BaCelLo reported to outperform all the
currently available state‐of‐the‐art methods for this prediction task.
BaCelLo was subsequently used to completely annotate 5 eukaryotic genomes, by integrating it
in a pipeline of predictors developed at the Bologna Biocomputing group by Dr. Pier Luigi Martelli and
Dr. Piero Fariselli. An online database, called eSLDB, was developed by integrating, for each
aminoacidic sequence extracted from the genome, the predicted subcellular localization merged with
experimental and similarity‐based annotations.
In the second part of the work a new, machine learning based, method was implemented for the
prediction of GPI‐anchored proteins. Basically the method is able to efficiently predict from the raw
aminoacidic sequence both the presence of the GPI‐anchor (by means of an SVM), and the position in
the sequence of the post‐translational modification event, the so called ω‐site (by means of an Hidden
Markov Model (HMM)). The method is called GPIPE and reported to greatly enhance the prediction
performances of GPI‐anchored proteins over all the previously developed methods. GPIPE was able to
predict up to 88% of the experimentally annotated GPI‐anchored proteins by maintaining a rate of
false positive prediction as low as 0.1%.
GPIPE was used to completely annotate 81 eukaryotic genomes, and more than 15000 putative
GPI‐anchored proteins were predicted, 561 of which are found in H. sapiens. In average 1% of a
proteome is predicted as GPI‐anchored. A statistical analysis was performed onto the composition of
the regions surrounding the ω‐site that allowed the definition of specific aminoacidic abundances in
the different considered regions. Furthermore the hypothesis that compositional biases are present
among the four major eukaryotic kingdoms, proposed in literature, was tested and rejected.
All the developed predictors and databases are freely available at:
BaCelLo http://gpcr.biocomp.unibo.it/bacello
eSLDB http://gpcr.biocomp.unibo.it/esldb
GPIPE http://gpcr.biocomp.unibo.it/gpipe
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Differenziamento macrofagico: gli effetti dei polimeri sinteticiScordari, Alessandra <1980> 23 May 2008 (has links)
Research for new biocompatible and easily implantable materials continuously
proposes new molecules and new substances with biological, chemical and physical
characteristics, that are more and more adapted to aesthetic and reconstructive
surgery and to the development of biomedical devices such as cardiovascular
prostheses.
Two classes of polymeric biomaterials seem to meet better these requirements:
“hydrogels” , which includes polyalkylimide (PAI) and polyvinylalcohol (PVA) and
“elastomers”, which includes polyurethanes (PUs). The first ones in the last decade
have had a great application for soft tissue augmentation, due to their similarity to
this tissue for their high water content, elasticity and oxygen permeability (Dini et
al., 2005). The second ones, on the contrary, are widely used in cardiovascular
applications (catheters, vascular grafts, ventricular assist devices, total artificial
hearts) due to their good mechanical properties and hemocompatibility (Zdrahala
R.J. and Zdrahala I.J., 1999).
In the biocompatibility evaluation of these synthetic polymers, that is important for
its potential use in clinical applications, a fundamental aspect is the knowledge of the
polymers cytotoxicity and the effect of their interaction with cells, in particular with
the cell populations involved in the inflammatory responses, i.e.
monocyte/macrophages.
In consideration of what above said, the aim of this study is the comprehension of
the in vitro effect of PAI, PVA and PU on three cell lines that represent three
different stages of macrophagic differentiation: U937 pro-monocytes, THP-1
monocytes and RAW 264.7 macrophages.
Cytotoxicity was evaluated by measuring the rate of viability with MTT, Neutral Red
and morphological analysis at light microscope in time-course dependent
experiments.
The influence of these polymers on monocyte/macrophage activation in terms of
cells adhesion, monocyte differentiation in macrophages, antigens distribution,
aspecific phagocytosis, fluid-phase endocitosis, pro-inflammatory cytokine (TNF-α,
IL-1β, IL-6) and nitric oxide (NO) release was evaluated.
In conclusion, our studies have indicated that the three different polymeric
biomaterials are highly biocompatible, since they scarcely affected viability of U937,
THP-1 and RAW 264.7 cells. Moreover, we have found that even though hydrogels
and polyurethane influences monocyte/macrophage differentiation (depending on the
particular type of cell and polymer), they are immunocompatible since they not
induced significantly high cytokine release. For these reasons their clinical
applications are strongly encouraged.
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An alternative B cell development programFossati, Valentina <1978> 23 May 2008 (has links)
Two major types of B cells, the antibody-producing cells of the immune
system, are classically distinguished in the spleen: marginal zone (MZ) and
follicular (FO). In addition, FO B cells are subdivided into FO I and FO II
cells, based on the amount of surface IgM. MZ B cells, which surround the
splenic follicles, rapidly produce IgM in response to blood-borne pathogens
without T cell help, while T cell-dependent production of high affinity,
isotype-switched antibodies is ascribed to FO I cells. The significance of FO
II cells and the mechanism underlying B cell fate choices are unclear. We
showed that FO II cells express more Sca1 than FO I cells and originate
from a distinct B cell development program, marked by high expression of
Sca1. MZ B cells can derive from the “canonical” Sca1lo pathways, as well
as from the Sca1hi program, although the Sca1hi program shows a stronger
MZ bias than the Sca1lo program, and extensive phenotypic plasticity exists
between MZ and FO II, but not between MZ and FO I cells. The Sca1hi
program is induced by hematopoietic stress and generates B cells with an
Igλ-enriched repertoire. In aged mice, the canonical B cell development
pathway is impaired, while the Sca1hi program is increased. Furthermore, we
showed that a population of unknown function, defined as Lin-c-kit+Sca1+
(LSK-), contains early lymphoid precursors, with primarily B cell potential
in vivo. Our data suggest that LSK- cells may represent a distinct precursor
for the Sca1hi program in the bone marrow.
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Marker sierici per la predizione precoce della preeclampsiaMorano, Danila <1973> 23 May 2008 (has links)
No description available.
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Novel potential molecular and biochemical markers for non-invasive prenatal screeningsBanzola, Irina <1974> 23 May 2008 (has links)
No description available.
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Studio di biomateriali usati come scaffold per Tissue Engineering e loro caratterizzazione con tecniche spettroscopiche vibrazionali e di analisi termicaDi Foggia, Michele <1980> 15 May 2008 (has links)
This research investigated someone of the main problems connected to the application of Tissue
Engineering in the prosthetic field, in particular about the characterization of the scaffolding
materials and biomimetic strategies adopted in order to promote the implant integration.
The spectroscopic and thermal analysis techniques were usefully applied to characterize the
chemico-physical properties of the materials such as
– crystallinity;
– relative composition in case of composite materials;
– Structure and conformation of polymeric and peptidic chains;
– mechanism and degradation rate;
– Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions).
This kind of information are of great importance in the comprehension of the interactions that
scaffold undergoes when it is in contact with biological tissues; this information are fundamental to
predict biodegradation mechanisms and to understand how chemico-physical properties change
during the degradation process. In order to fully characterize biomaterials, this findings must be
integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to
collaborations with biomedical engineers and biologists.
This study was focussed on three different systems that correspond to three different strategies
adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix
(PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote
biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the
interaction with osteoblasts.
The characterization of the PCL-PLLA composite underlined that the degradation started along
PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration.
Moreover it was found that some cellular lines are more active in the colonization of the scaffold.
In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an
essential role both in the degradation process and in the biomineralization of the material.
The study of self-assembling peptides allowed to clarify the influence of primary structure on
intermolecular and intermolecular interactions, that lead to the formation of the secondary structure
and it was possible to find a new class of oligopeptides useful to functionalize materials surface.
Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major
role, being non-destructive and non-invasive, two properties that make it suitable to degradation
studies and to morphological characterization. Also micro-IR spectroscopy was useful in the
comprehension of peptide structure on oxidized titanium: up to date this study was one of the first
to employ this relatively new technique in the biomedical field.
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