Annali online Unife (Università degli Studi di Ferrara)
Not a member yet
1987 research outputs found
Sort by
RNA interference for studiyng the role of fibrocystin/polyductin in human kidney cell lines
No full textMolecular mechanism and rnai correction of a dominant-negative Von Willebrand Factor gene deletion
No full textLab-on-a-chip and integrated strategies in tumor immunotheraphy
No full textBackground While conventional chemotherapy and radiation therapy have improved the survival of many cancer patients, there are still major disadvantages associated with these treatments such as high toxicity and drug-resistance. The possibility to manipulate the immune system to recognize and kill tumor cells is very attractive despite numerous obstacles remaining to be overcome. In particular, the ability of the immune system to destroy disseminated metastases in a specific way makes immunotherapy an attractive alternative to conventional therapies. Nevertheless, other unconventional technologies emerged in recent years seem to be very promising; in particular the analysis and monitoring of single cell-to-cell interactions and the capability to individually control single cells by Lab-on-a-chip devices have become of great interest in different areas of life sciences. These new technologies, in combination with progresses reached in anti-tumor vaccines, could be useful to improve immune T cell responses against tumor antigen for a more efficient immunotherapy. Aims This thesis focuses on two tumor immunotherapy issues: 1) design, realization and validation of innovative Lab-on-a-chip devices for immune system study, that allows single tumor cell and effector cell interaction, detection and isolation; 2) identification of molecular mechanisms that prevent EBV-associated tumors (e.g. Burkitt’s lymphoma) recognition by T cells and study of their potential correction by specific treatments. The main goal of this study remains indeed the evaluation of an integrated strategy for immunotherapy development enhancing for malignancies treatment. Methods Biocompatibility test, generation of memory CTL cultures, 51Cr release assay, IFN-Elispot, proteasomes purification, western blot assay, enzymatic assay, immunofluorescence, RT-PCR. Main Results As concerns the first part of the thesis, a main achievement was the design of Lab-on-a-chip platform that combined microfluidics and electronics together, consisting in a matrix of up to thousand microwells where living cells can be deposited. Subsequently, different materials have been evaluated to identify the most biocompatible ones for biosensor manufacturing. Once developed Lab-on-a-chip prototype, it has been tested from a functional point of view. In particular, it has been demonstrated that the biosensor is able to isolate and trap single cells inside microwells by dielectrophoresis, that recovered cells are still alive and that their biological functions and gene expression remain unaltered. Furthermore, tumor cell lysis by immune effector cells could be successfully monitored inside device microwells, showing that biosensor could be used for cell to cell interaction studies. Regarding the second aim of this thesis, it has been identified a new epitope-specific T cell response against EBV nuclear antigen 1 (EBNA1). It has also been demonstrated that CTLs specific for another EBNA1-derived epitope (referred as HPV) are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes (LCL) but not Burkitt’s lymphoma (BL). Afterwards LCL and BL have been compared for their antigen processing machinery, demonstrating that one of the major differences was at the proteasome level; indeed, proteasomes from BL cells have displayed a far lower chymotryptic and tryptic-like activities. Interestingly, it has also been shown that treatment with proteasome inhibitors partially restored the capacity of BL cells to present the HPV epitope. Conclusions The results achieved in single cell manipulation and cell to cell analysis interaction by Lab-on-a-chip technology, and the findings reached to improve BL immune recognition, represent an implementation of innovative tools that could allow important progresses in cancer diagnosis and immunotherapy.Background While conventional chemotherapy and radiation therapy have improved the survival of many cancer patients, there are still major disadvantages associated with these treatments such as high toxicity and drug-resistance. The possibility to manipulate the immune system to recognize and kill tumor cells is very attractive despite numerous obstacles remaining to be overcome. In particular, the ability of the immune system to destroy disseminated metastases in a specific way makes immunotherapy an attractive alternative to conventional therapies. Nevertheless, other unconventional technologies emerged in recent years seem to be very promising; in particular the analysis and monitoring of single cell-to-cell interactions and the capability to individually control single cells by Lab-on-a-chip devices have become of great interest in different areas of life sciences. These new technologies, in combination with progresses reached in anti-tumor vaccines, could be useful to improve immune T cell responses against tumor antigen for a more efficient immunotherapy. Aims This thesis focuses on two tumor immunotherapy issues: 1) design, realization and validation of innovative Lab-on-a-chip devices for immune system study, that allows single tumor cell and effector cell interaction, detection and isolation; 2) identification of molecular mechanisms that prevent EBV-associated tumors (e.g. Burkitt’s lymphoma) recognition by T cells and study of their potential correction by specific treatments. The main goal of this study remains indeed the evaluation of an integrated strategy for immunotherapy development enhancing for malignancies treatment. Methods Biocompatibility test, generation of memory CTL cultures, 51Cr release assay, IFN-Elispot, proteasomes purification, western blot assay, enzymatic assay, immunofluorescence, RT-PCR. Main Results As concerns the first part of the thesis, a main achievement was the design of Lab-on-a-chip platform that combined microfluidics and electronics together, consisting in a matrix of up to thousand microwells where living cells can be deposited. Subsequently, different materials have been evaluated to identify the most biocompatible ones for biosensor manufacturing. Once developed Lab-on-a-chip prototype, it has been tested from a functional point of view. In particular, it has been demonstrated that the biosensor is able to isolate and trap single cells inside microwells by dielectrophoresis, that recovered cells are still alive and that their biological functions and gene expression remain unaltered. Furthermore, tumor cell lysis by immune effector cells could be successfully monitored inside device microwells, showing that biosensor could be used for cell to cell interaction studies. Regarding the second aim of this thesis, it has been identified a new epitope-specific T cell response against EBV nuclear antigen 1 (EBNA1). It has also been demonstrated that CTLs specific for another EBNA1-derived epitope (referred as HPV) are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes (LCL) but not Burkitt’s lymphoma (BL). Afterwards LCL and BL have been compared for their antigen processing machinery, demonstrating that one of the major differences was at the proteasome level; indeed, proteasomes from BL cells have displayed a far lower chymotryptic and tryptic-like activities. Interestingly, it has also been shown that treatment with proteasome inhibitors partially restored the capacity of BL cells to present the HPV epitope. Conclusions The results achieved in single cell manipulation and cell to cell analysis interaction by Lab-on-a-chip technology, and the findings reached to improve BL immune recognition, represent an implementation of innovative tools that could allow important progresses in cancer diagnosis and immunotherapy
A Scalable Parallel Architecture with FPGA-Based Network Processor for Scientific Computing
No full textThis thesis discuss the design and the implementation of an FPGA-Based Network Processor for scientific computing, like Lattice Quantum ChromoDinamycs (LQCD) and fluid-dynamics applications based on Lattice Boltzmann Methods (LBM). State-of-the-art programs in this (and other similar) applications have a large degree of available parallelism, that can be easily exploited on massively parallel systems, provided the underlying communication network has not only high-bandwidth but also low-latency. I have designed in details, built and tested in hardware, firmware and software an implementation of a Network Processor, tailored for the most recent families of multi-core processors. The implementation has been developed on an FPGA device to easily interface the logic of NWP with the CPU I/O sub-system. In this work I have assessed several ways to move data between the main memory of the CPU and the I/O sub-system to exploit high data throughput and low latency, enabling the use of “Programmed Input Output” (PIO), “Direct Memory Access” (DMA) and “Write Combining” memory-settings. On the software side, I developed and test a device driver for the Linux operating system to access the NWP device, as well as a system library to efficiently access the network device from user-applications. This thesis demonstrates the feasibility of a network infrastructure that saturates the maximum bandwidth of the I/O sub-systems available on recent CPUs, and reduces communication latencies to values very close to those needed by the processor to move data across the chip boundary.This thesis discuss the design and the implementation of an FPGA-Based Network Processor for scientific computing, like Lattice Quantum ChromoDinamycs (LQCD) and fluid-dynamics applications based on Lattice Boltzmann Methods (LBM). State-of-the-art programs in this (and other similar) applications have a large degree of available parallelism, that can be easily exploited on massively parallel systems, provided the underlying communication network has not only high-bandwidth but also low-latency. I have designed in details, built and tested in hardware, firmware and software an implementation of a Network Processor, tailored for the most recent families of multi-core processors. The implementation has been developed on an FPGA device to easily interface the logic of NWP with the CPU I/O sub-system. In this work I have assessed several ways to move data between the main memory of the CPU and the I/O sub-system to exploit high data throughput and low latency, enabling the use of “Programmed Input Output” (PIO), “Direct Memory Access” (DMA) and “Write Combining” memory-settings. On the software side, I developed and test a device driver for the Linux operating system to access the NWP device, as well as a system library to efficiently access the network device from user-applications. This thesis demonstrates the feasibility of a network infrastructure that saturates the maximum bandwidth of the I/O sub-systems available on recent CPUs, and reduces communication latencies to values very close to those needed by the processor to move data across the chip boundary
Meccanismi di orientamento nella lucertola campestre: bussola a polarizzazione e ruolo dell’occhio parietale
No full textMy PhD research activity focused on the investigation of the role of polarized light in the directional orientation of the lacertid lizard Podarcis sicula. In particular, the aim of this work was to systematically analyze P. sicula orientation behaviour and to lay the foundation for future electrophysiological and molecular investigations of anatomical structures assigned to sky polarization perception. Another goal was to understand the evolutionary meaning of these structures and the mechanisms of polarization perception. For this purpose, the first experimental section (EXPERIMENT 1: Beltrami et al., 2010) examined whether ruin lizards P. sicula are able to orientate using the E-vector direction of polarized light. Lizard orientation was tested indoors, under an artificial light source: this device produced plane polarized light with a single E-vector, that provided an axial cue. These results showed that lizards can learn a training axis and that after 90° rotation of the E-vector direction of polarized light the lizards’ directional choices rotated correspondingly. The following step of the study aimed at elucidating whether the functioning of a sky polarization compass would be mediated by the lizard parietal eye. To test this, ruin lizards that met learning criteria were tested under polarized light after their parietal eyes were painted black. Lizards with black-painted parietal eyes were completely disoriented. These data showed for the first time that the parietal eye plays a central role in mediating the functioning of a putative sky polarization compass of lizards. Furthermore, the experimental apparatus used in this experiment emitted light that did not include wavelengths in the UV range. Thus, the UV range is not necessary for perceiving polarized light in ruin lizards, unlike other species as, for example, honey bees and desert ants that in the absence of UV are unable to use a sky polarization compass. That being so, the second experimental part (EXPERIMENT 2) was aimed at testing whether there is a preferential region of the light spectrum to perceive the E-vector direction of polarized light. The results showed that lizards can learn a training direction when trained under white light produced by an LCD and E-vector parallel to the training axis. Lizards that met the learning criteria were then tested under white light and E-vector perpendicular to the training axis, just to validate the new LCD equipment: as expected, lizards followed a 90° rotation of the Evector direction, confirming once again that they can use the polarized light for orientation. Thereafter, lizards were tested under coloured polarized light (blue, green, red and turquoise), initially with E-vector parallel to the training axis and then with E-vector perpendicular to the training axis, to examine whether P. sicula can perceive polarized light of a particular wavelength range and use it in orientation. Under both blue light and turquoise light lizards were able to orient in both E-vector’s conditions, otherwise under red light lizards were completely disoriented; under green light lizards were able to orient themselves only when the direction of the E-vector was the same as in training, whereas after a 90° rotation of the E-vector lizards were disoriented, in both spectral radiance level (high and low). Incorrect orientation after E-vector rotation under Green light was independent of (high or low) spectral radiance and one hypothesis to interpret these data is that the green stimuli could be barely discernible. These results, combined with the data under Green light previously discussed, demonstrate that the blue-turquoise part of the light spectrum is crucial for a correct functioning of sky polarization compass, whereas red wavelengths do not mediate perception of the E-vector. The third experimental section (EXPERIMENT 3) was performed outdoors. The first part of this experiment was aimed to validate the new set-up outdoors. Inside the experimental apparatus lizards had only the sky polarization pattern available for orientation. As expected, the results demonstrated that lizards can learn a training direction and the new set-up is well suited to investigate orientation mechanisms in lizards. The second part of this experiment was aimed at testing the time-compensated nature of the sky polarization compass. The results demonstrated that lizards can learn a training direction under blue sky with no sun’s disc, but surprisingly, they cannot retain the spatial information. Indeed, after 6 days without training, both control group and fast-shifted group, were disorientated. A possibility is that some aspects of the information necessary for the normal functioning of the sky polarization compass is not retained for a week with no training. Alternatively, it is possible that the sky polarization compass needs to be recalibrated almost daily by external cues, such as exposure of the lizards to the sun’s disc. Clearly, other explanations are possible, and further experiments outdoors are necessary to answer this important question. Results relating to my three year of PhD course show the way forward to new interesting questions and so to new possible experimental applications to finally clarify the evolutionary role of the sky polarization compass and its interactions with other orientation mechanisms (for example the sun compass or the magnetic compass). It should be very interesting to deepen the knowledge of the nature of the sky polarization pattern’s information, to elucidate the time-compensated mechanisms that set the information retention.My PhD research activity focused on the investigation of the role of polarized light in the directional orientation of the lacertid lizard Podarcis sicula. In particular, the aim of this work was to systematically analyze P. sicula orientation behaviour and to lay the foundation for future electrophysiological and molecular investigations of anatomical structures assigned to sky polarization perception. Another goal was to understand the evolutionary meaning of these structures and the mechanisms of polarization perception. For this purpose, the first experimental section (EXPERIMENT 1: Beltrami et al., 2010) examined whether ruin lizards P. sicula are able to orientate using the E-vector direction of polarized light. Lizard orientation was tested indoors, under an artificial light source: this device produced plane polarized light with a single E-vector, that provided an axial cue. These results showed that lizards can learn a training axis and that after 90° rotation of the E-vector direction of polarized light the lizards’ directional choices rotated correspondingly. The following step of the study aimed at elucidating whether the functioning of a sky polarization compass would be mediated by the lizard parietal eye. To test this, ruin lizards that met learning criteria were tested under polarized light after their parietal eyes were painted black. Lizards with black-painted parietal eyes were completely disoriented. These data showed for the first time that the parietal eye plays a central role in mediating the functioning of a putative sky polarization compass of lizards. Furthermore, the experimental apparatus used in this experiment emitted light that did not include wavelengths in the UV range. Thus, the UV range is not necessary for perceiving polarized light in ruin lizards, unlike other species as, for example, honey bees and desert ants that in the absence of UV are unable to use a sky polarization compass. That being so, the second experimental part (EXPERIMENT 2) was aimed at testing whether there is a preferential region of the light spectrum to perceive the E-vector direction of polarized light. The results showed that lizards can learn a training direction when trained under white light produced by an LCD and E-vector parallel to the training axis. Lizards that met the learning criteria were then tested under white light and E-vector perpendicular to the training axis, just to validate the new LCD equipment: as expected, lizards followed a 90° rotation of the Evector direction, confirming once again that they can use the polarized light for orientation. Thereafter, lizards were tested under coloured polarized light (blue, green, red and turquoise), initially with E-vector parallel to the training axis and then with E-vector perpendicular to the training axis, to examine whether P. sicula can perceive polarized light of a particular wavelength range and use it in orientation. Under both blue light and turquoise light lizards were able to orient in both E-vector’s conditions, otherwise under red light lizards were completely disoriented; under green light lizards were able to orient themselves only when the direction of the E-vector was the same as in training, whereas after a 90° rotation of the E-vector lizards were disoriented, in both spectral radiance level (high and low). Incorrect orientation after E-vector rotation under Green light was independent of (high or low) spectral radiance and one hypothesis to interpret these data is that the green stimuli could be barely discernible. These results, combined with the data under Green light previously discussed, demonstrate that the blue-turquoise part of the light spectrum is crucial for a correct functioning of sky polarization compass, whereas red wavelengths do not mediate perception of the E-vector. The third experimental section (EXPERIMENT 3) was performed outdoors. The first part of this experiment was aimed to validate the new set-up outdoors. Inside the experimental apparatus lizards had only the sky polarization pattern available for orientation. As expected, the results demonstrated that lizards can learn a training direction and the new set-up is well suited to investigate orientation mechanisms in lizards. The second part of this experiment was aimed at testing the time-compensated nature of the sky polarization compass. The results demonstrated that lizards can learn a training direction under blue sky with no sun’s disc, but surprisingly, they cannot retain the spatial information. Indeed, after 6 days without training, both control group and fast-shifted group, were disorientated. A possibility is that some aspects of the information necessary for the normal functioning of the sky polarization compass is not retained for a week with no training. Alternatively, it is possible that the sky polarization compass needs to be recalibrated almost daily by external cues, such as exposure of the lizards to the sun’s disc. Clearly, other explanations are possible, and further experiments outdoors are necessary to answer this important question. Results relating to my three year of PhD course show the way forward to new interesting questions and so to new possible experimental applications to finally clarify the evolutionary role of the sky polarization compass and its interactions with other orientation mechanisms (for example the sun compass or the magnetic compass). It should be very interesting to deepen the knowledge of the nature of the sky polarization pattern’s information, to elucidate the time-compensated mechanisms that set the information retention
Neurophysiological and histopathological evaluation of small fiber pathways as diagnostic characterization of neuropathic pain and autonom dysfunction syndromes
No full textThis manuscript is made up of two individual not related articles about peripheral neuropathic pain syndrome. The first article reports the effect on pain relief in patients with peripheral neuropathic pain after brachial plexus lesions or distal peripheral nerve injury using an implanted peripheral nerve stimulator applied directly on nerve branch using a peculiar surgical technique. Seven patients with post-traumatic lesion of brachial plexus or peripheral nerve complaining severe intractable pain were selected. Neuropathic pain diagnosis according with redefinition and the grading system of NEUPSIG (2008) was assessed. Conventional drugs for neuropathic pain and traditional surgical treatment were not effective. Patients underwent at baseline clinical evaluation with careful neuroalgological evaluation recording negative signs and positive phenomena, pain questionnaires, thermal-Quantitative Sensory Testing (QST). Surgical treatment consists in a new surgical technique for neurostimulator implant: quadripolar electrocatheters were placed directly on the sensory peripheral branch of nerve mainly involved into the ascellary cavity. To assess neuromodulation effect we perform clinical neuroalgological evaluation, pain scales and QST after 1 week and again after 1 month, and after each 6 months. No significant or unexpected adverse events occurred. The pain intensity dropped decrease from a NRS of 9±1.15 before surgery to 2.14±1.57 at 6-month of follow-up and to 2.57±1.13 at 12-months of follow-up (P < 0.001). We assessed after about 12 months with double-blind control with Neurostimulation turned off the restart of severe ongoing pain and paroxysms. These results expressed in details in the article show the safety and efficacy of this innovative technique in treatment of chronic and usually intractable severe pain in selected patients. The second article reported a peculiar phenotype of cold pain in patients with small fiber neuropathies. The aim of study was to characterize the distinct pattern of pain phenomena in these patients and to compare clinical, neurophysiological and histological features in order to assess the underlying pain mechanisms. 9 patients with painful small fiber neuropathy (SFN) complaining cold pain were selected and compared with patients with SFN complaining burning pain. A complete neuroalgological examination, nerve conduction studies, pain questionnary (NPSI), thermal-Quantitative Sensory Testing (QST) battery and skin biopsy at distal and proximal sites were performed. Then L-menthol and cinnamaldeyd (CA), TRPM8 and TRPA1 receptors agonists respectively, were topically applied to the calf in two different days and the effect on pain (recorded with 11-point Likart scale for 20 minutes), thermal sensation, tactile sensation and skin flare size (skin area mm2) were evaluated. We compared the results with 15 healthy subjects and 10 patients with SFN with burning pain as the main pain quality. At baseline evaluation cold-SFN showed a cold hyperalgesia or cold allodynia at lower limb in a disto-proximal fashion associated with severe cold and mild warm hypoaesthesia. L-menthol induced no sensation in 5 of 9 cold-SFN patients and burning pain sensation in 4. The L-menthol responses (vasodilatation and flare) were significantly reduced or nearly abolished in the allodynic area in cold-SFN pts. The CA effects were less significative, it produced a slight burning sensation in 3 pts, tactile allodynia and heat hyperalgesia in 2 pts affected by cold SFN. Skin biopsy showed in patients with cold painful SFN more severe denervation of dermal nerves compared with burning SFN and MBP-positive fibers were reduced compared with burning-SFN. All the other findings were detailed in the article. In conclusion, this study showed the existence of a peculiar neuropathic phenotype of cold pain that could be explained by selective or predominant dysfunction of TRPM8 receptor and A-delta thinly myelinated nerve fibers. Furthermore a selective group of patients with SFN complaining burning feet as exclusive painful syndrome shown a prevalent involvement of TRPA1 receptor afferent.This manuscript is made up of two individual not related articles about peripheral neuropathic pain syndrome. The first article reports the effect on pain relief in patients with peripheral neuropathic pain after brachial plexus lesions or distal peripheral nerve injury using an implanted peripheral nerve stimulator applied directly on nerve branch using a peculiar surgical technique. Seven patients with post-traumatic lesion of brachial plexus or peripheral nerve complaining severe intractable pain were selected. Neuropathic pain diagnosis according with redefinition and the grading system of NEUPSIG (2008) was assessed. Conventional drugs for neuropathic pain and traditional surgical treatment were not effective. Patients underwent at baseline clinical evaluation with careful neuroalgological evaluation recording negative signs and positive phenomena, pain questionnaires, thermal-Quantitative Sensory Testing (QST). Surgical treatment consists in a new surgical technique for neurostimulator implant: quadripolar electrocatheters were placed directly on the sensory peripheral branch of nerve mainly involved into the ascellary cavity. To assess neuromodulation effect we perform clinical neuroalgological evaluation, pain scales and QST after 1 week and again after 1 month, and after each 6 months. No significant or unexpected adverse events occurred. The pain intensity dropped decrease from a NRS of 9±1.15 before surgery to 2.14±1.57 at 6-month of follow-up and to 2.57±1.13 at 12-months of follow-up (P < 0.001). We assessed after about 12 months with double-blind control with Neurostimulation turned off the restart of severe ongoing pain and paroxysms. These results expressed in details in the article show the safety and efficacy of this innovative technique in treatment of chronic and usually intractable severe pain in selected patients. The second article reported a peculiar phenotype of cold pain in patients with small fiber neuropathies. The aim of study was to characterize the distinct pattern of pain phenomena in these patients and to compare clinical, neurophysiological and histological features in order to assess the underlying pain mechanisms. 9 patients with painful small fiber neuropathy (SFN) complaining cold pain were selected and compared with patients with SFN complaining burning pain. A complete neuroalgological examination, nerve conduction studies, pain questionnary (NPSI), thermal-Quantitative Sensory Testing (QST) battery and skin biopsy at distal and proximal sites were performed. Then L-menthol and cinnamaldeyd (CA), TRPM8 and TRPA1 receptors agonists respectively, were topically applied to the calf in two different days and the effect on pain (recorded with 11-point Likart scale for 20 minutes), thermal sensation, tactile sensation and skin flare size (skin area mm2) were evaluated. We compared the results with 15 healthy subjects and 10 patients with SFN with burning pain as the main pain quality. At baseline evaluation cold-SFN showed a cold hyperalgesia or cold allodynia at lower limb in a disto-proximal fashion associated with severe cold and mild warm hypoaesthesia. L-menthol induced no sensation in 5 of 9 cold-SFN patients and burning pain sensation in 4. The L-menthol responses (vasodilatation and flare) were significantly reduced or nearly abolished in the allodynic area in cold-SFN pts. The CA effects were less significative, it produced a slight burning sensation in 3 pts, tactile allodynia and heat hyperalgesia in 2 pts affected by cold SFN. Skin biopsy showed in patients with cold painful SFN more severe denervation of dermal nerves compared with burning SFN and MBP-positive fibers were reduced compared with burning-SFN. All the other findings were detailed in the article. In conclusion, this study showed the existence of a peculiar neuropathic phenotype of cold pain that could be explained by selective or predominant dysfunction of TRPM8 receptor and A-delta thinly myelinated nerve fibers. Furthermore a selective group of patients with SFN complaining burning feet as exclusive painful syndrome shown a prevalent involvement of TRPA1 receptor afferent
Memoria e psicoanalisi: 100 anni alla ricerca della memoria perduta
No full text  
Un’esperienza di insegnamento nella SSIS. Il caso della Logica matematica
Get PDFThis paper presents the rationale of a course of mathematical logic among the teaching activities for the Teachers’ Specialisation School, focusing on the main features of the discipline in its spin-off in teacher training.
Si presentano le ragioni e gli aspetti fondamentali dell’insegnamento di Logica matematica per la SSIS illustrandone le finalità e il valore formativo.