Mammalian telomeres and their partnership with lamins

Chromosome ends are complex structures, which require a panel of factors for their elongation, replication, and protection. We describe here the mechanics of mammalian telomeres, dynamics and maintainance in relation to lamins. Multiple biochemical connections, including association of telomeres to the nuclear envelope and matrix, of telomeric proteins to lamins, and of lamin-associated proteins to chromosome ends, underline the interplay between lamins and telomeres. Paths toward senescence, such as defective telomere replication, altered heterochromatin organization, and impaired DNA repair, are common to lamins' and telomeres' dysfunction. The convergence of phenotypes can be interpreted through a model of dynamic, lamin-controlled functional platforms dedicated to the function of telomeres as fragile sites. The features of telomeropathies and laminopathies, and of animal models underline further overlapping aspects, including the alteration of stem cell compartments. We expect that future studies of basic biology and on aging will benefit from the analysis of this telomere-lamina interplay

Barrier properties of polymer nanocomposites during cyclic sorption-desorption and stress-coupled sorption experiments

It is well known that layered silicate nanoparticles, when distributed within the matrix of a polymer, can retard the diffusion of small molecules and improve barrier properties. This has been demonstrated in both sorption and permeation experiments in the past. In order to assess the long-term retention of barrier properties of polymer-layered silicate nanocomposites, it becomes important to study their response to various environmental effects.;The main theme of this investigation is to study the effect of several water exposure cycles and exposure coupled with external tensile stress on the barrier properties of various polymers like vinyl ester, polyester and epoxy and their polymer-based nanocomposites. As expected, Montmorillonite clay decreased the diffusivity of moisture in the above polymers and retained its effect during the repeated cycles of sorption and desorption. However, in the presence of clay (5 wt%), diffusivities measured during absorption under external stress were larger than those without stress. SEM analysis on the 5 wt% clay loaded vinyl ester samples subjected to stress-coupled sorption revealed the presence of micro cracks which caused increase in diffusivities

Educating Parent English Language Learners on Healthy Routines for Children

The purpose of this project is to examine whether the linking of school music routines focused on literacy skills to home activities and programming is a viable and sustainable learning option for immigrant and refugee families, as well as to examine the sustainability of this program for non-musician staff at Learning in Style (LIS). (LIS, located in Minneapolis, a ministry of the Sisters of St. Joseph of Carondelet and Consociates, St. Paul Province.) The project serves the adult English language learning students who attend LIS and their children who attend the childcare program. It will also serve the staff who will potentially carry on some of the elements into future years to serve future LIS students. The goal of this project is to encourage parents to implement music routines with their children and assess the sustainable of these routines. The project also aims to develop literacy-based music routines at LIS that staff can sustain once the project is complete. OTs\u27 unique understanding of and role in the establishment and implementation of routines, as well as the evaluation and grading skills of OT, make OT students and practitioners uniquely qualified to contribute to this project. The remainder of this portfolio outlines the literature review, needs assessment process, interventions, assessment methods, and results of this Master’s Project

Characterization of AKTIP, a new protein involved in human DNA replication and telomere metabolism

Telomeres are nucleoprotein structures that protect the ends of linear chromosomes. They are composed of long tracts of TTAGGG repeats, telomere specific proteins that form the shelterin complex and several telomere accessory proteins that co-operate to telomere metabolism. Proper telomere maintenance is a crucial process to protect the genome against instability and telomere dysfunction has been linked to tumorigenesis and premature aging. AKTIP gene is the human homologue of Drosophila peo, a gene that was recently linked to telomere metabolism. The aim of this study was to understand if AKTIP could have a role in human telomere metabolism, in analogy with the telomeric function of its homologous in fly. For this purpose we have analyzed the phenotype of human cells in which AKTIP expression was downregulated by RNA interference. In human primary cells AKTIP downregulation triggered the reduction of the mitotic index, proliferation impairment and premature senescence. AKTIP reduction induced a strong DNA damage response proved by the accumulation of the phosphorylated form of proteins involved in DNA damage sensing and signaling such as ATM, p53 and Chk1, by the accumulation of p21 mRNA and by the formation of foci containing DNA damage response proteins. About half of these foci were located at telomeres (TIFs) indicating the presence of dysfunctional telomeres in AKTIP knocked down cells. These data were consistent with the accumulation of aberrant telomeres in MEFs p53-/- observed following the downregulation of murine homologue of AKTIP (named Ft1). AKTIP involvement in telomere metabolism was further suggested by its interaction with telomeric repeats observed by ChIP analysis. Altogether, these findings indicate that AKTIP takes part in telomere maintenance. Interestingly, immunostaining assays showed that AKTIP is not a stable component of telomeres but was found located in the nucleus, mainly at nuclear rim. This particular localization, in addition with the telomeric role outlined for AKTIP, suggest that AKTIP is a telomeric nonshelterin protein. Consistent with this hypothesis, we observed that Ft1 downregulation caused the formation of chromosomal aberrations in addition to telomeric abnormalities, indicating that AKTIP/Ft1 plays a role not only in telomere maintenance but also in the overall genomic stability, possibly contributing to DNA replication. Indeed, the most prominent telomeric aberration observed in Ft1 downregulated MEFs was the formation of multiple telomeric signals at the ends of chromosomes, also known as fragile telomeres, indicative of replication impairment. In addition, AKTIP downregulation was found to induce an S-phase block of cell cycle progression and a strong reduction of PCNA positive cells in primary fibroblasts, along with an increased sensitivity to drugs that impair DNA replication, as aphidicolin. Collectively, these data demonstrate that AKTIP is a protein needed for proper DNA maintenance in mammalian cells. In the telomeric context AKTIP likely is a telomeric accessory protein, rather than a shelterin-like protein, because it’s conserved in fly, differently from shelterin proteins, has a role in telomere maintenance but is not stably located at telomeres. AKTIP, in addition to its telomeric function, seems to have a more general role in cellular metabolism, as all the other telomeric nonshelterin proteins. In particular our data indicate that AKTIP could be involved in DNA replication. Considering all the collected data together, our current hypothesis is that AKTIP plays a role in replication of complex DNA structures, including telomeric repeats. Its downregulation could impair the replication fork progression through these DNA regions leading to chromosomal aberrations, DNA damage response and cell cycle alterations, the most prominent phenotypic traits of AKTIP knocked down cells

Synthesis of proton exchange membrane based on sulfonated polyphenylsulfone for fuel cell applications

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Engineering, 2017This dissertation presents a detailed study on the synthesis of nanocomposite membranes of sulfonated polyphenylsulfone embedded with carbon nanoball fillers. The effect of various synthesis parameters such as temperature, time, and concentration of the sulfonating agent on sulfonation of polyphenylsulfone, and the production of carbon nanoballs by non-catalytic chemical vapour deposition method were investigated. The synthesized carbon nanoballs were added to the polyphenylsulfone membrane in order to optimize the mechanical properties of the membrane. Furthermore, the effect of addition of low volumes of carbon nanoballs on the morphology and membrane properties was investigated. The sulfonation of polymeric membrane was characterized by Proton Nuclear Magnetic Resonance (1H NMR) which confirmed the sulfonation of polyphenylsulfone. The Transmission Electron Microscopy (TEM) analysis showed that produced CNBs had necklace structure with almost uniform size ranging between 40 to 60nm. Bruner-EmmettTeller (BET) analysis showed that CNBs had less impurities with pore volume and diameter of 0.0316 cm3/g and 16.7nm, respectively. From TGA result, it was observed that CNBs were thermally stable. Raman analysis indicates that CNBs were non conductive, a property which avoids unnecessary short circuits in the functioning of the fuel cells. Nano composite membranes with varying loading levels from 0.25 wt% to 4 wt% were prepared using ultrasonication at varying amplitudes of 20%, 60% and 75%, and simple evaporative casting technique. The TGA graph shows that the addition of carbon nanoballs has significantly increased the thermal stability of SPPSU membrane and all the composite membranes prepared with varying CNB loading showed similar decomposition profile. The nanocomposites prepared at 60% amplitude produced homogenous membranes; and the membrane with 1.75wt% CNB loading had high percentage resilience and satisfactory water uptake capacity than other membranes. The results confirmed that the addition of CNBs in low volumes increase the thermal stability and percentage resilience which are very crucial for fuel cell applications.XL201

Genomic instability and DNA replication defects in progeroid syndromes

Progeroid syndromes induced by mutations in lamin A or in its interactors – named progeroid laminopathies – are model systems for the dissection of the molecular pathways causing physio- logical and premature aging. A large amount of data, based mainly on the Hutchinson Gilford Progeria syndrome (HGPS), one of the best characterized progeroid laminopathy, has highlighted the role of lamins in multiple DNA activities, including replication, repair, chromatin organization and telomere function. On the other hand, the phenotypes generated by mutations affecting genes directly acting on DNA function, as mutations in the helicases WRN and BLM or in the polymerase polδ, share many of the traits of progeroid laminopathies. These evidences support the hypothesis of a concerted implication of DNA function and lamins in aging. We focus here on these aspects to contribute to the comprehension of the driving forces acting in progeroid syndromes and premature aging

Deep learning techniques for biological signal processing: Automatic detection of dolphin sounds

openConsidering the heterogeneous underwater acoustic transmission context, detecting and distinguishing vocalizations of cetaceans has been a challenging area of recent interest. A promising venue to improve current detection systems is constituted by machine learning algorithms. In particular, Convolutional Neural Networks (CNNs) are considered one of the most promising deep learning techniques, since they have already excelled in problems involving the automatic processing of biological sounds. Human-annotated spectrograms can be used to teach CNNs how to distinguish between information in the time-frequency domain, thus enabling the detection and classification of marine mammal sounds. However, despite these promising capabilities machine learning suffers from a lack of labeled data, which calls for the adoption of transfer learning to create accurate models even when the availability of human taggers is limited. In this thesis, we developed a dolphin whistle detection framework based on deep learning models. In particular, we investigated the performance of large-scale pre-trained models (VGG16) and compared it with the performance of a vanilla Convolutional Neural Network and several baselines (logistic regression and Support Vector Machines). The pre-trained VGG16 model achieved the best detection performance, with an accuracy of 98,9\% on a left-out test dataset.Considering the heterogeneous underwater acoustic transmission context, detecting and distinguishing vocalizations of cetaceans has been a challenging area of recent interest. A promising venue to improve current detection systems is constituted by machine learning algorithms. In particular, Convolutional Neural Networks (CNNs) are considered one of the most promising deep learning techniques, since they have already excelled in problems involving the automatic processing of biological sounds. Human-annotated spectrograms can be used to teach CNNs how to distinguish between information in the time-frequency domain, thus enabling the detection and classification of marine mammal sounds. However, despite these promising capabilities machine learning suffers from a lack of labeled data, which calls for the adoption of transfer learning to create accurate models even when the availability of human taggers is limited. In this thesis, we developed a dolphin whistle detection framework based on deep learning models. In particular, we investigated the performance of large-scale pre-trained models (VGG16) and compared it with the performance of a vanilla Convolutional Neural Network and several baselines (logistic regression and Support Vector Machines). The pre-trained VGG16 model achieved the best detection performance, with an accuracy of 98,9\% on a left-out test dataset

Stress management in composite biopolymer networks

Living tissues show an extraordinary adaptiveness to strain, which is crucial for their proper biological functioning. The physical origin of this mechanical behaviour has been widely investigated using reconstituted networks of collagen fibres, the principal load-bearing component of tissues. However, collagen fibres in tissues are embedded in a soft hydrated polysaccharide matrix which generates substantial internal stresses whose effect on tissue mechanics is unknown. Here, by combining mechanical measurements and computer simulations, we show that networks composed of collagen fibres and a hyaluronan matrix exhibit synergistic mechanics characterized by an enhanced stiffness and delayed strain-stiffening. We demonstrate that the polysaccharide matrix has a dual effect on the composite response involving both internal stress and elastic reinforcement. Our findings elucidate how tissues can tune their strain-sensitivity over a wide range and provide a novel design principle for synthetic materials with programmable mechanical properties

Increase of phenotypic variance in stressful environments

The hypothesis that genetic homeostasis breaks down to yield greater genetic variance in more stressful environments was examined. Environmental stress was measured by mean development time and by wing length, it being expected that more stress generally gives rise to longer development times and shorter wings. The correlations between mean values and genetic variance were predicted to be positive for development time and negative for wing length. The correlations were not always statistically significant, but were in the predicted direction in 7 out of 8 tests. Quite possibly this phenomenon contributes to observed increases of genetic variation in marginal environments and more rapid evolution during periods of special stres