Microbial Diversity in Raw and Pasteurized Milk with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Abstract In this thesis, a molecular PCR-based method was used to study the bacterial diversity in milk. The aim was to compare the microbiota of traditionally pasteurized milk with milk treated with a novel pasteurization technique, using terminal restriction fragment length polymorphism (T-RFLP). A second aim was to analyze the microbial composition of cheese produced from the two milk variants. Results of this molecular approach were compared with outcomes from traditional culturing on non-selective media, followed by 16S rRNA sequencing in order to evaluate the usefulness of the methods. Overall, the results demonstrated that T-RFLP is a powerful tool for analyzing microbiota in foods. In conclusion, both pasteurization techniques proved to be effective in reducing the number of bacteria. The initial hypothesis, that the two pasteurization techniques affect different parts of the microbiota of the raw milk, was confirmed. As expected, the molecular approach of DNA extraction direct from the milk detected a more differentiated microflora, compared to DNA extracted from cultivated bacteria from the milk. The results also indicated that the molecular approach was more reproducible between the sampling occasions. The cultivation and sequencing showed that the microbiota mainly consisted of Firmicutes and Actinobacteria, as well as Bacteriodetes. Popular science summary: Microbial Diversity in Raw and Pasteurized milk In this thesis, a molecular PCR-based method was used to study the bacterial diversity in milk. The aim was to compare the microbiota of traditionally pasteurized milk with milk treated with a novel pasteurization technique, using terminal restriction fragment length polymorphism (T-RFLP). A second aim was to analyze the microbial composition of cheese produced from the two milk variants. Results of this molecular approach were compared with outcomes from traditional culturing on non-selective media, followed by 16S rRNA sequencing in order to evaluate the usefulness of the methods. Much of the flavor and aroma of milk and cheese originates in its microbial content. Pasteurization is used to minimize the pathogens and extend the shelf-life for food. It decreases the number of microorganisms in the product by heating, followed by an immediate cooling. The treatment does not sterilize the product, but reduces the number of viable microorganisms. The awareness and control of the bacterial diversity in the milk is therefore a very important aspect in production of cheese, dairy product and other food products. Traditional methods to assess community diversity often include culturing on plates. In addition to being labor-intensive, it has proven to give a deceptive reflection of the original community structure. Molecular biology methods are based on variations in the DNA sequences between bacteria species. T-RFLP visualizes differences in the prokaryotic 16S rRNA. The analysis typically involve four steps: DNA isolation and purification, PCR amplification and restriction enzyme digestion, separation of digested products via capillary gel electrophoresis and finally analysis and clustering of data to generate a fragment profile for each sample. Identity of the terminal restriction fragments can be obtained by creation of a clone library. Effect of pasteurization The two pasteurization techniques affect different parts of the microbiota of the raw milk. Traditional pasteurization showed the greatest decrease in number of bacterial species. The cheese microbiota was considerably less diverse. As expected, the number of bacterial species found from the non-cultured population was greater than from the cultivatable population. The results also indicated that the molecular approach was more reproducible between the sampling occasions. A majority of the identified bacteria belonged to the Firmicutes phylum and were lactic acid bacteria (LAB). In conclusion, both pasteurization techniques proved to be effective in reducing the number of bacteria. The results confirmed the advantages of using molecular opposed to culture-based approaches to characterize microbiota. The study also verified the applicability of T-RFLP for analyzing microbiota in foods. Advisor: Klara Båth Degree Project 30 credits in Cell and Molecular Biology 2012 Department of Biology, Lund Universit

Molecular orientation in cellulose fibers and composites

The scope of this thesis is to quantitatively investigate the molecular orientation distribution of regenerated cellulose fibers and composites. The molecular orientation is known to affect macroscopic properties such as tensile strength of the fiber. In addition, the quality of a carbon fiber is, to a great extent, determined by the molecular orientation of the precursor. A plethora of techniques are paramount for materials characterization and a handful of these are suited for determination of molecular orientation. Since different methods have various experimental limitations, methodological awareness is crucial in the strive for quantitative data and in particular when cellulose fibers and other polymers are chemically modified, or a part of a composite. This work concerns three methods in order to investigate molecular orientation: rotor synchronized magic angle spinning solid-state nuclear magnetic resonance spectroscopy (ROSMAS), polarized Raman spectroscopy, and X-ray scattering. The latter is already a proven method for analyzing molecular orientation and was therefore used as a reference for the two first methods, which have never previously been applied on cellulose fibers. ROSMAS was used to investigate the chemical shift anisotropy, which relates to molecular orientation, on a bundle of Lyocell fibers. Polarized Raman spectroscopy was used to analyze the molecular orientation distribution from the Raman vibrational tensor on a single fiber. A new method was developed for polarized Raman spectroscopy by assuming a wrapped Lorentzian orientation distribution function, as measured from X-ray scattering patterns. The results from both ROSMAS and polarized Raman spectroscopy were in agreement with X-ray scattering on a highly oriented cellulose fiber bundle and on a single regenerated cellulose fiber, respectively, indicating that these methods are quantitative. The ROSMAS and X-ray methodologies were applied to a stretched fiber consisting of a regenerated cellulose-lignin composite intended as a carbon fiber precursor. Finally, ROSMAS was also used for determination of the complete chemical shift anisotropy in the molecular reference frame on regio-regular poly(3-hexylthiophene) fibers, in addition to elucidation of backbone and side chain orientation.In the grand perspective, resources have to be used efficiently to minimize environmental impact. Therefore, this work explores man-made environmentally benign cellulose alternatives to cotton and other polymers. These processes refine cellulose from plant life, typically trees, which can grow without pesticide on non-arable land

Delamination of Cross-laminated timber and its impact on fire development | Focusing on different types of adhesives

In the recent decade, the interest of building taller all-timber structures using engineered timber materials such as cross-laminated timber (CLT) has increased substantially. On the other hand, there’s also a significant resistance and fear concerning the fire safety of buildings with wood-based load-bearing structures. In this report, the aim is to investigate the potential deviations of the fire performance of CLT. More specifically the occurrence of losing entire layers due to fire exposure, delamination, and how this might affect the fire development. Fire tests were performed in a standard testing furnace with specimens of CLT manufactured with five different adhesives. For these tests, the fire conditions of a previous full-scale test were attempted to be replicated. After evaluating the results, it was concluded that the type of adhesive used, could have a significant impact on whether a CLT-element delaminates or not. Furthermore, delamination also has a severe impact on the fire development, increasing the temperatures and extending the duration of the fire. CLT as a future building material still faces a lot of challenges, and further research is needed to optimise the composition of the material and investigate complications other than fire performance as well.För framtidens träkonstruktioner är limträprodukter, så som korslimmat trä, helt avgörande för utvecklingen. Korslimmat trä kan tillämpas som prefabricerade element i såväl bärande väggar, som bjälklag och öppnar upp för helt nya möjligheter för snabbt, hållbart byggande. Samtidigt finns det ett kraftigt motstånd från många håll mot det ökande användandet av trä i bärande konstruktioner, inte minst ur ett brandsäkerhetsperspektiv. Tidigare forskning om brandbeteendet hos solitt trä är omfattande, men när det gäller eventuella avvikelser hos modernare limträprodukter, är beteendet mindre känt

Polarized Raman Spectroscopy Strategy for Molecular Orientation of Polymeric Fibers with Raman Tensors Deviating from the Molecular Frame

Polarized light is frequently used to identify molecular anisotropy in polymers, biological systems, and other materials. The influence of the Raman tensor by polarized light reveals not only the chemical structure but also information on the morphology of polymers. The orientation distribution of molecules in polymers has previously been determined for systems with the principal axis components of the Raman tensor parallel to the molecular frame. In many cases, the Raman tensor principal axis is not parallel to the molecular chain axis. Therefore, the orientation of the Raman tensor, relative to the molecular chain axis, is crucial if accurate information about the molecular orientation distribution is sought for. This work presents a strategy for separating the Raman tensor orientation angles from the molecular orientation angles for polymeric samples with fiber symmetry. Composite polymeric materials often experience signal overlap in the X-ray scattering wide-angle region, where the anisotropy is often resolved. While X-ray scattering investigates intermolecular distances, Raman spectroscopy resolves chemical information, and anisotropy, by the influence of Raman scattering. The quantitative principles presented here may aid in the evaluation of anisotropy in such composite materials

SGX-Bundler: speeding up enclave transitions for IO-intensive applications

Process-based confidential computing enclaves such as Intel SGX can be used to protect the confidentiality and integrity of workloads, without the overhead of virtualisation. However, they introduce a notable performance overhead, especially when it comes to transitions in and out of the enclave context. Such overhead makes the use of enclaves impractical for running IO-intensive applications, such as network packet processing or biological sequence analysis. We build on earlier approaches to improve the IO performance of work-loads in Intel SGX enclaves and propose the SGX-Bundler library, which helps reduce the cost of both individual single enclave transitions well as of the total number of enclave transitions in trusted applications running in Intel SGX enclaves. We describe the implementation of the SGX-Bundler library, evaluate its performance and demonstrate its practicality using the case study of Open vSwitch, a widely used software switch implementation

Faster enclave transitions for IO-intensive network applications

Process-based confidential computing enclaves such as Intel SGX have been proposed for protecting the confidentiality and integrity of network applications, without the overhead of virtualization. However, these solutions introduce other types of overhead, particularly the cost transitioning in and out of an enclave context. This makes the use of enclaves impractical for running IO-intensive applications, such as network packet processing. We build on ear- lier approaches to improve the IO performance of workloads in Intel SGX enclaves and propose the HotCall-Bundler library that helps reduce the cost of individual single enclave transitions and the total number of enclave transitions in trusted applications running in Intel SGX enclaves. We describe the implementation of the HotCall-Bundler library, evaluate its performance and demonstrate its practicality using the case study of Open vSwitch, a widely used software switch implementation

Paroxysmal Kinesigenic Dyskinesia

Background: Paroxysmal kinesigenic dyskinesia (PKD) is a rare condition associated with heterozygous mutations in the PRRT2 gene. Phenomenology shown: In this letter we illustrate the phenomenology of PKD in a man previously misdiagnosed as Tourette’s syndrome. Educational value: Regardless of underlying phenotype, PKD is highly responsive to some antiepileptic drugs

Multidimensional encoding of restricted and anisotropic diffusion by double rotation of the q-vector

Diffusion NMR and MRI methods building on the classic pulsed gradient spin echo sequence are sensitive to many aspects of translational motion, including time/frequency-dependence (&ldquo;restriction&rdquo;), anisotropy, and flow, which leads to ambiguities when interpreting experimental data from complex heterogeneous materials such as living biological tissues. Higher specificity to restriction or anisotropy can be obtained with, respectively, oscillating gradient or tensor-valued encoding which nevertheless both have some sensitivity to the property not being of direct interest. Here we propose a simple scheme derived from the &ldquo;double rotation&rdquo; technique in solid-state NMR to generate a family of modulated gradient waveforms allowing for comprehensive exploration of the two-dimensional frequency-anisotropy space and convenient investigation of both restricted and anisotropic diffusion with a single multidimensional acquisition protocol. The method is demonstrated by measuring multicomponent isotropic Gaussian diffusion in simple liquids, anisotropic Gaussian diffusion in a polydomain lyotropic liquid crystal, and restricted diffusion in a yeast cell sediment.</p

Distinct Lysosomal Network Protein Profiles in Parkinsonian Syndrome Cerebrospinal Fluid.

BackgroundClinical diagnosis of parkinsonian syndromes like Parkinson's disease (PD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) is hampered by overlapping symptomatology and lack of diagnostic biomarkers, and definitive diagnosis is only possible post-mortem.ObjectiveSince impaired protein degradation plays an important role in many neurodegenerative disorders, we hypothesized that profiles of select lysosomal network proteins in cerebrospinal fluid could be differentially expressed in these parkinsonian syndromes.MethodsCerebrospinal fluid samples were collected from PD patients (n = 18), clinically diagnosed 4-repeat tauopathy patients; corticobasal syndrome (CBS) (n = 3) and PSP (n = 8); and pathologically diagnosed PSP (n = 8) and CBD patients (n = 7). Each patient set was compared to its appropriate control group consisting of age and gender matched individuals. Select lysosomal network protein levels were detected via Western blotting. Factor analysis was used to test the diagnostic sensitivity, specificity and accuracy of the select lysosomal network protein expression profiles.ResultsPD, CBD and PSP were markedly different in their cerebrospinal fluid lysosomal network protein profiles. Lysosomal-associated membrane proteins 1 and 2 were significantly decreased in PD; early endosomal antigen 1 was decreased and lysozyme increased in PSP; and lysosomal-associated membrane proteins 1 and 2, microtubule-associated protein 1 light chain 3 and lysozyme were increased in CBD. A&nbsp;panel of lysosomal-associated membrane protein 2, lysozyme and microtubule-associated protein 1 light chain discriminated between controls, PD and 4-repeat tauopathies.ConclusionsThis study offers proof of concept that select lysosomal network proteins are differentially expressed in cerebrospinal fluid of Parkinson's disease, corticobasal syndrome and progressive supranuclear palsy. Lysosomal network protein analysis could be further developed as a diagnostic fluid biomarker in parkinsonian syndromes

Forest-edge effects on sea-salt aerosol deposition : a wind-tunnel study using living oak leaves

Landscape patchiness creates aerodynamic transition zones that affect the exchange of nutrients and pollutants between the atmosphere and vegetation. Using an artificially-generated NaCl aerosol (mass-versus-particle-size distribution with aerodynamic mean particle diameter 1.6 mu m; geometric standard deviation 1.9), we investigated the forest-edge effect on aerosol deposition within a model oak (Quercus robur) canopy in a wind tunnel with an emulated beach-to-forest transition. The deposition rate around the forest edge was 2-3 times higher than to the beach and 50%-60% higher than to the interior of the forest. The deposition velocity at the edge was 0.06 cm s(-1), which is 2-3 times higher than the beach-deposition velocity. Our results can help improve estimates of aerosol-borne inputs of nutrients or pollutants to forested landscapes that experience shifts in meteorological regimes due to changes in climate and forestry practices, in particular with respect to deciduous species in coastal environments