Filmes de Langmuir são usados como modelos simplificados de membranas celulares, cujas propriedades podem ser correlacionadas com efeitos fisiológicos de moléculas de interesse biológico, como os peptídeos antimicrobianos (PAMs). Nesta dissertação investigamos a interação do peptídeo Aureína 1.2, na forma de monômero (AU), dímero ((AU)2K) e com variações na porção N-terminal (KAU e DAU), com filmes de Langmuir obtidos do extrato lipídico da bactéria Escherichia coli. Todos os peptídeos injetados em concentrações de 20 a 200nM se incorporaram ao filme de Langmuir, causando expansão nas isotermas de pressão superficial, que foi significativamente maior para o dímero. O módulo de compressibilidade do filme de E. coli à pressão superficial correspondente à de uma membrana real praticamente dobrou, de cerca de 40mN/m para 80nM/m para o dímero, ao passo que para os outros peptídeos a alteração não foi significativa. Dos espectros de reflexão e absorção no infravermelho com modulação de polarização (PM-IRRAS), observou-se que todos os peptídeos interagiram tanto com as caudas quanto com as cabeças polares das moléculas do extrato de E. coli no filme de Langmuir. Diferentemente dos resultados de pressão e compressibilidade, não há tendência de um peptídeo ter interação mais relevante do que os outros. O maior efeito do dímero na expansão e compressibilidade do filme de Langmuir não se refletiu numa maior atividade bactericida contra E. coli, pois sabe-se da literatura que a atividade é maior para a Aureína 1.2 (AU). Provavelmente porque essa atividade deve depender da camada externa de lipopolissacarídeos de uma bactéria Gram-negativa. / Langmuir films are used as simplified cell membrane models whose properties can be correlated with physiological effects of molecules of biological interest, such as antimicrobial peptides (AMPs). In this dissertation we report on the interaction of Aurein 1.2 peptide as monomer (AU), dimer ((AU)2K) and modified peptide in the N-terminal portion (KAU and SAD), with Langmuir films obtained from a lipid extract of Escherichia coli. All peptides injected at concentrations from 20 to 200nM were incorporated into the Langmuir film, causing the surface pressure isotherm to expand, particularly for the dimer. The compressibility modulus of the E. coli Langmuir film at the surface pressure corresponding to an actual membrane nearly doubled, from about 40mN/m to 80nM/m for the dimer, whereas for the other peptides the change was not significant. From the polarization-modulated infrared reflection - absorption spectra (PM-IRRAS), we observed that all peptides interacted with both tails and polar heads of the molecules of E. coli extract in the Langmuir film. Unlike the results of pressure and compressibility, there was no tendency of a peptide having more relevant interaction than the others. The larger effect of the dimer in the expansion and compressibility of the Langmuir film was not reflected in a higher bactericidal activity against E. coli, since it is known from literature that the activity is higher for Aurein 1.2 (AU). Probably because this activity should depend on the outer layer of lipopolysaccharides of Gram-negative bacteria.

Humans are approximately 99% similar with inter-individual differences caused in part by single-nucleotide polymorphisms (SNPs), which poses a challenge for the effective treatment of disease. Bioinformatics resources can help to store and analyse gene and protein information to address this challenge, however these resources have limitations, so the collation and biocuration of gene and protein information is required. Using the large conductance calcium- and voltage-activated potassium channel, also known as the Big Potassium (BK) channel as an example, due to its ubiquitous expression and widespread varied role in human physiology, this study aimed to prioritise SNPs with the potential to affect the function of the channel. Using a BK channel resource created with bioinformatics tools and published literature, mSlo SNPs H55Q and G57A, located in the S0-S1 linker, were prioritised and selected for lab-based verification. These SNPs flank three cysteine residues proven to modulate channel cellular distribution via palmitoylation, a reversible process shown to increase protein association with the cell membrane. The SNPs alter the predicted palmitoylation status of C56, one of the cysteine residues located in the S0-S1 linker. The cellular distribution of BK channels incorporating the SNPs was assessed using confocal microscopy and revealed that the direction and magnitude of SNP mimetic cell membrane expression was closely related to the C56 predicted palmitoylation score; a 'C56 palmitoylation pattern' was observed. It was shown that exposure to metabolic substrates glucose, palmitate and oleate modulated SNP-mimetic cellular distribution and could invert the 'C56 palmitoylation pattern', indicating that there is interplay between the metabolic status of the cell and the amino-acid composition of the channel via palmitoylation. The creation of a novel BK channel resource in this thesis highlighted the limitations, and inter-dependency of bioinformatics and lab based experimentation, whilst SNP verification experiments solidified the link between S0-S1 cysteine residues and BK cellular distribution. BK channel function is linked with a number of physiological processes; thus, the potential clinical consequences of the SNPs prioritised in this thesis require further research.

Cell wounding, the loss of plasma membrane integrity, is a common event in the life of many cell types. Most cells are subjected to physiological events during normal functions that can lead to disruption of their plasma membranes, especially cells in the load bearing organs such as muscle, skin and bone. The capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. / In this thesis, we were trying to uncover single cell wound responses by applying the femtosecond laser (fs laser) technology. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscopic system. Combining real-time observations of the fs laser-induced wound and 3D reconstruction of the cells, post-damage cell and nucleus morphological transformation and wound dynamics were reported. The major findings of this study include: (1) Fs laser could induce a small hole on the plasma membrane of the targeted cell. With the same laser irradiation time, the initial hole size were positively correlated with the laser power. (2) Four typical hole evolution scenarios were reported. Hole resealing was a fast process mostly within 100 seconds in normal condition. Whether a cell could reseal the hole is dependent on the initial hole size. Cells had difficulty to cope with the bigger holes. Three ranges of hole size were given in the thesis to predict the hole resealing result. (3) After fs laser damage, the whole cell underwent a contraction. The post-damage nucleus area, footprint, and each section layer of the cell all shrank, only the thickness remained the same. The nucleus retreated a bit from the damage site after damage. (4) Oxidative stress altered some of the cellular responses to the laser damage. The fs laser- induced holes in oxidative groups were bigger than the normal condition. The cells underwent an overall swelling after fs laser damage instead of the contraction in the normal group. Section layer areas and the thickness of the cell increased after damage. But similar to the normal condition, nucleus shrinkage and retreat from the damage site were also found in the oxidative stress groups. (5) Although both acute and chronic oxidative stresses compromised the integrity of the plasma membrane, chronic oxidative stress compromised more severely with several critical post-damage cell transformations and low resealing ratio. Acute oxidative stress on the other hand may somehow promote the resealing ability of the cells. (6) The section layers closer to the bottom of the cells transformed less than the layers further away from the bottom. This probably suggested that the cell basal attachment provided a constraint force to the plasma membrane for morphological changes. / 細胞創傷，即細胞膜的完整性受損，是多種細胞生命週期中一種常見的現象。細胞在執行正常功能時可能遭遇不同程度的生理性損傷，其中大部分會導致細胞膜的破壞。這一現象對存在於承受壓力器官中的細胞更為頻繁，例如肌肉，皮膚和骨骼。細胞對於日常磨損性傷害以及意外創傷的修復能力，是維持組織完整性的基石。 / 在本論文中，我們通過使用飛秒鐳射技術模擬單細胞創傷，觀察並試圖揭示單細胞對於創傷的反應過程。在實驗中，參數可調的飛秒鐳射器與共軛聚焦顯微鏡整合為一個系統，用於在單細胞膜上進行定點損傷。我們結合了對損傷的即時觀測，細胞的三維結構重建技術，完整記錄了損傷前後的損傷部位，細胞整體以及細胞核的形態變化。以下是本研究的主要發現：（1）飛秒鐳射能夠在目標細胞的細胞膜上進行局部穿孔。在鐳射照射時間相同的情況下，鐳射穿孔的大小與鐳射的平均功率呈正相關。（2）我們發現了鐳射穿孔後穿孔部位有四種不同的變化情況。穿孔後細胞封孔是一相當快的過程，在細胞成功封孔的情況下，大部分細胞將在100秒以內將穿孔部位重新填滿。細胞是否可以將穿孔封住取決於鐳射照射後初始穿孔的大小。細胞很難修復較大的孔。我們將細胞初始穿孔大小分為三個範圍，根據這三個範圍可以利用初始孔的尺寸大致預測穿孔後細胞的封孔情況。（3）飛秒鐳射損傷細胞後，細胞將會收縮，並且細胞核的平面面積，細胞的平面面積（或稱細胞足跡），以及細胞各分層面積都有不同程度的縮小。僅細胞厚度未發生顯著變化。同時，細胞核的位置相對於損傷部位有所後退。（4）細胞在氧化應激過後，對於飛秒鐳射造成的損傷反應有所變化。具體表現為：鐳射穿孔的尺寸比正常情況下更大；穿孔後細胞將會整體腫脹而非收縮。各分層面積和細胞厚度都有不同程度的增大。但是細胞核的反應與正常情況類似，即細胞核將會收縮，並且後退以遠離鐳射損傷部位。（5）儘管急性氧化應激和慢性氧化應激都一定程度上損傷了細胞膜的完整性，但是從細胞對於鐳射創傷的反應觀察，長期慢性氧化應激對於細胞膜的損害更為嚴重，具體表現為鐳射損傷後細胞的嚴重形變和細胞膜修復比例的降低。而另一方面，急性氧化應激在某種程度上可以增強細胞對於鐳射穿孔的修復能力。（6）細胞膜穿孔後，細胞各層的面積變化不一，位置越靠近底層的分層面積變化越小。這可能表明細胞貼壁行為形成了一個對細胞形變的約束力。 / Duan, Xinxing. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 73-85). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

The objective of this work is to develop an innovative and quantitative method to study cell failure under fluidic pressure to understand cell membrane mechanical properties. Due to lack of experimental data related to cell failure property, the current research focuses on investigating the cell failure using a micro pipette aspiration experiment method to elaborate gradually increasing hydrostatic pressure to the cell causing the membrane to deform and eventually rupture. Based on our observation, the prostate cancer cells (PC-3) deformed into a deflated and flattened shape under higher hydrostatic pressure (249 Pa) while prostate epithelial cells (PrEC LH) cells generate a spherical and rounded shape. The stress along the cell membrane was estimated from the curvature data captured from the 2D microscopic images for each pressure magnitude to quantify the damage before rupture state. From the results, non-transformed prostate epithelial cells (PrEC LH) presented a stiffer and rupture resilient property compared to transformed prostate cancer cells (PC-3) which presented a softer and vulnerable property. Besides, the alteration of shape of the aspirated membrane directly affected the stress distribution over the membrane and as a result, provoked membrane failure. Multiple pieces of research have shown a higher stiffness of healthy cells compared to cancer cells including one of the previous studies done by our group which have also found that cancer cell tends to become stiffer after exposing to fluid shear stress. The discovery of this cellular behavior and novel numerical quantification method of cell failure could advance the study of cancer cell membrane failure, cellular matrix structure, response to mechanical loadings and potentially foundation in developing new treatment for cancer other than destructive chemical treatment.

Apoptosis, or programmed cell death, is essential for proper development and functioning of the body systems. During development, apoptosis plays a central role to sculpt the embryo, and in adults, to maintain tissue homeostasis by eliminating redundant, damaged or effete cells. Therefore, a tight regulation of this process is essential. Cell shrinkage associated efflux of K+ and Cl– through plasma membrane ion channels is an early event of apoptosis. However, little is known about these fluxes. The aim of this thesis was to investigate ion channels in the plasma membrane of neurons undergoing apoptosis. We studied differentiated (the mouse hippocampal cell line HT22, the human neuroblastoma cell line SK-N-MC, and rat primary hippocampal neurons) and undifferentiated (rat primary cortical neural stem cells cNSCs) cells with the patch-clamp technique. All cell types displayed a low electrical activity under control conditions. However, during apoptosis in differentiated neurons, we found an activation of a voltage-dependent anion channel. The conductance of the channel is 400 pS, the voltage dependence of the opening is bell shaped with respect to membrane voltage with a maximum open probability at 0 mV, and the Cl− to cation selectivity is >5:1. These biophysical properties remind about the voltage-dependent anion channel normally found in the outer mitochondrial membrane (VDACmt). Hence, we call our apoptosis-inducing plasma membrane channel VDACpl. The molecular identity of the channel was corroborated with the specific labelling of different anti-VDAC antibodies. Block of this channel either with antibodies or with sucrose prevented apoptosis, suggesting a critical role for VDACpl in the apoptotic process. VDACpl is a NADH (-ferricyanide) reductase in control cells. We found that the enzymatic activity is altered while the VDACpl channel is activated during apoptosis. Surprisingly, in cNSCs we did not find any activation of VDACpl, no VDACpl-specific labelling, no enzymatic activity, and no prevention of apoptosis with VDACpl-blocking strategies. Instead, we found an activation of a voltage-independent 37 pS ion channel, and that the Cl– channel blocker DIDS prevented apoptosis in cNSCs. Our finding that activation of VDACpl is critical for apoptosis in differentiated neurons hopefully can lead to new strategies in the treatment of several diseases related to apoptosis.

The aim for this research report is to categorize and describe students’ conceptions about the structure and function of the cell membrane from a phenomenographic and variation theory perspective. Students’ ability to understand different concepts depends on their ability to comprehend certain critical features of the content. The critical feature of understanding the structure of the cell membrane investigated here is the polar and non-polar properties of molecules. The critical feature of understanding the function of the cell membrane is transport through the cell membrane. Another aim is to investigate what animations, concerning cellular transport, can contribute to teaching and students understanding of the cell membrane. Furthermore, a subordinated aim is to distinguish whether there are any existing differences and similarities between South Africa and Sweden in consideration to students’ conceptions about the cell membrane. Two different methods of data collection, questionnaires and semi-structured interviews, were used in this investigation. 80 students participated in the questionnaire and 5 students participated in the interviews. Four categories of conceptions about the characteristics of polar and non-polar molecules have been identified. Furthermore, one of the most remarkable and notable findings discovered are that most teachers and students are not aware of the current scientific view on how water molecules are transported through the cell membrane. Knowledge about aquaporines, discovered by Agre in 1992, seems to be almost non-existing in science education in upper secondary school, in Sweden and South Africa as well. Furthermore, students experience animations to be complex and which in some cases seem to be regarded as messy representation. Simultaneously they strongly emphasise the need for animations to support learning and remembering. Animations can be seen as a source of variation in teaching. The conceptions described occurred both among the South African students as well among the Swedish students. Also similarities concerning students’ conceptions have been discerned between the two countries investigated. For example there could be that South African students possess a richer understanding for the concept of the cell membrane than the Swedish students, but find it more difficult to move between different contexts.

In this study, a new bacteriocin isolated from a Leuconostoc mesenteroides subsp. cremoris strain was purified to homogeneity by pH mediated cell adsorption-desorption, solid phase extraction with Amberlite XAD-16, cation-exchange chromatography and hydrophobic interaction chromatography. The purification resulted in an electrophoretically pure protein that was smaller than 6 kDa as judged by SDS-PAGE. The bacteriocin was found to be very hydrophobic and cationic and could be adsorbed by synthetic calcium silicate due to its cationic and especially hydrophobic nature. It was observed that this bacteriocin was sensitive to &amp / #945 / -amylase in addition to proteinase K, trypsine, pepsine and chymotrypsine and had a bactericidal mode of action with a concomitant cell lysis. The results indicated that bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was more stable to combined effect of pH and heat than nisin, lacticin 481, lacticin 3147 and lactococcin G and was bactericidal between pH 2.0-12. It was found that the bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was stable to organic solvents and could be extracted with chloroform containing solvents efficiently for purification. The bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was found to have a strong inhibitory activity against Listeria innoqua, Listeria monocytogenes, Bacillus cereus, Enterococcus faecalis, Lactobacillus delbrueckii, Lactobacillus cremoris, other Leuconostoc meenteroides strains and gram-negative bacterium Pseudomonas fluorescens. Some of the insensitive bacteria were observed to be sensitive when high concentration of the bacteriocin was used.

<p>The aim for this research report is to categorize and describe students’ conceptions about the structure and function of the cell membrane from a phenomenographic and variation theory perspective. Students’ ability to understand different concepts depends on their ability to comprehend certain critical features of the content. The critical feature of understanding the structure of the cell membrane investigated here is the polar and non-polar properties of molecules. The critical feature of understanding the function of the cell membrane is transport through the cell membrane. Another aim is to investigate what animations, concerning cellular transport, can contribute to teaching and students understanding of the cell membrane. Furthermore, a subordinated aim is to distinguish whether there are any existing differences and similarities between South Africa and Sweden in consideration to students’ conceptions about the cell membrane.</p><p>Two different methods of data collection, questionnaires and semi-structured interviews, were used in this investigation. 80 students participated in the questionnaire and 5 students participated in the interviews.</p><p>Four categories of conceptions about the characteristics of polar and non-polar molecules have been identified. Furthermore, one of the most remarkable and notable findings discovered are that most teachers and students are not aware of the current scientific view on how water molecules are transported through the cell membrane. Knowledge about aquaporines, discovered by Agre in 1992, seems to be almost non-existing in science education in upper secondary school, in Sweden and South Africa as well. Furthermore, students experience animations to be complex and which in some cases seem to be regarded as messy representation. Simultaneously they strongly emphasise the need for animations to support learning and remembering. Animations can be seen as a source of variation in teaching. The conceptions described occurred both among the South African students as well among the Swedish students. Also similarities concerning students’ conceptions have been discerned between the two countries investigated. For example there could be that South African students possess a richer understanding for the concept of the cell membrane than the Swedish students, but find it more difficult to move between different contexts.</p>

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.