| 1. |
- Byström, Roberth, 1971-
(författare)
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SOD1´s Law : An Investigation of ALS Provoking Properties in SOD1
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.
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| 2. |
- Vestergren, Johan, 1968-
(författare)
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Analysis and speciation of organic phosphorus in environmental matrices : Development of methods to improve 31P NMR analysis
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Phosphorus (P) is an essential element for life on our planet. It is central in numerous biochemical processes in terrestrial and aqueous ecosystems including food production; and it is the primary growth-limiting nutrient in some of the world’s biomes. The main source of P for use as agricultural fertilizer is mining of non-renewable mineral phosphate. In terrestrial ecosystems the main source is soil P, where the largest fraction is organic P, composed of many species with widely differing properties. This fraction controls the utilization of P by plants and microorganisms and influences ecosystem development and productivity. However, there is only scarce knowledge about the molecular composition of the organic P pool, about the processes controlling its bioavailability, and about its changes as soils develop. Therefore, the aim of this thesis was to develop robust solution- and solid-state 31P nuclear magnetic resonance spectroscopy (NMR) methods to provide molecular information about speciation of the organic P pool, and to study its dynamics in boreal and tropical soils. By studying humus soils of a groundwater recharge/discharge productivity gradient in a Fennoscandian boreal forest by solution- and solid-state NMR, it was found that P speciation changed with productivity. In particular, the level of orthophosphate diesters decreased with increasing productivity while mono-esters such as inositol phosphates increased. Because the use of solution NMR on conventional NaOH/EDTA extracts of soils was limited due to severe line broadening caused by the presence of paramagnetic metal ions, a new extraction method was developed and validated. Based on the removal of these paramagnetic impurities by sulfide precipitation, a dramatic decrease in NMR linewidths was obtained, allowing for the first time to apply modern multi-dimensional solution NMR techniques to soil extracts. Identification of individual soil P-species, and tracking changes in the organic P pools during soil development provided information for connecting P-speciation to bioavailability and ecosystem properties. Using this NMR approach we studied the transformation of organic P in humus soils along a chronosequence (7800 years) in Northern Sweden. While total P varied little, the composition of the soil P pool changed particularly among young sites, where also the largest shift in the composition of the plant community and of soil microorganisms was observed. Very old soils, such as found Africa, are thought to strongly adsorb P, limiting plant productivity. I used NMR to study the effect of scattered agroforestry trees on P speciation in two semi-arid tropical woodlands with different soil mineralogy (Burkina Faso). While the total P concentration was low, under the tree canopies higher amounts of P and higher diversity of P-species were found, presumably reflecting higher microbial activity.
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| 3. |
- Lidman, Martin, 1985-
(författare)
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The role of the mitochondrial membrane system in apoptosis : the influence of oxidative stress on membranes and their interactions with apoptosis-regulating Bcl-2 proteins
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis is a crucial process in multicellular organisms in sculpting them, especially during embryogenesis. In addition, apoptosis is responsible for the clearance of harmful or damaged cells which can otherwise be detrimental to the organism. The Bcl-2 family proteins are key players in the regulation of the intrinsic pathway of the apoptotic machinery. This family consists of three subfamilies with B-cell CLL/lymphoma 2 (Bcl-2) protein itself representing anti-apoptotic members, the Bcl-2-associated X protein (Bax), and pro-apoptotic BH3-only signaling proteins. The interplay between pro- and anti-apoptotic proteins on the mitochondrial membranes is central to the balance between the life and death decision of whether the membrane should be permeabilized or not. The cytosolic Bax protein can upon cellular stress translocate to the mitochondrial membrane where it can either carry out its action of forming homo-oligomers that cause outer membrane permeabilization or be inhibited there by the anti-apoptotic membrane protein Bcl-2. Upon mitochondrial outer membrane permeabilization (MOMP) apoptogenic factors leak out from the intermembrane space (IMS) of the mitochondria, leading to caspase activation and ultimately cell death. A common stress signal initiating apoptosis is an increased formation of reactive oxygen species (ROS in the mitochondria, who can cause oxidative damage to lipid membranes. This membrane damage presumably influences the lipid landscape and the membrane features and hence the interactions of the Bcl-2 family proteins with each other and the mitochondrial outer membrane (MOM). To investigate the significance of membrane oxidation on the behavior of the Bcl-2 family proteins, especially Bax, synthetically produced oxidized phospholipids (OxPls) were incorporated in MOM-mimicking vesicles. Differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy revealed a major perturbation in membrane organization in the presence of OxPls. These changes in membrane properties increase the affinity of Bax to its target membrane and enable its partial penetration and formation of pores, as fluorescence leakage assays confirmed. However, in the absence of BH3-only proteins these pores are not sufficiently large for the release of apopototic factors such as cytochrome C (CytC). To understand the inhibition of Bax by the full-length Bcl-2 protein, suitable detergent solubilizing conditions were carefully chosen to enable the measurement of their direct binding to each other outside the membrane, by an antimycin A2 fluorescence assay. The observed protein-protein interaction was confirmed by surface plasmon resonance (SPR). An established protocol for the reconstitution of Bcl-2 into stable proteoliposomes now paves the way for structural studies of this key protein, in its membrane environment near physiological conditions; information essential for understanding its function, on a molecular level, and its potential as a cancer drug target.
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| 4. |
- Ur-Rehman, Tofeeq, 1971-
(författare)
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Controlled release gel formulations and preclinical screening of drug candidates
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Simple gel formulations may be applied to enhance the systemic and local exposure of potential compounds. The aim of this thesis is the development and characterization of controlled release formulations based on thermo-reversible poloxamer gels, which are suitable for novel drug delivery applications. In particular co-solvents (DMSO, ethanol), mucoadhesive polymers (chitosan, alginate) and salts (sodium tripolyphosphate, CaCl2) have been used to enhance the applications of poloxamer 407 (P407) formulations in preclinical animal studies. The impact of these additives on the micellization and gelation properties of P407 aqueous solutions was studied by calorimetric methods, nuclear magnetic resonance spectroscopy (NMR) and “tube inversion” experiments. The drug release behavior of hydrophobic and hydrophilic drugs was characterized by using a membrane/membrane-free experimental setup. Finally, preliminary pharmacokinetic studies using a mouse model were conducted for screening of selected inhibitors of bacterial type III secretion and for evaluation of different formulations including P407 gel. All additives, used here, reduced the CMTs (critical micelle temperature) of dilute P407 solutions, with the exception of ethanol. The gelation temperature of concentrated P407 solutions was lowered in the presence of CaCl2, DMSO, TPP and alginate. 1H MAS (Magic Angle Spinning) NMR studies revealed that DMSO influences the hydrophobicity of the PPO segment of P407 polymers. Low concentrations of DMSO did not show any major effect on the drug release from P407 gels and may be used to improve the exposure of lead compounds in poloxamer gels. A newly developed in situ ionotropic gelation of chitosan in combination with TPP in P407 gels showed an enhanced resistance to water and reduced the release rates of model drugs. From preliminary pharmacokinetic studies in mice it was revealed that poloxamer formulations resulted in an increased plasma half-life of the lead compound.
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| 5. |
- Dingeldein, Artur
(författare)
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Bax and oxidized phospholipids - a deadly complex : Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are renowned for their vital role as cellular powerhouses because they provide ATP via cellular respiration. Additionally, these organelles also play an important role in other physiological processes, such as apoptosis. Apoptosis, or regulated cell death, is an important mechanism that regulates, for example, tissue homeostasis and embryonic development. Malfunctioning apoptosis can cause severe diseases such as various types of cancer and neurodegenerative diseases. The significance of mitochondria for apoptosis is that mitochondria host a variety of apoptogenic factors, such as cytochrome c. The release of these factors after the formation of an apoptotic pore can be regarded as a point of no return in the onset of apoptosis as these factors trigger the activation of caspases and consequently nuclear fragmentation.The mitochondrial outer membrane (MOM) is essential for deciding the cell’s fate, since the MOM provides an interaction surface for the pro- and anti-apoptotic proteins of the Bcl-2 protein family. Further, oxidized phospholipids (OxPls) within the MOM that are generated under oxidative stress conditions (a potent pro-apoptotic stimulus) can directly affect the equilibrium between pro- and anti-apoptotic proteins at the MOM surface, hence influencing the formation of apoptotic pores.To characterize the impact of different OxPls on membrane dynamics and organization, several MOM-mimicking systems were studied by solid-state magic angle spinning nuclear magnetic resonance spectroscopy (MAS NMR). These main experiments were accompanied by fluorescence spectroscopy and differential scanning calorimetry (DSC) studies to investigate the impact of OxPls and their interactions with the pro-apoptotic Bax protein at both the macroscopic and molecular levels. By combining several orthogonal methods, we were able to obtain a detailed description of the changes in MOM-mimicking vesicles induced by several types of OxPls. Moreover, we demonstrated how these changes impact the interaction between liposomes and the pro-apoptotic Bax protein.By using DSC, we were able to determine not only the macroscopic effect of two OxPls – PazePC and PoxnoPC – but also a concentration threshold. Both OxPls disrupted the membrane order such that the melting behavior of the MOM-mimicking vesicles became less cooperative. A decrease in cooperativity was detectable for OxPl concentrations of up to 5 mol%, after which the cooperativity remained constant. The addition of Bax resulted in an observable ordering effect, as some of the membrane disorder was negated by Bax and the melting process became more cooperative again. The ordering effect of Bax was subsequently confirmed by 31P MAS NMR experiments and cross polarization (CP) buildup curves. Analysis of the buildup curves revealed that at the molecular level, Bax enabled more efficient CP transfer, which indicated rigidification of the membranes after Bax insertion. Furthermore, the 31P NMR experiments provided the first molecular evidence of the importance of cardiolipin as a membrane contact site for Bax.Despite having similar disordering effects when studied with DSC, PoxnoPC and PazePC exhibited opposing effects on the pore formation ability of Bax. In studies with fluorescent dye-based leakage assays, Bax was able to form long-lived, stable pores in PazePC-containing giant unilamellar vesicles (GUVs). However, the observed leakage mechanism in PoxnoPC-containing GUVs could no longer be explained by an all-or-none leakage mechanism due to the brevity of the formed pores, leading to partially leaked vesicles, indicating a graded leakage mechanism instead.To investigate the possible reasons for the different leakage activities and to obtain mechanistic insights, we conducted 13C MAS NMR experiments. These experiments enabled us to pinpoint specific carbon sites in the different MOM-mimicking systems and to study their dynamic profile as a function of temperature. Though the OxPl-containing multilamellar vesicles (MLVs), compared with non-oxidized systems, also showed drastic dynamic changes, the molecular differences between PazePC- and PoxnoPC-containing vesicles were not significant enough to constitute a structural reason for the opposing leakage activities.In addition to investigating membrane dynamics, we were able to establish a novel strategy for producing cytotoxic Bax protein. This novel expression and purification strategy increased the obtained yields by an order of magnitude. By deploying a double fusion approach, we were able to inhibit both termini of the protein from exhibiting their disruptive, yield-diminishing interactions with the host cell membranes.In conclusion, over the course of this thesis we were able to develop fast, yet powerful tools to investigate the dynamic changes of MOM-mimicking vesicles under the influence of OxPls and pro-apoptotic Bax. In the future, these tools could be used to characterize the underlying protein-lipid interactions that are responsible for the opposing leakage activities. Due to the development of a novel Bax production strategy, future research could shift to protein-focused studies with the primary goal of determining the structure of the apoptotic Bax pore.
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| 6. |
- Wallgren, Marcus, 1981-
(författare)
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Insight into the mitochondrial apoptotic pathway : The interplay of the pro-apoptotic Bax protein with oxidized phospholipids and its counterplayer, the pro-survival Bcl-2 protein
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis plays a crucial role in multicellular organisms by preserving tissue homeostasis and removing harmful cells. The anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and the pro-apoptotic Bcl-2-associated X protein (Bax) act as major regulators of the mitochondrial apoptotic pathway. Activation of Bax via stress signals causes its translocation to the mitochondrial outer membrane (MOM). There, Bax forms homo-oligomeric pores, leading to the release of apoptogenic factors, caspase activation and ultimately cell death. However, the underlying mechanism for the recruitment and pore forming activity of Bax is still not elucidated. Nevertheless, the mitochondrial membrane system seems to play an active and crucial role, presumably being directly involved in the onset of the mitochondrial apoptosis. Since the formation of reactive oxygen species (ROS) is a common stress signal and one of the hallmarks of the mitochondrial apoptosis, direct damage can occur to these membranes by the generation of oxidized phospholipids (OxPls), whose presence can crucially influence the pro-apoptotic action of Bax there. To better understand the impact of OxPls on membranes as well as their potential role in the mitochondrial apoptotic process, defined OxPl species were incorporated into phospholipid vesicles and studied with various biophysical techniques. Differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy were used to gain insight into changes in membrane properties in the presence of OxPls. In addition to circular dichroism (CD) spectroscopy, DSC and solid state NMR were furthermore performed to elucidate the impact of OxPls on Bax-membrane interactions. The occurrence of OxPls gave rise to dramatic changes in membrane organization and dynamics, manifested as lateral phase separation into OxPl-rich and -poor domains and modified hydration at the membrane interface. The presence of OxPls also had a great impact on the interaction between Bax and mitochondria-mimicking vesicles, strongly promoting the association of the protein with the membrane.At the MOM, Bax is believed to be inhibited by Bcl-2. How this inhibition occurs is still a mystery due to the lack of biophysical information on Bcl-2, in particular on the full-length protein variant. Since Bcl-2 is also one of the main culprits in the progression of various forms of cancer, knowledge of the structural and mechanistic properties of the full-length protein is essential for a fundamental understanding of its function at a molecular level. To this end, a method for the production of full-length Bcl-2 was developed. By performing cell-free protein synthesis, preparative amounts of the protein were obtained, which enabled a biophysical characterization of the putative interaction between Bax and Bcl-2 using CD and fluorescence spectroscopy. A protocol for the reconstitution of Bcl-2 into proteoliposomes was also developed, promising for future studies of the full-length protein in its native membrane environment; a prerequisite to fully understand its pro-survival functions as well as providing crucial information for the design of novel anti-cancer drugs.
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| 7. |
- Bokvist, Marcus, 1975-
(författare)
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Membrane mediated aggregation of amyloid-β protein : a potential key event in Alzheimer's disease
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pathogenesis of Alzheimer’s disease (AD), the most common senile dementia, is a complex process. A crucial event in AD is the aggregation of amyloid-β protein (Aβ), a cleavage product from the Amyloid Precursor Protein (APP). Aβ40, a common component in amyloid plaques found in patients, aggregates in vitro at concentrations, much higher than the one found in vivo. But in the presence of charged lipid membranes, aggregations occurs at much lower concentration in vitro compared to the membrane-free case. This can be understood due to the ability of Aβ to get electrostatically attracted to target membranes with a pronounced surface potential. This electrostatically driven process accumulates peptide at the membrane surface at concentrations high enough for aggregation while the bulk concentration still remains below threshold. Here, we elucidated the molecular nature of this Aβ-membrane process and its consequences for Aβ misfolding by Circular Dichroism Spectroscopy, Differential Scanning Calorimetry and Nuclear Magnetic Resonance Spectroscopy. First, we revealed by NMR that Aβ40 peptide does indeed interact electrostatically with membranes of negative and positive surface potential. Surprisingly, it even binds to nominal neutral membranes if these contain lipids of opposite charge. Combined NMR and CD studies also revealed that the peptide might be shielded from aggregation when incorporated into the membrane. Moreover, CD studies of Aβ40 added to charged membranes showed that both positively and negatively membranes induce aggregation albeit at different kinetics and finally that macromolecular crowding can both speed up and slow down aggregation of Aβ.
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