Gene therapy can potentially treat a wide range of diseases ranging from inherited diseases to cancer. The successful use of nucleic acids to treat genetic diseases is limited by rapid capture and degradation of the nanoparticle by Kupffer cells in the liver. Scavenger receptors on the cell surface, capture both viral and non-viral nanoparticles leading to reduced efficacy. PEG-peptides were found to inhibit scavenger receptors on the surface of Kupffer cells by forming albumin nanoparticles when intravenously dosed. This work explores the development of potent, low-molecular weight PEG-peptide inhibitors. In order to study the in vivo activity of the nanoparticle, an in vivo assay was developed to directly assess the potency of inhibition. High molecular weight polylysine peptides (33.5 kDa) inhibited liver uptake with an IC50 of 18 μM. Incorporation of four leucine residues, to improve albumin binding, allowed for a decrease in PEG molecular weight and number of lysine residues, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys (7.4 kDa) that inhibited scavenger receptors with an IC50 = 20 μM. Further decrease in the PEG molecular weight resulted in the discovery of PEG2kDa- Cys-Tyr- (Leu-Lys4)3-Leu-Lys (4.4 kDa) with potency of 3 μM. The increase in potency could be attributed to a decrease in the zeta potential of the albumin nanoparticle resulting in more efficient scavenger receptor mediated uptake. Co- administration of PEG2kDa- Cys-Tyr-(Leu-Lys4)3-Leu-Lys with a stable PEGylated polyacridine DNA polyplex resulted in inhibition of rapid polyplex uptake by the liver with an IC50 = 11 μM. Other properties including spatial distribution of leucine, hydrophobicity and peptide length were also explored to determine their effect on liver uptake. Hydrophobic peptides resulted in the formation of micelles which were inactive as scavenger receptor inhibitors and exhibited increased liver uptake upon dose escalation. Reduction in the peptide length resulted in peptides that were not captured by the liver. Inhibition scavenger receptors has the potential to improve the efficacy of viral and non-viral nanoparticles. The findings of this work provide a framework for the development of PEG-peptide inhibitors capable of blocking live uptake of viral and non-viral nanoparticles.

Hydrogen gas is the cleanest fuel which produces only water as a combustion product with no greenhouse or toxic gases. The combustible hydrogen fuel is an energy carrier but not an energy source. As an element, hydrogen is widely available in nature as a component of water and of hydrocarbons. An energy source is needed to extract the element from these compounds and convert it to the combustible hydrogen gas. Today, the energy source for nearly all hydrogen production is fossil fuel, principally natural gas. The supply of natural gas is limited and its price is increasing. Greenhouse gas and air pollutants are emitted when natural gas is used. Electrolytic extraction of hydrogen from water can overcome these stated problems but is more expensive with the present price of natural gas. Manufacturing the hydrogen with a valuable co-product would address this cost disadvantage. Sulfuric acid is a valuable chemical that is produced in large quantities. This research project helps to develop a procedure for extracting hydrogen from water while producing sulfuric acid as a co-product. An electrochemical cell was designed and developed for the production of hydrogen which uses sulfuric acid as electrolyte. In this electrochemical cell with sulfuric acid as an electrolyte we produce hydrogen at the negative electrode while the positive electrode is bathed in sulfur dioxide which it oxidizes to sulfuric acid. The sulfuric acid is collected at the bottom of the cell as valuable co-product. The presence of SO2 to scavenge the anode substantially reduces the equilibrium voltage required for the direct dissociation of water into hydrogen and oxygen. Various design parameters and the fabrication of the reactor are discussed briefly in the thesis. Experimental results of hydrogen production and current voltage curves are discussed. Sulfuric acid corrosion of cell materials is also discussed.

<p>Abstract</p><p>It is estimated that up to 2 percent of the population in Third World countries survives on waste in one way or another. In Mexico City alone there exist 15,000 garbage scavengers called Pepenadores. The poverty and marginalization they experience is utterly linked to their work, and while they do much of the hard work their socio-economic situation seems stagnant. This paper explores the complexity of the waste management system in Mexico City which keeps them in this position, and how the current system is a manifestation of the existing symbiosis between the formal and informal sectors of the city.The main characters in the maintenance of this system are the leaders of waste management associations.Their struggle to maintain their powerful positions influences both the system’s relationship to the public sector and determines the socioeconomic situation of the Pepenadores.The paper also analyzes the effects of past efforts to change the system, and how policy changes always seem to work against the Pepenadores. Efforts to help the Pepenadores escape their vulnerable positions can be successful in the short-term, but the existing social structure in Mexico City make any permanent changes difficult to achieve.</p>

Abstract It is estimated that up to 2 percent of the population in Third World countries survives on waste in one way or another. In Mexico City alone there exist 15,000 garbage scavengers called Pepenadores. The poverty and marginalization they experience is utterly linked to their work, and while they do much of the hard work their socio-economic situation seems stagnant. This paper explores the complexity of the waste management system in Mexico City which keeps them in this position, and how the current system is a manifestation of the existing symbiosis between the formal and informal sectors of the city.The main characters in the maintenance of this system are the leaders of waste management associations.Their struggle to maintain their powerful positions influences both the system’s relationship to the public sector and determines the socioeconomic situation of the Pepenadores.The paper also analyzes the effects of past efforts to change the system, and how policy changes always seem to work against the Pepenadores. Efforts to help the Pepenadores escape their vulnerable positions can be successful in the short-term, but the existing social structure in Mexico City make any permanent changes difficult to achieve.

Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted. This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Scavenger receptors are a large family of structurally diverse molecules that have been implicated in a range of biological functions. In this work, a newly identified multi-domain scavenger receptor-serine protease IrSRP-1 from the tick Ixodes ricinus is characterized. IrSRP-1 is related to the serine protease Sp22D from the mosquito Anopheles gambiae. IrSRP-1 is expressed mainly in the tick gut but also in hemocytes, Malpighian tubules, tracheas and ovaries of fully fed females. This was confirmed with Western blots and immunohistological labeling with antibodies raised against recombinantly expressed IrSRP-1 trypsine-like domain. According to acquired qRT-PCR profiles relative expression of IrSRP-1 is strongly up-regulated during female feeding and remains unchanged in ticks experimentally injected with various microbes. Functional characterization by RNA interference revealed that lowering IrSRP1 expression leads to a higher mortality rate during tick female feeding.

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spα, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by β-glucan but not by mannan. The K d of the rshCD5/(1→3)-β-D-glucan phosphate interaction is 3.7 ± 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1→3)-β-D- glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

Over the past decades, wolverines in Sweden have made a recovery from near extinction to recolonization large part of their historic range. Effective conservation of large carnivores, which inhabit extensive territories, necessitates adaptative management that considers the diverse ecological and societal factors spanning their entire range. This report contributes to our understanding of wolverines in the southern periphery of their recolonized area, focusing on their scavenging behaviour. I utilize data from 14 wolverines, tracked with GPS-collars in Värmland, Dalarna and Jämtland over five years (2018-2022). The monitoring covered 19 three-week periods, during spring, early summer, and autumn. My thesis focusses on the wolverine’s utilization of two type of scavenging sites: anthropogenic food resources and carcasses from wild ungulates. The results show that females exhibit higher visit frequencies to both types of scavenging sites during spring and summer, but this difference diminishes in autumn when males visit scavenging sites more frequently than in other seasons. Anthropogenic feeding sites had more visits during autumn, compared to wild carcasses, whereas wild carcasses are more commonly utilized in spring and summer. The presence of large predators influences wolverine scavenging behavior, as evidenced by shorter visits to feeding sites in Jämtland (with high bear density) compared to Värmland and Dalarna. Nevertheless, these low-conflict areas have all benefited wolverines by increasing their reproductive rate, offering hope for the ongoing recolonization. Moreover, my results show that human activities in this region can have a positive impact on wolverines, by acting as and apex predator providing a stable food source, which should further facilitate recolonization success.