Familial hypercholesterolemias (FH) are inherited mutations that cause elevated total cholesterol and low-density lipoprotein cholesterol levels (LDL-C) which lead to premature coronary heart diseases. Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect the patient’s response to the drug. Single Nucleotide Morphism (SNP) mutations in the LDLR, apoB, LDRAP1, and PCSK9 genes are linked to familial hypercholesterolemia. The mutations in the LDLR gene are the most common while mutations in the apoB and PCSK9 genes are the least common in hypercholesterolemia patients. My research will study how pharmacogenetics can be used to diagnose and prescribe patients with FH who have apoB and PCSK9 double gene mutations. I will genotype and sequence the PCR amplified gene segments of the patients with FH to identify any of the 6 apoB SNPs and any of the 3 PCSK9 SNPs that are known. Then, I will provide 4 different treatments: placebo, antisense therapy (mipomersen), PCSK9 inhibitor (alirocumab), and a combination of mipomersen + alirocumab, and I will measure the LDL-C levels before and after a 12-week trial. I hypothesize that individuals with both apoB and PCSK9 gene mutations with the known SNPs that cause loss of function will be more responsive when given both treatments by observing a significant decrease in LDL-C levels.

Respiratory Syncytial Virus (RSV) is a potentially life-threatening respiratory pathogen that infects approximately 64 million children and immunocompromised adults globally per year. Currently, there is a need for prophylactic and therapeutic approaches effective against primary and secondary RSV infections. This project focuses on the development of a simple, smart, and scalable anti-RSV nanotherapeutic that combines novel cellular antiviral defense mechanisms targeting the inhibition of viral fusion and replication. An ICAM-1 targeted liposomal nanocarrier will be synthesized and coated with a layer of chitosan containing the anti-fusion HR2-D peptide as an extracellular defense mechanism. Additionally, chitosan complexed to dual expressing short hairpin RNA (shRNA) recombinant plasmids will be encapsulated within the nanocarrier, and provide an intracellular defense mechanism that will interfere with the expression of the NS1 and P proteins. In combination, both defense mechanisms are expected to induce a synergistic anti-RSV effect that will surpass those of conventional therapeutics. Through this research, the NS1 and P containing plasmid (pSH-NS1-P) was cloned, and the nanotherapeutic was successfully synthesized. Based on the acquired results, pSH-NS1-P was shown to express anti-RSV effects, and it was also concluded that both inserts were producing active shRNA. Additionally, the anti-RSV efficiency of HR2-D was confirmed. Overall, this research will lead to development of a dual-mechanistic anti-viral nanotherapeutic.

Purchase management of medical supplies is a critical and important process that affects the services provision quality. Nonetheless, it is facing a growing pressure to provide visibility and traceability of the purchase, to reduce fraud, to improve flexibility and to ensure communication between everyone involved. Currently, private health institutions in Peru choose to implant different software products within the same company with restricted visibility access to other concerned parties and based on information from a single source. A new alternative is Blockchain technology, since it provides a single source of shared truth to all participants and ensures that the information cannot be altered, thus offering high levels of transparency that, together with IoT technology, creates not only visibility about where things are, but also traceability, showing the current state of things. / Revisón por pares

<p>Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of <i>Salmonella enterica</i> serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic <i>Escherichia coli</i>.</p><p>The immune response elicited using secreted proteins from <i>S. enterica</i> serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant <i>Salmonella</i> atypical fimbriae (SafB/D) complexes, a conserved structure within <i>Salmonella</i> species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. </p><p>Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. </p><p>Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.</p>

<p>The subject of this thesis was to study two intestinal barriers to oral drug bioavailability, drug efflux proteins of the ABC-transporter family, and in particular ABCB1/P-glycoprotein (Pgp), and the drug metabolizing enzyme cytochrome P450 (CYP) 3A4. At the onset of this thesis, similarities between CYP3A4 and Pgp in terms of their tissue distribution and gene regulation, along with overlapping substrate specificities, had generated the hypothesis that CYP3A4 and Pgp may have a complementary function and thus form a coordinated intestinal barrier to drug absorption and gut wall metabolism.</p><p>In the first part of this thesis, a cell culture model of the intestinal epithelium that expressed both functional Pgp and CYP3A4 was developed. This model was then used to investigate the steroselective drug efflux and metabolism of R/S-verapamil. In summary, the results indicated that the two barriers in the cell culture model were in agreement with those in the human intestine.</p><p>Both ABC-transporters and CYPs are regulated by drugs that interact with nuclear receptors. However, while the regulation of CYPs is quite well understood, less is known about how repeated drug administration regulates the most abundantly expressed ABC-transporters. Therefore, in the second part of this thesis, the effects of repeated drug administration on the gene regulation of four ABC-transporters and CYP3A4 were studied in intestinal epithelial cell lines in vitro and in the perfused human jejunum in vivo. The in vitro studies revealed that the ABC-transporters are induced by drugs that interact with slightly different sets of nuclear receptors. The in vivo study showed that repeated oral administration of St John’s wort decreased the bioavailability of verapamil, predominantly by induction of intestinal CYP3A4. This part of the thesis provides new information about the regulation of ABC-transporters, shows that the intestinal metabolism is the most significant barrier to oral bioavailability of verapamil and provides evidence for a clinically significant interaction between verapamil and St John’s wort in vivo.</p>

<p>The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems <i>in vitro</i> and after lung administration <i>in vivo</i> were established for the first time.</p><p>Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells <i>in vitro</i> and after lung administration <i>in vivo</i>. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for <i>in vivo</i> gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI <i>in vitro</i> and <i>in vivo</i>. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for <i>in vivo</i> gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.</p>

<p>Most pathogens enter the body via mucosal surfaces. In contrast to parenterally administered vaccination, mucosal vaccination has the advantage of eliciting both a systemic and a local mucosal immune response. An oral biodegradable adjuvant with these features would have great potential. </p><p>This thesis has focused on the development of a new oral vaccine against diphtheria. Biodegradable polyacryl starch microparticles were used as a mucosal adjuvant. Diphtheria toxin or cross-reacting material of diphtheria toxin (CRM197) was covalently conjugated to the microparticles and fed to mice by oral gavage. Formaldehyde treatment was also studied as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations. All formulations given to mice orally or parenterally, but not intranasally, induced a strong systemic immune response and diphtheria toxin neutralising antibodies. Only formulations administered orally induced a mucosal IgA response as well. </p><p>The non-toxic recombinant protein CRM197 proved to be a promising antigen candidate in an oral diphtheria vaccine when conjugated to the microparticles. Mild treatment of CRM197 with formaldehyde before conjugation to the starch microparticles potentiated the immunogenicity of the formulation. However, no immune response was detected in healthy volunteers after administration of this vaccine in a phase I trial. The possible reasons for the difference in response between mouse and man are discussed.</p><p>The use of cDNA expression macro array technology was also evaluated as a tool in vaccine-related research. Tetanus toxoid and aluminium phosphate were used as model parenteral antigen and adjuvant. It was concluded that the antigen modulates the molecular mechanisms of the aluminium phosphate adjuvant to a greater extent than previously recognised.</p>

<p>Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response.</p><p>When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. </p><p>Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. </p><p>Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.</p>

<p>Cellulose powders from various sources were manufactured and characterized to investigate the influence of their crystallinity index, surface area, and pore volume on sorption phenomena and the relevant pharmaceutical functionality. The influence of the cellulose crystallinity index on moisture sorption was important at low and intermediate relative humidities. At high relative humidities, properties such as surface area and pore volume took precedence in governing the moisture sorption process.</p><p>The theory of physical adsorption of gases onto fractal surfaces was useful for understanding the distribution of water in cellulose and the inner nanoscale structure of cellulose particles. It was found that, as a consequence of swelling, moisture induces a fractal nanopore network in cellulose powders that have a low or intermediate degree of crystallinity. On the other hand, no swelling occurs in highly crystalline cellulose powders and moisture sorption is restricted to the walls of the open pores.</p><p>No correlation was found between the cellulose crystallinity index and the incorporation and release of nicotine in cellulose mixtures. By loading nicotine in highly porous matrices of the Cladophora sp. algae cellulose, higher stability against oxidative degradation, higher loading capacity, and more steady release into an air-stream was achieved than when commercially available microcrystalline cellulose was loaded.</p><p>It was also shown that, by manipulating the structure of cellulose, the undesired hydrolysis of acetylsalicylic acid in mixtures with cellulose can be avoided. It was suggested that a broad hysteresis loop between the moisture adsorption and desorption curves of isotherms at low relative humidities could be indicative of an improved compatibility between acetylsalicylic acid and cellulose.</p><p>In all, this thesis demonstrates how the pharmaceutical functionality of microcrystalline cellulose can be improved via engineering of the structure of native cellulose powders.</p>

Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of Salmonella enterica serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic Escherichia coli. The immune response elicited using secreted proteins from S. enterica serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant Salmonella atypical fimbriae (SafB/D) complexes, a conserved structure within Salmonella species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.