Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: Impact for allograft rejection

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell-matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

High incidence of contaminating maternal cell overgrowth in human placental mesenchymal stem/stromal cell cultures: A systematic review

Placenta is a readily accessible translationally advantageous source of mesenchymal stem/stromal cells (MSCs) currently used in cryobanking and clinical trials. MSCs cultured from human chorion have been widely assumed to be fetal in origin, despite evidence that placental MSCs may be contaminated with maternal cells, resulting in entirely maternally derived MSC cultures. To document the frequency and determinants of maternal cell contamination in chorionic MSCs, we undertook a PRISMA-compliant systematic review of publications in the PubMed, Medline, and Embase databases (January 2000 to July 2013) on placental and/or chorionic MSCs from uncomplicated pregnancies. Of 147 studies, only 26 (18%) investigated fetal and/or maternal cell origin. After excluding studies that did not satisfy minimal MSC criteria, 7 of 15 informative studies documented MSC cultures as entirely fetal, a further 7 studies reported cultured human chorionic MSC populations to be either maternal (n=6) or mixed (n=1), whereas 1 study separately cultured pure fetal and pure maternal MSC from the same placenta. Maternal cell contamination was associated with term and chorionic membrane samples and greater passage number but was still present in 30% of studies of chorionic villous MSCs. Although most studies assume fetal origin for MSCs sourced from chorion, this systematic review documents a high incidence of maternal-origin MSC populations in placental MSC cultures. Given that fetal MSCs have more primitive properties than adult MSCs, our findings have implications for clinical trials in which knowledge of donor and tissue source is pivotal. We recommend sensitive methods to quantitate the source and purity of placental MSCs.

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

The cross-talk of LDL-cholesterol with cell migration: Insights from the Niemann Pick Type C1 mutation

Cholesterol is considered indispensible for the recruitment and functioning of integrins in focal adhesions for cell migration. However, the physiological cholesterol pools that control integrin trafficking and focal adhesion assembly remain unclear. Using Niemann Pick Type C1 (NPC) mutant cells, which accumulate Low Density lipoprotein (LDL)-derived cholesterol in late endosomes (LE), several recent studies indicate that LDL-cholesterol has multiple roles in regulating focal adhesion dynamics. Firstly, targeting of endocytosed LDL-cholesterol from LE to focal adhesions controls their formation at the leading edge of migrating cells. Other newly emerging literature suggests that this may be coupled to vesicular transport of integrins, Src kinase and metalloproteases from the LE compartment to focal adhesions. Secondly, our recent work identified LDL-cholesterol as a key factor that determines the distribution and ability of several Soluble NSF Attachment Protein (SNAP) Receptor (SNARE) proteins, key players in vesicle transport, to control integrin trafficking to the cell surface and extracellular matrix (ECM) secretion. Collectively, dietary, genetic and pathological changes in cholesterol metabolism may link with efficiency and speed of integrin and ECM cell surface delivery in metastatic cancer cells. This commentary will summarize how direct and indirect pathways enable LDL-cholesterol to modulate cell motility.

Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Alamethicin and related membrane channel forming polypeptides

Alamethicin and several related microbial polypeptides, which contain a high proportion of agr-aminoisobutyric acid (Aib) residues, possess the ability to modify the permeability properties of phospholipid bilayer membranes. Alamethicin induces excitability phenomena in model membranes and has served as an excellent model for the study of voltage sensitive transmembrane channels. This review summarizes various aspects of the structural chemistry and membrane modifying properties of alamethicin and related Alb containing peptides. The presence of Aib residues in these sequences, constrains the polypeptides to 310 or agr-helical conformations. Functional membrane channels are formed by aggregation of cylindrical peptide helices, which span the lipid bilayer, forming a scaffolding for an aqueous column across the membrane. After consideration of the available data on the conductance characteristics of alamethicin channels, a working, hypothesis for a channel model is outlined. Channel aggregates in the lipid phase may be stabilized by intermolecular hydrogen bonding, involving a central glutamine residue and also by interactions between the macro-dipoles of proximate peptide helices. Fluctuations between different conductance states are rationalized by transitions between states of different aggregation and hence altered dimensions of the aqueous core or by changes in net dipole moment of the aggregate. Ion fluxes through the channel may also be affected by the electric field within the aqueous core.

Multi-Disciplinary Design and Simulation of a Fuel Cell Engine for a UAV

This report describes a methodology for the design and coupling of a proton exchange membrane (PEM) Fuel Cell to an Unmanned Aerial Vehicle (UAV). The report summarizes existing work in the field, the type of UAV and the mission requirements, design the fuel cell system, simulation environment, and compares endurance and range to when the aircraft is fitted with a conventional internal combustion engine (ICE).

The effects of slip velocity at a membrane surface on blood flow in the microcirculation

Closed-form solutions are presented for blood flow in the microcirculation by taking into account the influence of slip velocity at the membrane surface. In this study, the convective inertia force is neglected in comparison with that of blood viscosity on the basis of the smallness of the Reynolds number of the flow in microcirculation. The permeability property of the blood vessel is based on the well known Starling's hypothesis [11]. The effects of slip coefficient on the velocity and pressure fields are clearly depicted.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Effect of lutein on the transport of Ca2+ across phospholipid bilayer and mitochondrial membrane

Lutein (3,3'-dihydroxy alpha-carotene), a xanthophyll present in plant chloroplasts, increases the permeability of phospholipid vesicles to Ca2+, even though the pigment does not bind the metal ion. Energy-dependent uptake of Ca2+ by mitochondria is inhibited by lutein, which permits a rapid efflux of the ion from Ca2+-loaded mitochondria. These results are consistent with the view that the deleterious action of lutein on mitochondrial oxidative phosphorylation results from its destabilizing action on membrane structure.

Bending the Rules of Cell Protrusions : Molecular Mechanisms and Biological Roles of Inverse-BAR Proteins in Cell Morphogenesis

Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.

DNA Packaging and Host Cell Lysis: Late Events in Bacteriophage PRD1 Infection

The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.