his thesis focuses on optimization of Linux-based operating system for a accumulator-powered device Raspberry Pi 4. Compared to other devices commonly used in Internet of Things projects, the Raspberry Pi 4 offers many functions within one device. However, the disadvantage is the high consumption of electricity. The aim of this thesis is to achieve greatest possible savings in electricity consumption of the Raspberry Pi 4 device, with regard to functionality of the device as a server for data collection from sensors.

The number of discovered vulnerabilities rapidly increases. For example in 2019 there were discovered 20 362 vulnerabilities. The probability of cyber-attacks realization is high. Therefore it is necessary to propose and implement automated and low-cost Intrusion Prevention or Intrusion Detection Systems (IPS/IDS). This implemetation can focus on home use or small corporate networks. The main goal of the system is to detect or mitigate cyber-attack impact as fast as possible. The master's thesis proposes IPS/IDS based on Raspberry Pi that can detect and prevent various cyber-attacks. Contents of this thesis are focus on description of cyber-attacks based on ISO/OSI model's Link and Network layers. Then there is description of IPS/IDS systems and theirs open source representatives. The practical part is focus on experimental workspace, hardware consumption of choosen detection systems, cyber-attacks scenarios and own implementation of detection program. Detection program is based on these chosen systems and puts them together to be easily manageable.

En kostnadseffektiv, liten robotbil som är fjärrstyrd via Wi-Fi harutformats och testats för att möjliggöra inspektion och identifiering avpotentiella faror vid olycksplatser. Robotbilenär avsedd att användasför rekognosering och inspektion och kan bidra till att utforma enadekvat insatsplan. Hjulupphängning och hjul konstruerades medhjälp av en 3D-skrivare. Den färdiga robotbilen är utrustad medmikrokontrollern Raspberry Pi som har flera funktioner som gör denanvändbar i olika scenarier. Det finns en kamera som möjliggörfjärrinspektion av bilens omgivning. Utöver kameran finns det tresensorer som är kopplade till Raspberry Pi-enheten, nämligen engassensor för att upptäcka farliga gaser, en ultraljudssensor för attmäta avståndet till närmaste objekt och en temperatursensor för attmäta omgivningstemperaturen. Robotbilen använder enmotorstyrningsmodul för att styra bilens rörelse och två servomotorerför att möjliggöra att rotera kameran i vertikal och horisontell led.Robotbilens strömförsörjning kommer från ett batteri och tvåspänningsomvandlare används för att reglera spänningen tillmotorstyrningsmodulen och Raspberry Pi-enheten. Genom attintegrera dessa komponenter i en enda enhet och programmeraRaspberry Pi-enheten för att styra dem, kan robotbilen effektivt hjälpaatt undersöka och hantera potentiella faror. / A cost-effective small robot car that is remote-controlled via Wi-Fi hasbeen designed and tested for inspecting and identifying potentialhazards at accident sites. The robot car is intended for reconnaissanceand inspection purposes and can contribute to formulating anadequate action plan. Wheel suspension and wheels were constructedusing a 3D printer. The finished robot car is equipped with theRaspberry Pi microcontroller, which has severalfeatures that make ituseful in various scenarios. There is a camera that allows remoteinspection of the car's surroundings. In addition to the camera, thereare three sensors connected to the Raspberry Pi unit: a gas sensor todetect dangerous gases, an ultrasonic sensor to measure the distanceto the nearest object, and a temperature sensor to measure theambient temperature. The robot car uses a motor control module tocontrol its movement and two servo motors to enable the rotation ofthe camera in the vertical and horizontal directions. The robot car ispowered by a battery, and two voltage converters are used to regulatethe voltage to the motor control module and the Raspberry Pi unit. Byintegrating these components into a single unit and programming theRaspberry Pi unit to control them, the robot car can effectively assistin investigating and managing potential hazards.

A mesh network is a technology that is being repopularized and becoming commonly used by the general public. As this increase in use is observed, technologies such as Bluetooth are being adapted to create mesh variants. In this thesis, a Bluetooth mesh network is created and tested using raspberry pi 4’s and the Bluetooth interface, btferret. This thesis attempts to approach the limits of this technology using accessible tools, outlining the performance the network possesses to serve as a guideline to determine if it suitable for use for tasks at hand. Experimentation is split into two overarching methods where a test for latency and throughput is conducted. The thesis goes on to expose these tests to different stressors, categorized as either internal or external. The data collected aims to show the impacts of internal properties, in this case size of the packets transmitted, the size of the network, and finally the number of hops a packet is able to make within the network. The external factors tested for consists of various environmental properties in the form of obstacles and interference. Walls and a microwaves were used as obstacles while WiFi and other Bluetooth signals were used for interference. The results show that Bluetooth Low Energy (BLE) mesh networks are clearly affected by several internal and external factors. From the experimentation conducted, the thesis illustrates the relative effects of each property the tests are exposed to. / Ett mesh nätverk är en teknik som blivit populär igen och används ofta av allmänheten. Eftersom denna ökade användning observeras, tekniker som Bluetooth anpassas för att skapa mesh nätverksvarianter. I denna avhandling skapas och testas ett Bluetoothnätverk med Raspberry pi 4’s och Bluetoothgränssnittet, btferret. Denna uppsats försöker nå gränserna för denna teknik med hjälp av tillgängliga verktyg, definiera nätverks prestandan som en riktlinje för att avgöra om det är lämpligt för användning för uppgifter till hands. Resultaten visar att BLE mesh nätverk har tydliga begränsningar som avslöjar sig i olika sammanhang. I denna raport så undersöks paket storlek och antal hopp som ett paket kan göra inom nätverket utan signifikant prestandafall. Dessutom har olika andra faktorer, såsom väggar och andra störande radiofrekvenser visat sig påverka nätverket. Från alla experiment som genomförts så illustreras relativa effekt av det olika faktorer.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

Automatic Passenger Counting (APC) systems are some of the many Internet-Of-Things (IoT) applications and have been increasingly adopted by public transportation companies in recent years. APCs provide valuable data that can be used to give an real time passenger count, which can be a convenient service and allow customers to plan their travels accordingly. The provided data is also valuable for resource streamlining and planning, which potentially increases revenues for the public transportation companies. This thesis briefly studies and evaluates different APC technologies, highlights the advantages and disadvantages of these, and presents an Edge-prototype based on Computer Vision and Object Detection. The presented APC was tested in a lab environment and with recordings of people walking in and out of a designated area in the lab. Test results from the lab environment show that the presented low-cost APC efficiently detects passengers with an accuracy of 98.6% on pre-recorded videos. The APC was also tested in real time and the results show that the low-cost APC only achieved an accuracy of 66.7%. This work has laid the ground for further development and testing in a public transport environment.

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