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In this episode, we switch from base ten to binary as we introduce twos complement representation and show how computers store and manipulate signed integers.

In this episode, we continue our discussion of twos complement binary representation by covering overflow and how shifting left and right can be used to perform multiplication and division by powers of two.

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The central nervous system (CNS) is protected by a vascular blood-brain barrier that prevents many types of pathogens from entering the brain. Still, some pathogens have evolved mechanisms to traverse this barrier and establish a long-term infection. The apicomplexan parasite, Toxoplasma gondii, is one such pathogen with the ability to infect the CNS in virtually all warm-blooded animals, including humans. Across the globe, an estimated 30% of the human population is infected with Toxoplasma, an infection for which mounting evidence suggests increases the risk for developing neurological and neuropsychiatric disorders, like seizures and schizophrenia. In my dissertation, I investigate the telencephalic neural circuit changes induced by long-term Toxoplasma infection in the mouse brain and the neuroimmune signaling role of the complement system in mediating microglial remodeling of neural circuits following parasitic infection. While there has been keen interest in investigating neural circuit changes in the amygdala – a region of the brain involved in fear response and which Toxoplasma infection alters in many species of infected hosts – the hippocampus and cortex have been less explored. These are brain regions for which Toxoplasma also has tropism, and moreover, are rich with fast-spiking parvalbumin perisomatic synapses, a type of GABAergic synapse whose dysfunction has been implicated in epilepsy and schizophrenia. By employing a range of visualization techniques to assess cell-to-cell connectivity and neuron-glia interactions (including immunohistochemistry, ultrastructural microscopy, and microglia-specific reporter mouse lines), I discovered that longterm Toxoplasma infection causes microglia to target and ensheath neuronal somata in these regions and subsequently phagocytose their perisomatic inhibitory synapses. These findings provide a novel model by which Toxoplasma infection within the brain can lead to seizure susceptibility and a wider range of behavioral and cognitive changes unrelated to fear response. In the Toxoplasma infected brain, microglia, along with monocytes recruited to the brain from the periphery, coordinate a neuroinflammatory response against pathogenic invasion. This is characterized by a widespread activation of these cells and their increased interaction with neurons and their synaptic inputs. Yet, whether T. gondii infection triggers microglia and monocytes (i.e. phagocytes) to target, ensheath, and remove perisomatic inhibitory synapses on neuronal somata indiscriminately, or whether specificity exists in this type of circuit remodeling, remained unclear. Through a comprehensive assessment of phagocyte interactions with cortical neuron subtypes, I demonstrate that phagocytes selectively target and ensheath excitatory pyramidal cells in long-term infection. Moreover, coupling of in situ hybridization with transgenic reporter lines and immunolabeling revealed that in addition to phagocytes, excitatory neurons also express complement component C3 following infection (while inhibitory interneurons do not). Lastly, by employing targeted deletion of complement components, C1q and C3, I show that complement is required for phagocyte ensheathment of excitatory cells and the subsequent removal of perisomatic inhibitory synapses on these cells (albeit not through the classical pathway). Together, these studies highlight a novel role for complement in mediating synapse-type and cell-type specific circuit remodeling in the Toxoplasma infected brain. / Doctor of Philosophy / Parasites are microorganisms that rely on other living organisms (called hosts) for their survival. Although some parasites only live on their hosts, others have developed ways to establish infections and obtain the nutrients that keep them alive from host cells. My Ph.D. research has focused on studying one of these parasites, Toxoplasma gondii (commonly referred to as Toxo), that has evolved the unique ability to establish brain infections in almost all animals around the world, from rodents to humans. Recent discoveries show that brain infection with this parasite can cause seizures, an imbalance in the way that specialized cells of the brain (called neurons) communicate with each other, causing harmful hyperactivity within the brain. Toxo infection can also cause behavioral and cognitive changes in infected animals, making them more susceptible to predation. In humans, infection with Toxo increases their risk for developing different types of mental illness, such as schizophrenia. The focus of my Ph.D. research has been in trying to understand, at the cellular and molecular level, how infection with this parasite can lead to seizures and behavioral changes, by using mice as a model. Mice have a similar brain structure to humans, and over the years, scientists have developed many tools that allow us to visualize and study the connections between neurons (called synapses). I'm interested in understanding how changes in these connections affect how neurons communicate with each other, and ultimately, how we behave and think. I have been studying a type of connection that, if lost or damaged, can lead to seizures and some types of mental illness. These connections are called 'perisomatic inhibitory synapses', and they form on many distinct types of neurons, but specifically on the cell bodies of these neurons. They act as a traffic light, informing neurons when and for how long to 'slow down' their activity. I discovered that after the parasite enters the brain, it causes another type of cell in the brain, called microglia, to extensively interact with neurons in the cortex and hippocampus (areas of your brain important for thinking, executing behavior, and learning). Microglia are immune cells of the brain that inspect the brain for anything damaged or that doesn't belong (like parasites) and removes them from the brain. By performing experiments where I delete individual immune molecules from mice, I found that one immune molecule, called 'complement component C3' acts as cue for microglia to find these cells, wrap around them, and permanently remove these important connections. Surprisingly, however, microglia don't remove these connections from all neurons, indiscriminately, they do so only on one specific cell type called 'excitatory pyramidal neurons,' and as the name implies, they're the ones who drive activity in the brain. My half-a-decade's worth of research helps us understand parasitic infections in the brain in a couple of ways: First, I have discovered one of the mechanisms by which neuronal connections are lost in the Toxo-infected brain (which is a mechanism that leads to loss of neuronal connections in the injured and aging brain as well). This is significant because it might provide insight into why some people who are infected with Toxo develop seizures or mental illness, while others don't. More importantly, Toxo-infection causes changes in the brain that are very specific, in terms of both the type of neuronal connection that is affected and the type of cell that is affected. Why these changes are so specific remain to be uncovered, but it suggests that Toxo can either a) trigger a unique immune response in the brain that leads to very precise changes in neuron-toneuron connections and signaling or b) the parasite, while hiding inside of neurons, may hijack the machinery of certain cell types in a way that helps them survive longer.

Yes / Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country. / Erasmus Mundus Joint Doctorate Program of the European Union through a training grant to WS and the European Research Council-INTERRUPTB starting grant (nr.311725) to BdJ.